ANIMAL GENETIC ENGINEERING RESEARCH SECTION, SHANGHAI KEY LABROARTORY OF AGRICULTRUAL GENETIC AND BREEDING

1. 研究方向
根据上海都市农业发展要求和畜牧兽医科技服务都市公共卫生工作的需要，利用现代生物技术进行动物及人畜共患病免疫诊断制剂的研制。同时追踪国内外动物生物技术的最新进展，结合现代免疫、生物信息及动物转基因克隆技术，选育和开发抗病动物品种
1. Research orientation
Zoonotic diseases and new types of animal vaccines, diagnosis reagents and recombinant proteins for enhancing animal growth or immunology. Research on animal transgene and cloning were also carried out.
2. 在研项目进展
2. progress of current research project 
2.1动物基因工程重组蛋白微球注射剂缓控释技术的研发
(1) 建立重组牛生长激素基因工程大肠杆菌高密度发酵工艺，发酵菌体密度OD550达到60以上；目标蛋白表达量在25％左右。
(2) 确立包含体提取及变复性工艺，包含体纯度在80％左右，复性重组牛生长激素纯度达90％以上，生物活性检测表明，可使去垂体大鼠每天增重1－1.5g。
(3) 建立重组牛生长激素原料冻干工艺，采用聚乙二醇（PEG）为赋形剂效果最好。
(4) 以PLGA50/50，1.4万作为微球骨架材料，S/O/W和W/O/W两种方法载药量均可达到10%以上，包封率可达95%以上。体外释放试验表明S/O/W制备的微球突释小，释放较W/O/W均匀，可缓释两周左右。但两者体外释放累积量不超过50%。
(5) 采用W/O/W 和S/O/W时稳定剂如糖类、表面活性剂对牛生长激素的稳定性均无明显提高。而采用S/O/O时，对牛生长激素的稳定性均有一定的提高，其中以甘露醇的效果最好，使90%的蛋白可以复性；有机试剂二甲亚砜、丙酮对蛋白活性的影响较其他有机试剂小。
(6)采用5%蜂蜡作为增稠剂，以PEG作为致孔剂，制备的重组牛生长激素油混悬制剂首日释放量为20.3%，且能持续释放至31天。
(7)重组牛生长激素油混悬制剂去垂体大鼠动物试验表明此种油混悬注射剂在体内至少能缓释31天，其有望发展成为新型长效制剂。
2.1 Study on the injectable microsphere dose of animal recombinant protein
(1) .High cell density fermentation of recombinant bovine growth hormone E.coli. had finished, cell density is about OD550=60, the rate of recombinant protein to total cell protein is about 25%. 
(2) Recombinant bovine growth hormone (rBGH) had been successfully extracted from host cell in inclusion bodies. The technology to denature and renature rBGH had finished. The renaturation ratio is about 40%,and the purity is about 85%. Injection experiment of hypophysectomized rats proved that we have obtained r-pST with high native bioactivity.
(3) The lyophylization technology of recombinant bovine growth hormone have been finished and found the protein power has 93% activity by adding PEG as protection reagent.
(4)  The carrying ability of S/O/W and W/O/W using PLGA50/50 are at least 10%, packing ratio is more than 90%. In vitro tests indicate that the release of the microglobule prepared by S/O/W is more gradually than that prepared by W/O/W, but the total release rate is less than 50%.
(5) Stabilizing agent such as saccharide or surfacent has no effect on the stability of BST when prepared by S/O/W and W/O/W, but could improve the stability of BST prepared by S/O/O. DMSO and acetone have less maleficence on the protein than other organic solvent.
(6) The percentage of the medicine release of the recombined bovine somatotropin (rbST) oil suspension, with beeswax as dope and PEG as poriform, was 20.3% on the first day, and then released successively for 31 days.
(7) The study on the release effect of recombined bovine somatotropin (rbST) oil suspension with male hypophysectomized rats indicates that the oil suspension can release successively in vivo for about 31 days and it has the potential to be developed successfully as a new long active rbST formulation．
2.2 猪源性戊型肝炎阻断技术研究
猪戊型肝炎病毒（HEV）主要有1，2，3，4四个基因型，此前，中国大陆仅发现基因1型和4型。本研究从上海郊区的5个猪场收集了65份猪粪便样品，通过扩增HEV ORF2部分区域的150nt基因片段进行HEV RNA RT-PCR检测，结果发现15份样品为阳性，进化树分析显示其中8株为基因4型，7株HEV为基因3型，此基因3型与美国的猪US-SW HEV毒株同源性较高，这是迄今为止在中国大陆首次发现猪的HEV基因3型。随后采用相同方法对分布在上海9个区36个猪场的414份猪粪便样品进行猪HEV流行病学调查，结果显示111份为HEV RNA阳性，不同区的HEV感染率为8.3% to 41.7%。对其中的32份阳性样品的150nt基因片段进行测序，进化树分析显示10株为基因4型，22株为基因3型，此研究结果提示基因3型HEV已在上海各区猪场普遍流行。由于大陆地区至今尚未有人感染HEV基因3型的报道，因此本研究分离到的猪基因3型是否具有人畜共患性及其来源需要进一步研究。
2.2 The effective prevention of HEV infection in swine
HEV isolates have been classified tentatively into four major genotypes, genotype 1, genotype 2, genotype 3 and genotype 4. Only strains of HEV genotype 1 and genotype 4 have been isolated so far in china mainland. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. The genotype 3 isolate was most closely related to one swine HEV strain, US-SW, which was isolated from pigs in the United States. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland. To estimate accurately the prevalence of the HEV infection in shanghai , 414 swine feces from 36 different pig farms located all 9 different districts of Shanghai suburb were detected for HEV RNA by using the same method. The result of RT-PCR revealed that 111 HEV positive samples were found among 414-pig fecal specimens. The prevalence of HEV infection in different districts was varied from 8.3% to 41.7%. Among the 111 HEV positive samples, 32 samples were sequenced. Phylogenetic analysis of the amplified sequences indicated that 10 isolates belonged to genotype 4 and the other 22 isolates belonged to genotype 3. The result indicated that genotype 3 HEV has been widespread in pig farms of shanghai suburb. To our knowledge, the human infection of genotype 3 in china mainland has not been reported，so the zoonotic potential and the source HEV genotype 3 in shanghai need further study.  
2.3 口蹄疫注苗与感染牛（血清）鉴别诊断试剂盒中试生产研究
(1)确定的检测抗原制备工艺可用于该抗原蛋白的中试生产；项目已完成该指标
(2)使用确定的工艺生产的NSP-ELISA试剂盒保存期为6个月；本项目组装的试剂盒保存期可达10个月，但规定可使用的期限为6个月，以保证检测结果可靠性。
(3)修正的临界值对注苗与感染牛（血清）的检测特异性高于90%，本研究组装的试剂盒对阴、阳性样品检测特异性达91%以上；
(4)完成试剂盒2万头份检测量的中试生产，抽样检查试剂盒的检测误差不高于10%（与UBI公司产品比较检测）；项目完成近5万头份检测量的抗原生产，抽样检测误差低于10%。
(5)完成试剂盒1万头份检测量的推广应用，使用报告证明试剂盒可用于口蹄疫注苗与感染牛（血清）的鉴别诊断；本项目已完成18000余份样品检测工作，并且使用报告证明可用于临床FMDV鉴别诊断。
(6)提交试剂盒制造规程和半成品、成品检验标准；已撰写了生物制品申报规程，准备提交。
(7)提交农业部新兽药（生物制品）申报材料。在进行中
与国内外同类技术比较：研制的试剂盒与国际同类产品性能相当，达与国际先进水平（见查新检索报告）
成果应用前景和范围、存在问题及改进意见：产品可用于畜牧养殖场FMDV野毒感染检测，净化畜群；进出口活畜检验检疫，防止携带FMDV野毒动物进入我国；消费市场检测，防止携带FMDV的畜产品进入流通。据农业部统计，猪存栏4.8亿头（2006上半年），牛存栏14157.5万头（2005年），羊存栏达37265.9万只（2005年），总计近10亿头。按照我国口蹄疫防制计划和建立无规定疫病区的要求，口蹄疫易感动物的免疫接种密度必须达到80%，即注苗动物总数为8亿头（只），其中牛11326万头，按10%的抽检比例计算，每年应检测1132万头。按本项目所研制的试剂盒每头份销售价格为5元计算，则一年的产值约为7140万元（人民币）
2.3 Studies on serological detection methods in differentiating of bovine vaccinated with those of infected. 
(1) The technology of antigen preparation can be used in the pilot scale production
(2) The test for NSP-ELISA kit preservation is done for more than ten months, and six month preservation was the suggestion, so that the detection result is reliable.
(3) The detection specificity of immunized bovines and infected ones is higher than 90% to the revised critical value; the detection specificity of the kit is higher than 91% fot detecting of the negative and positive sample.
(4) More than 20000 doses of detection kit have finished by pilot scale production; the relative detecting accuracy of the kit is less than 10% comparing with the control of UBI co., we have detected more than 50,000 cattle serum using the kit, the relative accuracy of sample drawing and detection is lower than 10%.  
(5) Application of the kit for 10,000 bovines serum detection, and the using report displays that the kit can be used for the differential diagnosis of immunized bovines and infected ones; our project have detected 18,000 samples, and the report of detection displays that the kit can be used for the clinical differential diagnosis of FMDV. 
(6) The procedure of the kit production and the test criterion of the semi-product and final product will be; we have prepared the procedure of biological product, we will refer it.
(7) We are referring the materials of the new veterinary drugs to the Ministry of Agriculture.
Comparing to the same trade: 
Our technology has achieved the advanced level of the world.
The outlook of application and the problems: 
The kit can be used to detect the FMDV street strain of the domestic animal; it can be used in the quarantine of the importing livestock; it also can be used to detect the FMDV in the livestock circulation. According to the statistics of the Ministry of Agriculture, the number of breeding pigs in the farm is 4.8×108(2006 .1-6), the number of breeding cattle in the farms is 14157.5×104 in the year of 2005, the number of breeding sheep in the farms is 37265.9×104 in 2005, the total number of the three kind of domestic animal is nearly to 10×108. According to the plan of FMDV prevention and cure, the inoculation rate of the susceptible animal must be 80%, so the total number of inoculated animal is 8×108, including 11326×104 cattle. According to the sampling inspection rate of 10%, 1132×104 cattle should be inspected. Our kit can be sold 5 yuan/cattle, so the value of output will be 7140×104 yuan per year(RMB).
2.4猪流感监测诊断技术研究
(1) SIV血清学检测方法建立：
利用原核系统成功表达了猪流感病毒NP蛋白；SDS-PAGE电泳检测其分子量为60ku，经western blot分析，表达产物被SIV抗血清能特异性识别。
完成了重组NP蛋白小量发酵工艺条件的优化，用表达产物作琼脂扩散试验进行活性鉴定，证实该融合表达的蛋白具有生物学活性，且只与SIV血清有免疫反应，而与其他几种猪病毒血清不发生反应，可作为检测用抗原。
完成了表达蛋白小量纯化条件摸索，采用试剂盒纯化，获得高纯度重组蛋白。经过对抗原包被浓度、ELISA板的封闭方法、样品稀释方法、二抗、底物的选择、结果判定等进行试验和筛选，确定ELISA检测试剂盒基本组成和操作。
进行了试剂盒敏感性、特异性、重复性及符合率试验，结果，猪流感重组NP抗原ELISA检测法比HI、AGP方法敏感性高；与美国IDEXX公司SIV抗体检测试剂盒比较，阳性血清中的检出率即为相对灵敏度为91.6%，两者对样品检测的符合率为94%，两种系统的检测结果无显著差异（P>0.05）。试剂盒批内重复试验、批间重复试验无显著性差异（P＞0.05）；撰写了申报规程草案，并通过初审。
(2)猪流感病毒分型检测技术研究
SIV 各型通用NP基因通用引物设计筛选：下载Genebank各种亚型NP基因模板，H1、H3、H5、H9、N1、N2序列模板，采用Aligment软件进行类比分析，筛选保守区域，设计引物，按照扩增片段大小、引物GC含量百分比，再通过模拟PCR（Spcr）筛选与主要血清亚型（H1/H3、H5/H9）基因模板PCR扩增产物情况，筛选到理论上符合条件的引物。
HA分型多重PCR分型检测体系初步建立 根据NP/H1/H3亚型及NP/H5/H9亚型引物扩增片段大小不同，反应条件的差异，将各引物配比组合，形成不同的配组，再用混合模板（H1/H3；H5/H9）进行扩增，验证引物组合扩增单一模板、混合模板的检测效果，获得针对NP/H1/H3、NP/H5/H9、N1/N2模板的两体系多重引物组合。
根据试验筛选到的引物组合，组建多重RT-PCR试剂盒，试剂盒含有从RNA 进行RT及PCR过程所需各组分。
对试剂盒灵敏性和特异性检测：将RNA模板按比例稀释，分别用建立的体系做RT-PCR检测，分析本试剂盒检测灵敏度，结果：H1/H3组合对H1模板最低检出量为0.1ng，对H3模板最低检测量为2ng。而扩增PRRS、PPV、PRV、PCR病毒核酸，结果均为阴性，表明试剂盒特异性好，与几种常见猪病原无交叉反应，可用于SIV检测。
2.4 Detection and diagnosis of swine influenza virus
(1) Studies on SIV serology detection method:
The NP protein of SIV has been successfully expressed in the prokaryotic system; the protein’ molecular weight is 60 kd which is displayed through the SDS PAGE. The protein can be recognized by the SIV antiserum in the western blot.
 We have optimised the minor fermentation technology of the fused NP protein. The protein has the biological activity in the agar diffusion reaction, and the SIV serum can only recognize the protein, the other virus’ serum cannot recognize the protein, so the protein can be used to detect the SIV.
