新城疫疫苗对SPF鸡和商业肉鸡的免疫学效力分析

将不同新城疫病毒疫苗免疫SPF鸡和商业用肉鸡，对免疫后抗体水平变化和对野毒株攻击的保护率进行了监测。利用Clone 30、F48E8和NXF3单联苗和联苗免疫过的SPF和商业肉鸡，对10^5EID50两次攻毒保护率均为100％；而只用La Sota疫苗免疫的SPF鸡保护率为40％。试验数据表明，分离株NXF3具有较好的免疫原性和对强毒攻击的保护，可以作为候选疫苗用于商业肉鸡。     

鸡传染性法氏囊病免疫复合物疫苗的安全性和免疫效力试验

用鸡法氏囊病免疫复合疫苗(IBDV-Icx)和常规IBD活疫苗分别免疫1日龄商品鸡和SPF鸡,进行疫苗的安全性及免疫效力比较.免疫后8d检测,IBDV-Icx免疫组鸡的法氏囊未见明显萎缩,而传统弱毒疫苗免疫组鸡的法氏囊萎缩明显;免疫后28d各组用IBDV标准强毒株进行攻击,IBDV-Icx免疫组鸡的攻毒保护率均为10/10,常规IBD活疫苗对照组分别为8/10及9/10.免疫后3个月,IBDV-Icx免疫组血清抗体可达AGP1∶32,攻击强毒仍为10/10保护,而常规IBD活疫苗免疫后2个月抗体AGP为0,攻毒保护率为1/10,免疫后3个月时攻毒10/10发病.     

鸡传染性鼻炎(三价)和新城疫二联灭活疫苗的研究

利用Page A型、B型、C型副鸡禽杆菌和新城疫病毒La Sota株,研制鸡传染性鼻炎(三价)和新城疫二联油乳剂灭活疫苗。用3个批次疫苗进行单剂量(0.5mL/次)3次接种和大剂量(2.0mL)单次接种的安全性试验、SPF鸡的免疫效力试验、商品鸡的免疫持续期试验和疫苗的保存期试验。结果表明,3批疫苗对试验鸡安全无副作用;免疫接种SPF鸡只30d后对A、B、C型副鸡禽杆菌攻毒的保护率≥80%,用20μL/只疫苗免疫接种SPF鸡21d后新城疫的平均HI抗体24;商品鸡42日龄首免,110日龄二免,二免后9个月对A、B、C型副鸡禽杆菌攻毒的保护率≥70%,对新城疫病毒强毒100%保护;用4℃~8℃保存12个月和15个月的3批疫苗进行了SPF鸡的近期免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥70%,用20μL/只疫苗免疫SPF鸡,免疫接种后21d新城疫病毒的平均HI抗体24,对新城疫病毒强毒的攻毒保护率70%。说明研制的疫苗安全有效。     

鸡新城疫重组鸡痘病毒活疫苗的免疫持续期和加强免疫研究

本研究旨在评价表达新城疫病毒（NDV）血凝素-神经氨酸酶（HN）基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗的免疫持续期和加强免疫对疫苗免疫效力的影响。用rFPV-12LSHN活疫苗免疫14日龄SPF鸡,103PFU/羽,7d即可检测到NDV HI抗体应答,对NDV强毒F48E8株攻毒保护率达100%。一次免疫18周后,对NDV强毒攻击依然提供完全保护。鸡痘病毒（FPV）疫苗免疫4周,再接种rFPV-12LSHN活疫苗,攻毒保护率降低至50%。相反,rFPV-12LSHN免疫4周,随后二次免疫可显著提高对NDV的体液免疫应答水平（P〈0.01）,对NDV强毒攻击的保护率仍然为100%。结果表明,表达NDV HN基因的重组鸡痘病毒（rFPV-12LSHN）活疫苗,能够快速建立坚强免疫力,免疫持续期至少可达18周,rFPV-12LSHN的二次免疫可以提高疫苗的免疫力。     

传染性喉气管炎新城疫鸡痘重组病毒免疫效力的研究

在表达鸡传染性喉气管炎病毒(ILTV)糖蛋白gB基因和新城疫病毒(NDV)F基因的重组鸡痘病毒(rF-PV-gB-F)安全性检验合格后,以5.0×101~5.0×104PFU不同含量按0.1mL/鸡的剂量免疫100只30日龄SPF鸡,30d后分组分别用ILTVWG株和NDVF48E9株强毒进行攻击。免疫鸡抗鸡痘病毒抗体都转为阳性,痘反应和接种剂量有关,重组疫苗的最小反应剂量为50PFU。重组疫苗可以诱发对新城疫和传染性喉气管炎的保护,0.1mL/鸡的接种量在500~5000PFU浓度范围内的免疫效果最好,对于ILTV攻击的发病保护率在70%以上,对NDV强毒攻击的抗死亡保护率可以达到80%,这为进一步考察疫苗的免疫效力试验以及进行田间试验奠定了基础。     