 We have finished the optimization of the minor purification technology of the NP protein, and we harvested the pure protein. We have ascertained the composition and procedure of the ELISA detection kit through coating antigen, method of blocking, the assessment of the result, and the choosing of the substrate and the antibody against the anti-serum.
  We have done the experiment of the kit’s sensitivity, specificity, repeatability, and coincidence. The result displays that the method of NP protein Elisa detection is more sensitive than the method of HI and AGP. The positive serum’ detection rate of the kit is 91.6%, compare to the SIV antibody detection kit of the American company IEXX, the coincidence rate of the two kits is 94%, the two system don’t have a significant deviation (p> 0.05). There is not a significant deviation in an experiment and the different times’ experiments (p> 0.05). We have finished the regulations of the kit, and it has been qualified in the first inspection.
(2) The genotyping diagnosis of the SIV:
 The design of the NP gene universal primer of the deferent type of SIV:
The deferent subtype NP gene templates, and the H1, H3, H5, H9, N1, N2 gene sequences have been downloaded from Genbank. We designed the optical primers by analyzing in the software of Aligment, screening the conservative region, screening the length of the PCR product, comparing the percentage of GC, screening in the simulation PCR, and the PCR amplification of the subtype H1/H3、H5/H9.
(3) The preliminary construction of the HA multi-PCR genotyping system:
 According to the length of NP/H1/H3, NP/H5/H9 amplification product and the condition of PCR, we mix the deferent primers and templates in deferent ratio to amplify the product. We verified the detection result of single template and multi-templates, obtained two systems of multi-primers groups for NP/H1/H3, NP/H5/H9, and N1/N2 templates.
According to the screened primer groups, we construct the multi-RT-PCR kit, which contains the composition for the reverse transcription and the PCR.
The sensitivity and the specificity of the kit:
We used the constructed kit to do the RT-PCR to detect the RNA template, which was diluted in proportion. The result displayed that the minimum value of the subtype H1 template can be detected is 0.1 ng by the H1/H3 primer group, and the minimum value of the subtype H3 template is 2ng. We used the kit to detect the PRRS, PPV, and PRV, and the result is negative. It displays that the specificity of the kit is well, it don’t have the cross-reaction with the universal swine’s pathogen, so it can be used to detect the SIV.
2.5猪种种质资源长期保存技术研究
(1) 猪精子冷冻技术的研究：筛选并研制出高效、低毒（无毒）的冷冻保存液，进一步优化了冷冻程序；建立了猪精子冷冻保存的实用化技术--精液大管（5ml）冷冻技术，克服了传统颗粒、细管法有的局限，实现一支冷冻精液输精一头母猪，填补了这一技术在国内的研究、使用空白，使猪精液冷冻保存技术在地方猪种种质资源长期保存中更具可操作性。冻精解冻后精子活力达0.4级以上,受胎率达70%以上。
(2) 猪卵母细胞玻璃化冷冻保存：GMP法是一种冷冻猪卵母细胞的有效方法，与普通麦管法相比，该法能显著提高猪MII期卵母细胞冻后存活率（34.5% vs 63.3%）。对卵母细胞的冷冻保存、体外受精、冻卵的超微结构和细胞骨架变化等进行了系统的研究，表明冷冻过程中细胞骨架的破坏是影响猪卵母细胞(胚胎)冻后复苏的主要原因。本实验首次报道猪GV期卵母细胞冷冻保存后，经体外受精获得桑椹胚发育；在国内率先报道冷冻猪卵母细胞内细胞骨架的变化，
(3) 猪胚胎冷冻保存的研究：研究冷冻保存液、胚胎包装方法、冻前胚胎预处理技术等对猪胚胎冻后存活力的影响；比较程序法、OPS法（开放式拉长麦管）和GMP法（玻璃微细管）的冷冻保存效果；建立了猪胚胎玻璃化冷冻技术。在我国首次获得猪胚胎超低温（–196℃）冷冻后代。GMP法玻璃化冷冻法产仔率30%(3/10)。
在完成上述研究的基础上，成功保存了3个地方品种（系）的冷冻精子和冷冻胚胎。
2.5Research on techniques of long-term conservation for pig breeds
(1) Study on the cryopreservation of boar spermatozoa: According to a series of experiments, one kind of high efficiency and low-noxious(innocuity) cryopreservation solution was developed; the techniques of 5mL maxi-straws for the freezing of boar spermatozoa, a practical method, which overcomed the limit of conventional pellet and straw methods and successfully realized using only one 5mL maxi-straw in each insemination.of sow. The techniques padded an item of domestic blank in the area of sperm freezing, which made the cryopreservation of boar spermatozoa become more maneuverability in the long-term conservation of pig breeds. The sperm motility after warming could reach 0.4 and the pregnant rate could get more than 70%.
(2) Cryopreservation of porcine oocytes by vitrification: GMP(glass micropipette) is an effective method for vitrification of porcine oocytes. Compared with conventional straw method, this method could significantly improved the survival rate(34.5% vs 63.3%) of porcine MII stage oocytes after warming. The systemic research on the cryopreservation, in vitro fertilization and the ultrastructure & cytoskeletal of vitrified oocytes showed that the damage of cytoskeletal structure was one of the key reasons that caused the low efficiency of the cryopreservation of porcine oocytes. In our experiment, we first got the morula development after in vitro fertilization porcine vitrified GV stage oocytes in the world and first reported the cytoskeletal change of porcine vitrified oocyes at home. 
(3) Cryopreservation of porcine embryos: Studies on the effects of different cryopreservation solution, vitrified carriers, and different pre-treatments to the viability of porcine embryos; Comparison the different freezing methods on the effect of the embryo cryopreservation, such as the programmed freezing with straw, OPS(open pulled straw) method and GMP method; Establish a reasonable technique of porcine embryo vitrification method and got the first example with alive piglets by vitrification in China. The litter rate of GMP vitrification method was 30%.
Based on those research of porcine breed resources, we successfully cryopreserved three kinds of local pig sperms and embryos.
2.6大肠杆菌热敏性肠毒素作为疫苗黏膜免疫佐剂功能研究
利用体外PCR扩增、重叠延伸方法结合，获得热敏性肠毒素6个突变体：三个位点单点突变体3个、3个双位点突变体。分别克隆到T载体中，进行测序，以确定突变位点的正确性。将连接到T载体上的外源基因亚克隆到原核表达载体，经PCR鉴定和序列分析证实，成功获得了6个LT表达突变体。
分别采用不同的表达条件：温度、培养基、宿主表达菌、不同诱导时间等表达重组蛋白，筛选最佳表达条件，SDS-PAGE电泳证表达了两种不同分子量的蛋白LTA、LTB。
FPLC纯化LT突变体表达蛋白，经用HIStag抗体和抗CTB抗体进行Western检测，结果两条蛋白带均可与抗His-tag抗体产生免疫学反应，而LTB还可与具有相似免疫原性的抗CTB抗体发生反应。
利用patent-mouse小鼠毒性试验、ADP-核糖转移酶活性试验筛选毒性、酶活性也降低的LT突变体，目前已成功筛选到具有较低毒性的LT突变体，为后续免疫佐剂功能研究奠定了基础。
2.6 Adjuvant function of E. coli. Heat labile enterotoxin through mucosal immunization.
Six heat-labile enterotoxin mutants by PCR amplification and overlap extension were obtainede, which include 3 single site mutation and 3 mutants of two site mutation. The mutate gene were cloned into the T-vector, and sequence it to verify the mutante site mutation. Then the mutate gene were sub cloned into pET vector separately, and abstained 6 mutants of LT. The positive clones were verified by sequencing and PCR. 
 The condition of expression was optimized, which included the temperature, the nutrient medium, the expression bacteria, and the induction time of the experiment. The two deferent molecular weight proteins LTA and LTB were displayed in the SDS-PAGE.
 The mutant protein LT was purified by FPLC. The two proteins both can react with the anti-His tag antibody in the western blotting, and the LTB protein can react with the anti-CTB antibody.
  We have screened the low virulent LT mutant by the patent-mouse toxicity experiment and the ADP-ribosyltransferase activity experiment; it has established the base for the research of the immunoadjuvant function
2.7猪流感及新城疫病毒检测方法研究
(1) SIV多重PCR技术研究及标准制订研究进展
病毒增殖培养：将保存的SIV 亚型流感病毒毒种取出，接种鸡胚进行增殖，收获病毒后，HA/HI测定病毒效价及血清亚型。
引物设计：采用Primier Primer 5.0 设计了一系列引物组合，针对流感病毒的NP基因，分型检测的H1、H3亚型引物组合。
核酸提取及单一PCR体系建立与引物对筛选：采用Trizol方法提取病毒基因组RNA，分别采用H1、H3亚型病毒RNA筛选NP基因引物组合。再分别用不同亚型引物扩增模板，筛选型特异性引物对。已筛选到NP基因2对，H1亚型特异性引物对3对，H3亚型特异性引物对3对。且扩增片段大小不同，可用于多重PCR体系组合。
对不同引物组合进行了初步筛选，已经获得较好的H1/H3亚型组合，N1/N2组合，并组装了鉴别诊断试剂盒。对试剂盒灵敏性特异性检测表明，可用于SIV分型检测。
 起草了上海市地方标准申报书，并获得上海市质量技术监督局批准，制订上海市地方标准“猪流感病毒及H1/H3亚型多重RT-PCR检测方法”，目前正撰写送审稿。
(2) 出口家禽新城疫病毒检测方法研究
通过对大量不同毒力新城疫病毒Ｆ基因核苷酸序列分析，根据毒力和序列间的相关规律，已分别设计与合成了两组引物。
已完成对大批量临床样品整理及病毒分离工作， 建立了PCR-ELISA检测新城疫病毒方法，并获得上海市质量技术监督局批准，与上海出入境检验检疫局合作制定上海市地方标准地方标准。
2.7 Detection method of swine influenza virus and Newcastle disease virus
 (1) Study on multi-PCR detection method of SIV and the standard of the method
   The viral multiplication cultivation: different subtype influenza virus were inoculated into the chicken embryos, and detected the titer of the virus and the subtype of the serum by the HA/HI.
   The design of the primers: We designed a series of primer groups, included that the primers for the NP gene and the primers for the subtype H1, H3.
RNA extractions, construction of single PCR system and screening of the primers: genome RNA of the virus was extracted by Trizol reagent, and screened the NP primer pairs of the subtype H1, H3 virus. Then we screened the specific primer pairs by the deferent amplification template of the subtype.
Specific H1/H3 and NP primers pairs were obtained, and a serious primer mixture were gained to construct the diagnosis kits. It is proved that it can be use to detect the SIV by the sensitivity and specificity experiment.
After the method was established, local standard on multiplex RT-PCR for detection and subtyping SIV was drafted, and the Shanghai Municipal Bureau of Quality and Technical Supervision licensed it. Now we are soliciting the suggestion and writing the illustration of the standard.
(2) The research of the detection of Necastle Disease Virus of the exporting poultry:
Two pairs of primers have designed and synthesized by analysing the deferent NDV strain’ s F gene sequence.
The disposal of the lots of clinical samples and the isolation of the NDV have finished by using 9-day old SPF chichen embryos.
We have established the NDV PCR-ELISA detection method, and the Shanghai Municipal Bureau of Quality and Technical Supervision has licensed it. We cooperated with the Entry-Exit Inspection and Quarantine Bureau to institute the local standard.
2.8 猪核移植胚DNA甲基化改变与“印迹”基因表达特征研究
（1）建立了猪核移植供体细胞培养技术。
首先选定合适的猪核移植所需的体细胞。通过比较成纤维细胞、卵丘细胞、颗粒细胞、输卵管上皮细胞等体细胞。表明成纤维细胞、卵丘细胞是较合适的供体细胞，取材方便，形成重构胚后囊胚发育率较高。
（2）改进了猪卵母细胞去核方法。
猪卵母细胞去核是影响猪体细胞核移植的关键技术之一。本实验室对传统的McGrath-Solter、Willadsen去核法技术进行了改进，建立了二步挤压去核法，在猪体外成熟卵母细胞上的去核操作表明，无论在去核操作成功率（73.9%）、还是在去核效率（81.2%）都显著好于McGrath-Solter去核法（42.5%、67.4%，p<0.05）。
（3）建立了猪体细胞核移植重构胚的融合、激活和核移植重构胚体外培养方法。
细胞融合液组成为：0.3mol/L甘露醇，0.4mol/LMgSO4, 0.5mol/L CaCL2和3% BSA；融合条件为：100 V/mm DC，30μs刺激一次；电刺激后的重构卵在NCSU-23（加4 mg/mL BSA）培养液中培养30min。                   
激活和核移植重构胚体外培养方法：将重构胚置于10μmol/L钙离子载体A23187的NCSU-23培养液中培养5min 或1个直流脉冲（125 V/mm，70μs），随后置于NCSU-23（含2mmol/L 6-DAMP 和4 mg/mL BSA）培养液中培养5h，培养条件39℃,5％CO2。
核移植重构胚的体外发育囊胚率最高可达11.7%。
(4) 猪核移植胚DNA甲基化改变与“印迹”基因表达特征研究
在获得各阶段正常受精胚、体外受精胚、孤雌激活胚及体细胞核移植重组胚的基础上，采用免疫荧光技术研究体细胞克隆胚各时期DNA甲基化的变化；采用定量RT-PCR
（real-time）技术比较U2afbp-rs基因的表达。表明，体细胞核移植重组胚体外发育过程中DNA甲基化、U2afbp-rs基因的表达发生了显著的变化。
该研究初步阐明了猪体细胞核移植重组胚早期发育过程中死亡率高的分子生物学机理，从而为提高体细胞克隆猪成功率奠定基础。
2．8 DNA methylation and the expressing characteristics of “imprinting gene” in porcine nuclear transfer reconstituted embryos
（1）The techniques of the culture of donor cells for nuclear transfer
First the different donor cells was examined to establish a preliminary procedure for porcine cloning. Fibroblast cells(FC), oviduct epithelial cells(OEC), granulose cells(GC) and cumulus cells(CC) were taken in this experiment. The FC and CC were considered as the better donor cells because of its convenient sources and high blastocyst developmental rate of reconstructed embryos.
（2）Improve of the enucleated method of porcine oocytes
Enucleated method is one of the key techniques for animal cloning. The results show that the modified Willadsen’s method in our lab for enucleation, two step-squeezing method is significantly better than McGrath-Solter in the enucleated rates(73.9％, 42.5%, P<0.05) and the rates of absolutely removing nuclei matter(81.2%, 67.4%, P<0.05). 
（3）The methods of electronic fusion, activation and the in vitro culture of reconstructed embryos have been successfully established in our lab.
The composing of the electronic fusion were 0.3mol/L D-mannitol, 0.4mol/LMgSO4, 0.5mol/L CaCL2and 3% BSA. The condition of the fusion were 100 V/mm DC, 30μs and only one times. The electronic activated embryos were cultured in the medium of NCSU23(4 mg/mL BSA) for 30 min. ………………
The method of activation and the in vitro culture of reconstructed embryos were: put the reconstructed embryos into the medium of NCSU23 contained with 10μmol/L A23187 for 5 min and one time of 125 V/mm and 70μs DC, then the embryos were transferred into the NCSU23(with 2mmol/L 6-DAMP and 4 mg/mL BSA) for 5 min.
The blastocyst developmental rate of reconstructed embryos could got 11.7% in vitro.
（4）DNA methylation and the expressing characteristics of “imprinting gene” in porcine nuclear transfer reconstituted embryos
In this experiment, different stage embryos from normal fertilized, in vitro fertilized, parthenogenetically activated and nuclear transferred embryos were got; The Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 was adopted to investigate the change of DNA methylation of different developmental nuclear transferred embryos; The expression of the U2afbp-ra gene was compared by a real-time PCR techniques. The results showed that the DNA methylation and the genic expression of the U2afbp-ra changed significantly with the development of reconstructed embryos cultured in vitro.
This study partly clarified the molecular biological mechanism of the high mortality rate of reconstructed embryos in the early development, and layed a foundation for improving the efficiency of porcine nuclear transfer.
2.9 猪流感病毒表位疫苗研究
根据流感病毒HA、NA基因核酸序列与蛋白结构预测，采用在线软件筛选疏水区域，选择不同亚型HA、NA抗原位点1-2个，串联形成表位基因片段。经PCR扩增后酶切连接到原核表达载体中，并通过诱导表达获得了重组蛋白，SDS检测蛋白表达分子量21.0 kd。经Western 检测，该蛋白可与SIV  H1、H3亚型血清发生免疫反应，通过变性条件纯化，获得了纯化蛋白，正在进行大量表达与蛋白活性分析试验。
通过在N、C端分别连接免疫佐剂分子，构建了表位基因片段与分子免疫佐剂基因串联表达载体，并成功获得原核表达。期望通过分子内佐剂作用来提高表位疫苗免疫效果，通过SDS-PAGE及Western blot检测分析了表达产物免疫活性，证实了表达蛋白的生物学活性，目前正进动物免疫试验。
2.9 Study on epitope vaccine of swine influenza virus
 We analyzed the HA, NA gene sequence, anticipated the protein structure, and screened the hydrophobic region in the online software. After that, we selected 1-2 antigen sites of the subtype gene, and constructed the tandem epitope gene. The epitope gene was cloned into the prokaryotic expression vector by PCR and enzymolysis. The protein was expressed, and its molecular weight is 21.0 kd. The protein can react with the serum of the SIV subtype H1/H3.We purified the protein under the denaturation condition, now we are massively purifying the protein, and analyzing the activity of the protein.
We constructed the tandem epitope and immunoadjuvant gene vector by ligating the immunoadjuvant gene to the end of N’and C’, and it was successfully expressed. We hope that the internal molecular immunoadjuvant can enhance the immunizing potency of the epitope vaccine. The protein is active which is proved by the SDS-PAGE and western blot. Now we are massively purifying the protein so that the animal immunity test can be carried out in the next step
2.10猪体细胞克隆技术的建立和优化
探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后，将经血清饥饿（0.5%FBS）培养2-9d、0.1mg/L Aphidicolin(APD)培养+0.5% FBS培养2-9d或一般培养法（10%FBS）培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞，直接注射到去核的卵母细胞质中，或注射到卵周隙中，再经电融合（100V/mm， 30μs，电脉冲1次）构建重构胚。重构胚以钙离子载体A23817 或电脉冲结合6-DMAP 激活处理，体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1 mg/L APD + 0.5% FBS培养处理后的重组胚卵裂率，均高于血清饥饿和一般培养处理的同种供体细胞（P<0.01）。卵丘细胞、颗粒细胞经0.1 mg/L APD + 0.5% FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞（P<0.05）。以猪颗粒细胞为核供体时，电融合法的重构胚分裂率显著高于胞质内注入法（P<0.05），但囊胚发育率无显著差异（p>0.05）。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞，其具有正常二倍染色体的细胞比例均无显著差异（P>0.05）；以这2种细胞不同培养代数做供体进行核移植时，各代之间核移胚的体外分裂率、囊胚发育率无显著差异（P>0.05）。这些结果表明：（1）猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定；（2）0.1 mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果，血清饥饿培养则无明显效果；（3）猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞，核移植后都能得到体细胞克隆的囊胚，但前者的效果略优于后者，且其核移植效果不受供核细胞培养代数的影响；（4）电融合核移植胚胎的发育率高于胞质内直接注入法，但两者的总体效率相近。
在完善体细胞核移植重构胚构建方法的基础上，课题组从10月份开始，在巴马小型猪、上海白猪上进行重构胚的移植，目前正在观察移植效果，以决定下一步的研究工作重点。
2.10 The establishment and optimization of porcine cell cloning technique
This study was undertaken systematically to examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44 h and then enuleated in manipulation medium containing 5μg/mL cytochalasin B Fibroblast cells(FC), oviduct epithelial cells(OEC), granulose cells(GC) and cumulus cells(CC) after 3~9passages in TCM+10%FBS for2~9 days or continue culturing in 10%PBS for 2~9 days, then, transferred into enucleated oocytes by microinjection or electronic fusion(100V/min, 30μs and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of reconstructed embryos with FC and GC pretreated with 0.1μg/mL APD+0.5%PBS were significantly higher than that of serum starvation group and control group(P<0.01). There were significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC, and FC, OEC pretreated with 0.1μg/mL APD+0.5%PBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection(P<0.05), 3="" 6="" 10="" but="" no="" difference="" was="" found="" in="" their="" proportion="" developing="" to="" blastocysts.="" of="" gc="" at="" and="" fc="" passages="" had="" normal="" which="" did="" not="" show="" significant="" among="" p="">0.05) When GC at G3, G4, G5 and G6 of passages were as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1μg/mL APD to treat donar cells to nuclear transfer can improve the efficiency of somatic cell muclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GCcells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is high than that by microinjection, but there is no difference in the total efficiency between the two methods.
Based on the efficient nuclear transfer system, our research group operated the reconstructed embryo transfer in the Bama minipigs and Shanghai White pigs from last October. Now we are observing the result of nuclear transfer and decide the emphases in our next research work
2.11重组毕赤酵母高密度发酵表达猪α干扰素的研究
本项目已经完成。在单因素试验的基础上，利用Plackett-Burman实验设计优化重组毕赤酵母产猪IFNα的生长和诱导条件，采用ANN-PR-控制方式建立了重组毕赤酵母在5L发酵罐中高密度分泌表达重组猪α型干扰素的工艺。                   
 and high density fermentationa2.11 Experssion conditions of porcine IFN- for genetically engineered Pichia Pastoris
This project has been finished. The growth phase and induce phase conditions of the genetically engineered Pichia Pastoris have been established according to Plackett-Burman which based on single factor experiment. We have established the expression technology of  in Pichia Pastoris using ANN-PR controllingarecombinant porcine IFN- method.
2.12 重组猪α干扰素的本体动物实验
重组α猪干扰素的发酵液通过离心、透析、过滤等步骤进行了处理。采用WISH细胞-VSV系统，以病变抑制法检测了重组猪α干扰素的生物学活性，效价为1.2×106IU/mL，利用BCA法检测了重组α猪干扰素的蛋白含量，含量为1.6mg/ml。现已初步选定甘露醇、甘氨酸、牛血清白蛋白、右旋糖苷作为重组α猪干扰素的冻干配方，已进行了共熔点的测定，下一步将对以上配方进行筛选。
2.12 aThe animal experiment of porcine recombinant IFN-
Porcine recombinant (PoIFNα) fermentation has been dealt with according to centrifugal,aIFN-   hasadialysis and filtration . The biology activity of porcine recombinant IFN- been detected using WISH-VSV system and cytopathic effect method. The anti-virus activity of PoIFNα is1.2×106IU/mL. The production of PoIFNα concentration is 1.6mg/ml using the method of BCA. Four lyoprotective protocol have been tested to select the optimum lyophilized formation of PoIFNα. Their common melting point has been detected.
2.13新疆阿克苏地区肉牛的改良与提高
课题组已于2006年7月、8月，赠送黑白花牛、南德温牛精液各1000支给新疆维吾尔自治区温宿县畜禽品种改良站，按照课题要求在当地有计划地进行杂交改良工作。
2.13The improvement of the meat cattle in Xinjiang Akesu region
Our research group has sent 1000 numbers of freezing sperm of Holstein bull and South Devon cattle to the Wensu county rescue station of Xinjiang Uygur Autonomous Region for free. Now we are designedly processing the improve work according to the request of the task.

猪IFNα在毕赤酵母中分泌表达条件
的优化及高密度发酵
蓝胜芝1,2于瑞嵩2,3，俞明月1,2，董世娟2,3，曹祥荣1，李震2,3*
(1)．南京师范大学生命科学学院，江 南京210097)
2)．上海市农业遗传育种重点实验室．上海20l106)
3)．上海市农业科学学院畜牧兽医研究所，上海20l106)
摘要  从发酵时间、接种量、pH值、诱导剂量等方面对重组基因工程(毕赤酵母甲醇利用缓慢型)的摇瓶发酵条件进行了优化，进一步探索了酵母菌表达猪IFNα的发酵工艺，包括种细胞密度对初始发酵期的影响，补加甘油、甲醇速率条件的控制等．结果表明，摇瓶发酵时，诱导最佳时间为96 h，甲醇最适浓度为8 g／L，发酵pH范围为6.4～9.0．最适接种量2:1．在分批发酵、接种量为10ck且种子细胞光密度(0D600)为5～6时，最有利于细胞的高密度培养，在补料发酵时，根据溶氧控制甘油流加速率与时机，细胞干重达到11 8.75g／L，在48h重组猪IFN-α表达量达到1.304 g/ L ．
关键词  猪IFNα，毕赤酵母，摇瓶发酵，高密度发酵
Studies on Expression Conditions of PoIFNα and High•density Fermentationfor Genetically Engineered Pichia Pastoris
Lan Shengzhi 1,2，Yu Ruisong2,3，Yu Mingyue 1,2，Dong Shijuan2,3 ，Cao Xiangrong1 ，Li Zhen2,3* 
(1)，School of Life Sciences，Nanjing Normal University，Nanjing 2 1 0097，China
2)．Shanghai Key Laborator3． Agricultural Breeding，Shanghai 201 106．China  
3)．Institute of Shanghai Key Laboratory Livestock Breeding and Veterinarian，Shanghai 201106．China)
Abstract：The optimum fermentation conditions of genetically engineered Pichia pastoris in shake-flask cultivation and in fed-batch fermentation are reported respectively in this paper．Experimental results revealed that the fermentation period induced with methanol is 96 h．The optimum methanol is 8 g／L． pH range is 6。4-9．0 and the seed inoculum amount is 2：1 in shake-flask fermentation．Although the seed inoculum amount increases，the target protein concentration decreases． The 10％ inoculum and 5 -6 OD600 of seed are benefit to the high density cultivation．With glycerol feeding strategy based on DO leve1．The cell dry weight of broth reaches 118.7g/L and the IFN concentration is 1.034g/L by methanol inducement for 48 hours at the fed-batch fermentation phase．
Key words：PoIFNα，pichia pastoris，shake-flask fermentation，high-density fermentation
原载于：《南京师大学报》 2006，29（3）：76～80

尿素浓度梯度复性重组牛白细胞介素-2
于瑞嵩1,2 ，黄建珍2,3，李震1,2
(1). 上海市农业科学院畜牧兽医研究所，上海201106；
2). 上海市农业遗传育种重点实验室，上海201106；
 3). 江西农业大学动物科技学院，南昌330045)
摘要：尿素浓度梯度已成功用于多种重组蛋白的复性，对尿素浓度梯度复性rblL-2进行研究，结果表明：调整变性rblL-2的浓度在4 mg/mL左右，添加DTT使其达到10 mmol／L，4℃搅拌过夜，4℃ 分别对4、3、2、1 mol／L尿素，50 mmol／L Tris•Cl，pH 8.5透析12 h。然后再对pH 8.5 的PBS透析12 h，可成功对rblL-2复性，复性率达70％左右。生物活性测定表明得到了具有较高生物活性的rblL-2。
关键词：rblL-2；复性；尿素浓度梯度
Renaturation of rblL-2 by dialysis against urea concentration gradient
YU Rui-song1,2 , HUANG Jian-zhen2,3 , Li Zhen 1,2
(1).AnimalHusbandry andVeterinaryResearch Institute．SAAS，Shanghai 201106，China;
2). Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding，Shanghai 201106．China;
3). College of Animal Science，Jiangxi agriculture University，Nanchang 330045，China)
Abstract：Several kinds of recombinant protein had been successfully renatured by dialysis against urea gradient．The study on the renaturation of rbIL-2 hy urea gradient method indicated that adjusting the denatured protein(rhIL-2) concentration around 4mg/mL，adding DTT up to 10 mmol/L , and stirring the solution overnight at 4℃ ，and then dialyzing the denatured mlution against 4，3，2 and 1 mmol／L urea with 50mmol/L  Tris’HCI at pH 8.5 in turn for 12 hours at 4℃ could successfully  renature the rblL-2．and the renaturation rate reached about 70%.The determination of  biological activity showed that the renatured rbIL-2 had a rather high biological activity(1.2×104 IU/mL)．
Key words：rbIL-2；Renaturation；Urea concentration gradient

原载于《上海农业学报》 2006，22(4)：33-36

重组牛白细胞介素-2对猪瘟及猪繁殖呼吸综合征疫苗免疫抗体的影响
李 震1,2周宗清1,2 于瑞嵩1,2 黄建珍3 李祥瑞3孙景元3 赵 磊3 
(1上海市农业科学院畜牧兽医研究所 201106)
(2上海市农业遗传育种重点实验室 动物遗传工程分室 201106)
(3南京农业大学 动物医学院 南京210095)
摘要：为确定重组牛白细胞介素-2（rbIL-2）对猪瘟及猪蓝耳病（猪繁殖与呼吸综合征）疫苗的免疫增强作用，分别利用杜长上杂交肉猪或巴马小型猪（断奶初期）进行了猪瘟及猪蓝耳病弱毒疫苗接种时的牛IL-2注射试验。实验证明连续5天注射牛IL-2制品可有效提高猪瘟和猪蓝耳病疫苗的免疫抗体水平。对于猪蓝耳病所进行的试验证明：rbIL-2制品可使初免后正常免疫剂量组抗体水平提高164%，二免时可使疫苗剂量减半组抗体水平提高54%。利用猪瘟疫苗所进行试验的结果显示：rbIL-2制品对于猪瘟疫苗初次免疫作用不明显；在猪瘟疫苗二免时对疫苗剂量减半组具有良好的免疫增强作用，可使二免后10-20天的免疫抗体水平提高25%~52%，而且对免疫抗体高水平的维持具有良好作用。
关键词：免疫 牛白细胞介素-2 猪瘟 猪繁殖呼吸综合征 
Effect of Recombinant Bovine Interleukin-2 on Swine Sera Antibodies against Viruses of Classical Swine Fever and Swine Reproductive and Respiratory Syndrom
Li Zhen1,2 Zhou Zongqing1 Yu Ruisong1,2 Huang Jianzhen3 Li Xiangrui3 Sun Jingyuan3  Zhao Lei3
1．Institute of Animal Sciences and Veterinary Medicine, Shanghai Acedemy of Agricultrual Sciences  Shanghai 201106
2．Animal Genetic Division, Shanghai Municipal Laboratory of Agricultural Genetic and Breeding  Shanghai 201106
3．College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095
Abstract: To confirm the immunology enhancing effect of recombinant bovine interleukin-2 (rboIL-2) on vaccination of classical swine fever (CSF) and swine reproductive and respiratory symdrom(PRRS), pigs were immunized with the vaccines and at the same time injected with rboIL-2. The experiment showed that 5d injection of rboIL-2 could raised the sera antibody levels of the two vaccines. rboIL-2 could increase the sera antibody levels by 164% and 54% at the first and second PRRS vaccination. Intead of imposing an effect on the first CSF vaccination, rboIL-2 could enhance the 2nd CSF vaccination, increasing antibody levels by 25% at the 10th day and 52% at 20th day of the 2nd CSF vaccination.

Key words: Immunology; bovine interleukin-2; classical swine fever; swine reproductive and respiratory syndrome

原载于: 《西北农林科技大学学报》（2006）第34卷。

Study on renaturation of recombinant
bovine interleukin-2 expressed in Escherichia coli
Huang Jian-zhen 1,3,YU Rui-song1,2 ，SHEN Qiu-gu3, LI Xiang-rui4 , LI-Zhen1,2
(1), Animal Husbandryand Veterinary Research Institute，Shanghai Academy of Agricultural Science ，Shanghai 201106，China ;
 2), Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai 201106,
China; 
3), College of Animal Science, Jiangxi Agricultural University, Nanchang 330045, China; 
4), Faculty of Veterinary Medicine，Nanjing Agricultural University，Nanjing 210095, China )
Abstract：Recombinant bovine interleukin-2 forms insoluble inclusion body when over expressed in Escherichiacoli，and must be denatured and renatured in vitro be fore acquiring biological activity．We systematically studied such conditions of renaturing  rBolL-2 as pH，protein concentration，DTT concentration，ratio of oxidizer(GSSG) to reducer(GSH)，and additions of Cu2+，L-Arginine and Guandine-HCl of low concentration as well as renaturation methods．The results showed that adding 20mmol/L DDT to denaturation solution could improve the efficiency of the subsequent rBolL-2 renaturation，and the refolding rate of 3.5mg/mL  rBolL-2 protein solution could reach 50％ by means of urea gradient renaturation．In which the bioactivity of 9.6×104 IU/mL could be obtained．
Key wolds：Recombinant bovine interleukin-2(rBoIL-2)；Inclusion body；Renaturation
大肠杆菌表达重组牛IL．2复性技术的研究
黄建珍1,3 ，于瑞嵩1,2 ，沈秋姑3，李祥瑞4 ，李震1,2
(1). 上海市农业科学院畜牧兽医研究所，上海201106；
2).上海市农业遗传育种重点实验室，上海201106；
3).江西农业大学动物科技学院，南昌330045；
4).南京农业大学动物医学院，南京210095)
摘要： 研究了重组牛白介素2复性条件，如pH，蛋白浓度，DTT浓度，氧化剂(GSSG)和还原剂(GSH)的比例，Cu2+及L-Arg，低浓度的盐酸胍的添加，复性方法等。结果表明，复性液中添加20mmol/mL DTT可以提高rBolL-2的复性效率；利用尿浓度梯度复性法3.5mg/mL 的rBolL-2蛋白溶液复性率可达50％以上，测定活性效价达9.6×104 IU/mL。
关键词：重组牛白细胞介素2；包含体；复性

原载于 <上海农业学报> 2006，22(4)：27—32

上海地区鸡传染性支气管炎病毒S2基因的检测及分析
刘惠莉1,2 ，陆承平1*，朱伟云1
(1)．南京农业大学动物医学院，江苏南京210095；
2)．上海市农业科学院畜牧兽医研究所  上海市农业遗传育种重点实验室，上海201106)
摘要：参考GenBank发表的传染性支气管炎病毒(infectious bronchitis virus，IBV)S基因序列，设计合成了1对引物，模拟PCR表明，该引物能扩增现已发表的所有IBV 的S2基因部分核酸序列，扩增片段493 bp。建立的RT-PCR检验体系检测灵敏度达10～50 ELD50，检出限量为0.1 ng。采集上海地区12个鸡场疑似IBV 禽样品34份进行检洲，阳性为32份，序列分析表明均为肾变型IBV S2基因片段，S2基因同源性较高(≥99 )，与其他地区肾变型IBV 也有较高同源性，而与腺胃型和呼吸型相比较，序列差异较大。进化分析表明，尽管IBV S2片段变异率明显低于S1基因，但二者表现相同的进化趋势，S2基因亦可反映出IBV的分子变异及毒株间的亲缘关系。
关键词:鸡传染性支气管炎；S2基因；进化分析
Molecular Epidem ics of Infectious Bronchitis Virus in Shanghai District andAnalysis of Its S2 Fragment
LIU Hui—li1，2 ，LU Cheng—ping1 ，ZHU Wei—yun1
(1)．College of Animal Veterinary Medicine ,Nanjing Agricultural University，Nanjing 210095，China；
2)．Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences; Shanghai Municipal Key Laboratory of Animal Geneticsand Breeding，Shanghai 
201106．China)
Abstract：A pair of specific primer was designed and synthesized in the conserved region of S2 gene according to the infectious bronchitis virus(IBV) S sequences at GenBank．A 493 bp am plicon was abtained from sim ulating PCR using softwareSPCR3.0．The PCR system was specific with IBV while no reaction with other avian virus tested in the study．10-50 ELD50 of the virus was detected．By using this method to detect the clinical samples，which were collected from l 2 chicken factories．32out of 34 suspicious samples were positive．Further analysis showed all the IBV-positive belonged to the nephropathogenic viruses．The S2 genes of the epidemic viruses share high identities with each other，while has much more difference with the proventriculopathogenic IBV and other viruses causing respiratory  symptom in China．Phylogenic analysis showed though S2 fragments have less divergence among isolates than S1 genes，it shows the same phylogenic tendency with S gene among the viruses．
Key words：avian infectious bronchitis virus；S2 gene；phylogenic analysis

原载于《 中国兽医学报》2006，26（2）126～132

口蹄疫病毒3AB试剂盒的检测及比较
潘　洁, 陈　波, 邢继兰, 饶　忠, 徐泉兴, 尤永进
(上海市农业科学院畜牧兽医研究所,上海市农业遗传育种重点实验室,上海 201106)
摘　要:以重组口蹄疫病毒非结构蛋白3AB 为检测抗原,研制了口蹄疫病毒非结构蛋白ELISA 检测试剂盒,该试剂盒可用于判定被检动物是否感染口蹄疫病毒。重组口蹄疫病毒非结构蛋白3AB 作为检测抗原包被酶标板反应孔,以辣根过氧化物酶标记的重组蛋白A/ G作为第二抗体,建立了抗非结构蛋白抗体检测的方法。结果表明,在1746 份免疫猪血清中,3AB-ELISA 对免疫猪血清的特异性为98.68%;在1077 份健康非免疫猪血清中,3AB-ELISA 对健康非免疫猪血清的特异性为98.68%；在36份人工感染猪血清中,3AB-ELISA 对阳性检出率为100 %。而牛的特异性的各项指标和阳性检出率分别为94.04%,97.96%和100%。猪、牛口蹄疫病毒非结构蛋白酶联免疫吸附试验检测试剂盒与国外同类试剂盒比较,阳性检出率高出12.5 个百分点。
关键词: 非结构蛋白3AB ;试剂盒; 检测
Detection and comparison of diagnostic kits for FMDV-3AB
PAN Jie , CHEN Bo , XING Ji Lan , RAO Zhong , XU Quan xing , YOU Yong jin
( Animal Husbandry and Veterinary Research Institute , Shanghai Academy of A gricutural Sciences ;Shanghai Key Lab of A gricultural Genetics and Breeding , Shanghai 201106 , China)
Abstract :In order to judge if the detected animal is infected with foot and mouth disease virus(FMDV) ,a FMDV- NS 3AB EL ISA diagnostic kit was developed by using the recombinant non-structural protein 3AB as a detecting antigen. The reaction microplate wells were coated with the detecting antigen based on the non- structural protein 3AB , the recombined protein A/ G marked with horseradish peroxidase was used as the second antibody , and the anti-non-structural protein antibody detecting method was established. The specificity of FMDV-NS 3AB EL ISA diagnostic kit was 96. 68 % in 1 746 immune swine serums and 7. 96 % in 1077 normal swine serums ; it s positive detection rate was 100 % for 36 swine serums artificially infected with FMDV. And for bovine serums the above indexes were respectively 94. 04 % , 97. 96 % and 100 %. The positive detection rate of the enzyme-linked immunosorbent assay kit for FMDV-3AB of swine / bovine was 
12. 5 percentage points higher than that of the similar kits abroad.
Key words : Non-structural protein 3AB ; FMDV-NS 3AB EL ISA kit ; Detection

原载于<<上海农业学报>>　2006 ,22 (2) : 20 - 23
　

猪胚胎开放式拉长细管玻璃化冷冻保存研究
张德福1,2 ，刘 东1,2 ，吴华丽1,2 ，郑筱峰3 ，王昭凯4 ，王少兵4
(1)上海农业科学院畜牧兽医研究所，上海 201106
2)上海市农业遗传育种重点实验室动物遗传工程研究室，上海 201106
3)南京农业大学动物医学院，南京 210095
4)南京农业大学动物科技学院，南京 210095)
摘 要 从20头供体母猪获得的291枚可用胚胎(囊胚／桑葚胚)，采用二步法开放式拉长细管(OPS，open pulled straw)玻璃化冷冻技术进行保存，即胚胎首先在冷冻液I(TCM199+20％FBS+10％EG+10％DMSO)中平衡3min，然后立即转入冷冻液II(TCM199+20％FBS+20％EG+20％DMSO+0.4mol／L SUC)中并在lmin内装管，直接投入液氮保存；3个月后解冻移植给8头受体母猪，其中1头怀孕产仔(8头活仔)，在我国首次获得猪胚胎超低温(一196℃)冷冻后代。
关键词 猪，冷冻胚胎，玻璃化冷冻
Study on Vitrification of Porcine Embryos by Open Pulled 
Straw Method
ZHANG De—Fu1,2 , LIU Dong1,2, W U Hua-Li1,2, ZHENG Xiao—Feng3
WANG Zhao—Kai4, WANG Shao—Bing4
(1) Animal Husbandry and Veterinary Research Institute，Shanghai Academy ofAgricultural Scierwes，Shanghai 201106，China
2) DivisionofAnimal Genetic Engineering，ShanghaiMunicipalKey Laboratory ofAgri-Genetics and Breeding，Shanghai 201106，China
3) AnimalMedicine College，Nanjing Agriculture University，Nanjing 252100，China
4 )Animal Sci&Technology College，Nanjing Agriculture University，Nanjing 252100，China)
Abstract  291 embryos(Blastocyst／Morula)from 20 donor SOWS were vitrified by two step method with OPS(open pulled straw) in solution I(TCM199+20％FBS+10％EG+10％DMSO)for 3min, and solution II(TCM199+20％FBS+20％EG+20％DMSO+O、4mol／L SUC)for 1min，stored in liquid nitrogen for 3 months,and transferred into 8 recipient SOWS after warming，one recipient SOW was pregnant and 8 alive piglets were born．This is the first paper to report getting alive piglets by vitrification inChina．
Key words  pig，frozen embryos，vitrification
原载于<生物工程学报>2006, 22, (5):845-849

猪体细胞核移植重构胚发育异常及印迹
基因表达特征研究的进展
张德福 1,2，吴华莉1,2 ，刘东1,2 ,陈学进3，陈莹3，王凯3
(1) 上海市农业科学院畜牧兽医研究所，上海201106；
2) 上海市农业遗传育种重点实验室，上海201106；
3) 上海第二医科大学，上海市发育生物学重点实验室，上海200025)
摘 要 由于猪的器官极可能成为人类未来器官的供应源，猪体细胞核移殖及转基因等方面的研究已成为全球的热点。近几年来，猪体细胞核移植研究取得了一定的进展，但总体来看核移植效率还很低(1 ～2％)，还需要不断完善核移植程序及对一些基础问题的深入研究。本文主要对猪体细胞核移植重构胚发育异常及“印迹” 基因表达特征作一综述与分析， 旨在为研究者提供一些有益的启示
关键词 猪；核移植；印迹基因
Abnormal development and expressing characteristics of
‘‘imprinting gene” in reconstituted embryos from
porcine somatic nuclear transfer
ZHANG De—fu1,2，WU Hua—li1,2 ，LIU Dong1,2，CHEN Xue—jin3，
CHEN Yin3，WANG Kai3
(1)Animal Husbandry and Veterinary Research Institute，Shanghai Academy of Agricultural Sciences，Shanghai 201106，China；
2)Shanghai Municipal Key Laboratory of Agri—Genetics and Breeding，Shanghai 201106，China；
3)Shanghai Municipal Key Laboratory of Developmental Biology，Shanghai No．2 Medical University，Shanghai 200025，China)
Abstract Research in the fields of somatic cell nuclear transfer(SCNT)and transgenic cloning in pigs has become global highlight，because porcine organs would probably become the first source of donor organs for human xen0transplantation.Recently,great progress has been made in porcine SCNT．However，the efficiency of nuclear transfer remains very low (1 ～ 2 )．It is necessary to improve the procedure Of nuclear transfer and to further investigation Of some basic problems．In this paper，we mainly focus on the abnormal development and expressing characteristics of “imprinting gene” in reconstituted embryos from porcine somatic nuclear transfer， including its applications and approach of increasing the efficiency of SCNT．
Key words  pigs；nuclear transfer；imprinting gene
原载于<<广西农业生物科学>>2006,25(增刊):145-149

猪卵母细胞冷冻保存研究
张德福1,2 ，朱良成3 ，刘 东1,2 芮 荣3汤琳琳1,2 项智峰3
( 1)上海市农业科学院畜牧兽医研究所，上海201106；
2)上海市农业遗传育种重点实验室／动物遗传工程研究室，上海201106
3)南京农业大学动物医学院，南京252100)
摘 要 【目的】本研究试图通过比较猪卵母细胞超低温冷冻保存方法、冷冻承载工具、冷冻卵母细胞的类型，从而有效地保存猪卵母细胞；【方法】利用屠宰场卵巢采集的卵母细胞，以台盼蓝染色、二乙酸荧光素(FDA)染色鉴定卵母细胞冷冻后的成活率，以体外成熟和体外受精鉴定卵母细胞冷冻后的发育能力，研究了不同冷冻方法和不同冷冻保护剂对猪卵母细胞的冷冻效果【结果】(1)程序化法冷冻保存中，9％乙二醇(EG)，l0％二甲基亚砜(DMSO)，10％甘油(Giy)均对猪MII期卵母细胞有冷冻保护作用，极显著高于对照组(FDA染色成活率33.8％，25.8％，23.5％vs 2.5％，P<0.01)，且以9％EG的效果最好，显著高于另外两组(33.8％VS 25.8％，2 3.5％，P<0.05)。(2)玻璃微细管(GMP)法是猪卵母细胞超低温冷冻的较好方法，以普通的麦管(Straw)法进行的程序化冷冻为对照组，GMP管法显著提高猪卵母细胞的冷冻成活率(分别为63.3％和34.5％，P。(3)在玻璃化冷冻方法中，不同的冷冻液载体对猪卵母细胞冷冻成活率有影响以EFS40为冷冻液，Straw和GMP管作冷冻液载体，卵母细胞的成活率分别为45.0％和65.9％，二者差异显著(P<0.05)(4)用straw的程序化冷冻法和用GMP管的玻璃化冷冻法对猪GV期卵母细胞冷冻均有效，但二者差异显著(成活率分别为30.0％和59.7％，P冷冻后继续培养，分别有2.8％和6.3％的卵母细胞周围颗粒层发生扩散。(5)冷冻对MII期卵母细胞的发育潜能影响较大，用新鲜精液使其受精，仅有4.9％的受精卵分裂，极显著低于对照组的49.5％(P；发育至4一细胞期的比例为1.7％，但未能发育至8一细胞期以上胚胎。【结论】选用MⅡ期卵母细胞、以GMP管为冷冻承载工具、应用玻璃化冷冻方法能够较好地保存猪卵母细胞，为进一步完善猪卵母细胞超低温冷冻保存技术体系提供了参考依据。
关键词 猪；卵母细胞；冷冻保存；体外受精
Studies on Cryopreservation tudes G of Porcine 0oocyte
ZHANG De-fu1,2,ZHU Liang-cheng3 , LIU Dong1,2,RUI Rong3,TANG Lin-1in1,2 ,
XIANG Zhi-feng3
(1)Animal Husbandary and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106；
2)Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai 201106；
3) Animal Medical College, Nanjing Agriculture University, Nanjing 252100)
Abstract 【Objective】The aim of the present study was to try to cryopreserve porcine oocytes efficiently, and to investigate the effect of cryopreservation method，cryopreservation tool and types of cryoprotectant on pig oocyte survival and its in vitro maturation and cleavage following IVF and IVC．【Method】Experiments in pig oocyte cryopreservation and in vitro fertilization(IVF) were conducted using oocytcs collected from a slaughterhouse．The efects of diferent methods an d diferent cryoprotectant solution on cryopreservation of porcine oocytes were examined．Survival was assessed by trypan blue(TB)staining，fluorescein diacetate(FDA) staining，maturation in vitro and cleavage after IVF．The results were showed as follows：【Results】(1)Cryoprotectant solution (9％EG，IO％DMSO，10％Gly)were efective to cryopreserve pig MII oocytcs when in programmed freezing．The survival rates (FDA dyeing)of pig oocytes after frozen—thawed were very significantly higher than that of control(33.8％，25.8％，23.5％VS 2.5％，P<0.01)．Among these three cryoprotectant solution，9％EG Was significantly superior to the other two in survival rate(33.8％VS 25.8％，23.5％,P<0.05)．(2)The freezing method by GMP(glass micropipette)was suitable and eficient to cryopreserve  pig oocytes．Compared with programmed freezing method using Straw，GMP method could significantly promote the survival rate of pig oocytes (63．3％and 34．5％，respectively，P<0．05)．(3)The vitrification solution carrier had a positive efect on survival rate of pig oocytes．When EFS40 was used as vitrification solution，Straw and GMP led to different survival rates，which were 45.0％ and 65.9％，respectively(P<0.05)．(4)The programmed freezing method by Straw and vitrification method by G were available in cryopreservation of pig oocytes，but these two methods resulted in different survival rates(30.0％and 59.7％，respectively, P<0.05)．(5)Cryopreservation had a great effect on the developmental ability of pig oocytes after IVF and IVC．Vitrified MII oocytes were
fertilized with fresh spermatozoa an d only 4．9％ eggs cleaved after 48 culture．which was very significan tly lower than the cleavage rate of control(49.5％，P<0.01)．the subsequent developmental rateto4-cell stage was 1.7％，but none to 8-cell stage．【Conclusion】The cryopreservervation of porcine oocytes with MII oocytes．vitrification and GMP were efficient．
Key Words Pig；Oocyte；Cryopreservation；in vitro fertilization( IVF)

原载于<中国农业科学>2006，39(6)：1233-1240

体细胞的组织来源及培养代数对猪核移植
重构胚发育的影响
张德福1,2 刘 东1,2 汤琳琳1,2 王英1,2 陈茵3 王凯3  Karl ScheHande4 LIN Cai Lu4
(1)上海市农业科学院畜牧兽医研究所，上海201106
2)上海市农业遗传育种重点实验室，上海201106
3)上海第二医科大学，上海市发育生物学重点实验室，上海200025；
4)Animal Breeding Institute，Bonn University，Bonn 53121，Germany)
摘 要  本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40—44h的猪卵母细胞去核后，将经血清饥饿(0.5％FBS)培养2-9d、0.1mg／L Aphidicolin (APD)培养+0.5％ FBS培养2-9d或一般培养法(10％FBS)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞，直接注射到去核的卵母细胞质中，或注射到卵周隙中，再经电融合(100V/mm,30s，电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6-DMAP激活处理．体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1mg／L APD+0.5％ FBS培养处理后的重组胚卵裂率，均高于血清饥饿和一般培养处理的同种供体细胞(P<0.01)。卵丘细胞、颗粒细胞经0．1mg／L APD+0.5％ FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P<0.05)。以猪颗粒细胞为核供体时，电融合法的重构胚分裂率显著高于胞质内注入法(P<0.05)，但囊胚发育率无显著差异(p>0.05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞，其具有正常二倍染色体的细胞比例均无显著差异(P>0.05).以这2种细胞不同培养代数做供体进行核移植时。各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P>0.05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定；(2)0.1mg／L APD预培养处理供体细胞能提高猪体细胞核移植的效果，血清饥饿培养则无明显效果；(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞，核移植后都能得到体细胞克隆的囊胚，但前者的效果略优于后者，且其核移植效果不受供核细胞培养代数的影响；(4)电融合核移植胚胎的发育率高于胞质内直接注入法，但两者的总体效率相近。
关键词 猪体细胞 核移植 细胞周期
EFFECTS OF DⅡ FERENT DONOR CELLS AND PASSAGES ON
THE DEVELOPM ENT OF NUCLEAR
TRANSFER PORCINE EM BRYOS
ZHANG De Fu1,2  LIU Dong1,2  TANG Lin Lin1,2  WANG Ying1,2  CHEN Yin3
WANG Kai3 Karl Schellander4  LIN Cai Lu4
( 1)Anima! Husbandry and Veterinary Research Institute，Shanghai Academy of Agricultural Sciences，Shanghai 201106；
2)Division of Anima! Genetic Engineering，Shanghai Municipal Key Laboratory of Agri—Genetics and Breeding，Shanghai 201106；
3) Shanghai Municipal Key Laboratory of Developmental Biology，Shanghai No．2 Medical University，
Shanghai 200025； Anima! Breeding Institute，Bonn University，Bonn 53121，Germany)
ABSTRACT  This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos，so as to establish a preliminary procedure for porcine cloning．Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5μg/mL cytochalasin B．Fibroblast cells(FC)，oviduct epithelial cells(OEC)，granulosa cells(GC) and cumulus cells(CC)after 3-9 passages in TCM + 10％ FBS were treated by serum starvation ( 0．5％ FBS for 2-9 days)，0.1μg/mL aphidiconlin (APD) for 1 day and 0.5％ FBS for 2-9 days or continue culturing in 10％ FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion(100 V/mm，30μS and 1 pulses)．Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP.And cultured for 6 days, to evaluate their cleavage and embryonic development．The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1g/mL APD +0.5％FBS were significantly higher than that of serum starvation group and control group (P<0．01)． There was significantly μdifference in the cleavage rate and embryonic development among embryos derived from GC,CC and FC,OEC pretreated with 0.1mL APD + 0.5％FBS. The cleavage rate of embryos reconstructed with GC by electro fusion was significantly higher than that by microinjection(P<0.05),but 3="" 6="" 10="" no="" difference="" was="" found="" in="" their="" proportion="" developing="" to="" blast="" of="" gc="" at="" and="" fc="" passages="" had="" normal="" karyotype="" which="" did="" not="" show="" significant="" among="" them="" p="">0.05)．When GC at G3，G4，G5 and G6 of passages were used as donor cells， the cleavage rate and blastocyst rate was similar. Moreover, FC at G6,G7,G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development． These results indicate that:(1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2)Using0.11μg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency (4) The development of reconstructed embryos by electro fusion is higher than that than microinjection，but there is no difference in the total are not influenced by the culture passages; by electro fusion is higher than that by efficiency between the two methods．
Key words：Pigs．Somatic cells nuclear transfer．cell cycle
原载于<分子细胞生物学报>2006,39(2):145-151

猪卵母细胞去核方法的改进

张德福1,2 ，刘 东1,2 ，汤琳琳1,2 ，陈晓宇3，Lin Cailu4 
(1)．上海市农业科学院畜牧兽医研究所，上海201106；
2)．上海市农业遗传育种重点实验室动物遗传工程研究室，上海201106；
3)．西北农林科技大学动物科技学院，陕西杨凌712100；
4)．Animal Breeding Institute，Bonn University，Bonn 53121，Germany)
摘 要 对猪体细胞克隆技术中关键步骤之一的去核方法作了较深入的研究。结果表明，由本实验室改进的Willadsen去核法-二步挤压去核法，无论在去核操作成功率(73.9% )上还是在去核效率(81.2% )上都明显好于McGrath—
Soher 去核法(42.5%、67.4%，P<0.05)；同时，本试验还发现，卵母细胞成熟培养36 h后去核组去核效率(89.1% )显著地高于成熟培养44 h后去核组(55.8%，P<0.05)，而核移植重构胚的发育率2个组间无显著差异。
关键词 猪；卵母细胞；去核；核移植
A Modified Enucleation Method of Porcine Oocytes
ZHANG De—fu 1,2，LIU Dong1,2，TANG Lin—lin1,2 ，CHEN Xiao—yu3，LIN Cai—lu4 
(1)．Animal Husbandry and Veterinary Research Institute，Shanghai Academy Agricultural Sciences，Shanghai 201106，China；
2)．Laboratory of Animal Genetic Engineering，Shanghai Municipal Key Laboratory of Agri—
genetics and Breeding，Shanghai 201106，China；
3)．College of Animal Science and Technology，North—west Sci—tech University of Agriculture and Forestry，Yangling，Shanxi 7121 00，China；
4)．Animal Breeding Institute，Bonn University，Bonn 53121，Germany)
Abstract  This experiment was conducted to study enucleated method，which iS one of the key techniques for animal cloning．The results show that the modified Willadsen’ S method for enucleation，two—step—squeezing method is significantly better than McGrath—Soher’S method in the enucleated rates(73.9％,42.5% , P<0.05)and the rates(81.2%,67.4%,P<0.05) of absolutely removing nuclei matter．It was also found that the rates(89.1% )of absolutely removing nuclei matter for 36 h —matured—in—vitro oocytes is significantly higher than that for 44 h (55.8%,P<0.05)．
Key words pigs；oocytes；enucleation；nuclear transfer

原载于<中国兽医学报>2006,26(5):574-577

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动物研究分室与南京师范大学生命科学院、南京农业大学、江南大学、四川农业大学、山东农业大学联合培养硕士研究生19名，与南京师范大学、南京农业大学、四川农业大学共同培养的8位硕士研究生2007年6月以优异的成绩通过硕士论文答辩毕业。