鸡传染性法氏囊病免疫复合物疫苗对SPF雏鸡的免疫效果

用鸡传染性法氏囊病免疫复合物疫苗和活疫苗分别免疫1日龄SPF雏鸡,免疫后9d观察法氏囊病变,免疫后14、21、28d对各组鸡采血测定IBDV中和抗体,21d及28d时用强毒攻击,计算各组疫苗保护率。结果表明,免疫后9d免疫复合物疫苗组鸡法囊正常,活疫苗组鸡法氏囊全部明显萎缩;免疫后14、21、28d,免疫复合物疫苗组鸡IBDV中和抗体分别为2.9±0.99、8.1±0.88和10.6±1.78,活疫苗组IBDV中和抗体分别为3.3±1.14、8.3±1.16和9.2±1.23,免疫后21d及28d疫苗免疫组均100%保护。试验证明免疫复合物疫苗比活疫苗更为安全,免疫复合物疫苗免疫SPF雏鸡产生比活苗更高的IBDV中和抗体,保护率达100%。     

鸡传染性鼻炎三价灭活疫苗安全性试验和效力检验初报

利用PageA、B、C型副鸡禽杆菌,研制鸡传染性鼻炎(IC)三价灭活疫苗。用6批IC疫苗进行了大剂量单次接种和单剂量多次接种的安全性试验,结果表明,IC三价灭活疫苗安全无副作用;用3批疫苗进行SPF鸡和普通鸡的免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;商品鸡42日龄首免,110日龄二免,二免后约9个月对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;用4℃～8℃保存一年的3批疫苗进行了SPF鸡的近期免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%。     

鸡传染性法氏囊病免疫复合物疫苗对1日龄中等母源抗体水平雏鸡免疫效果的研究

将鸡传染性法氏囊病免疫复合物疫苗和BX株活疫苗分别免疫1日龄中等母源抗体水平京红雏鸡,同时免疫1日龄SPF雏鸡作对照,免疫后第9天观察法氏囊病变,免疫后第28天采血测定IBDV中和抗体,并用强毒攻击,计算各组疫苗保护率。结果显示,免疫后第9天,免疫复合物疫苗组SPF雏鸡和京红雏鸡法氏囊均正常,BX株活疫苗组SPF雏鸡法氏囊出现了萎缩病变;免疫后第28天,对照SPF雏鸡和京红雏鸡的免疫复合物疫苗组、BX活疫苗组IBDV中和抗体效价分别为9.2log2、8.5log2和 7.2log2、4.4log2,攻毒保护率均为100%。试验结果表明免疫复合物疫苗免疫1日龄中等水平母源抗体雏鸡效果很好,能产生较高的中和抗体,攻毒保护率能达到100%。     

H9N2亚型禽流感疫苗株的筛选及其免疫效果评价

通过交叉血凝试验,疫苗备用毒株免疫SPF鸡后再用同源和异源毒株攻毒,评价疫苗备用毒株对SPF鸡保护效果。结果显示,H9N2疫苗备用株免疫SPF鸡后,再用H9亚型流感病毒流行株攻毒,SPF鸡咽喉和泄殖腔排毒量大大降低。结果表明,本试验筛选的疫苗备用毒株对H9N2亚型流感病毒具有一定的保护率,完全符合疫苗株要求。     

鸡新城疫油乳剂灭活苗的试验研究

利用Laso ta系鸡新城疫病毒(NDV)接种无特定病原(SPF)鸡胚获取的含毒尿囊液,加入一定比例的矿物油佐剂制成鸡新城疫油乳剂灭活苗,和Laso ta 系弱毒疫苗联合使用免疫不同日龄不同品种的鸡群。获得了抗体滴度上升均匀在体内持续时间长,抗ND 强毒攻击的满意效果。疫苗效价高,免疫原性好,免疫保护率明显高于应用ND—Ⅰ系疫苗免疫的鸡群。对首免用Laso ta 系活苗和油乳剂灭活苗皮下注射0.1ml 的35日龄、70日龄肉仔鸡攻以ND 强毒,保护率为百分之百。对在120日龄免疫用新城疫油苗肌肉注射0.5ml 的蛋用种鸡在免疫后11个月以ND 强毒进行攻击,保护率亦为百分之百。     

Response of bovine gammadelta T cells to activation through CD3

Since the T cell receptor of γδ T cells is associated with CD3 molecules, it is a reasonable postulate that signal transduction through CD3 would occur in γδ T cells as it does in β T cells. However, while a small percentage of bovine γδ T cells divided in cultures of peripheral blood mononuclear cells (PBMCs) in response to stimulation by anti-CD3 monoclonal antibody (mAb) the majority of viable γδ T cells at the end of the culture period had not. This was assessed by carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis here and previously [Res. Vet. Sci. 69 (2000) 275]. When intracytoplasmic staining for interferon-γ (IFN-γ) was also used here to assess activation through CD3, a small proportion of γδ T cells (approximately 14%) produced IFN-γ during the first 4 h of culture and by 72 h of culture that number had doubled. By comparison, a much larger proportion of CD4 and CD8 T cells stimulated with anti-CD3 mAb divided and although the percentage of CD4 and CD8 T cells that produced IFN-γ at 4 h was similar to that of γδ T cells, by 72 h the majority of CD4 and CD8 T cells were IFN-γ⁺. Addition of IL-2 did not increase the proportion of γδ T cells that responded to anti-CD3 stimulation by cell division. To test the hypothesis that γδ T cells were inhibited from responding by other mononuclear cell populations within PBMC, monocytes were removed from the PBMC or γδ T cells were purified by magnetic-bead sorting. Only a small distinct population of the sorted cells underwent multiple cell divisions in response to anti-CD3 mAb and removal of monocytes resulted in only a moderate increase in γδ T cell replication. The anti-CD3 mAb stimulation system may provide a useful system to evaluate the difference in the requirements for activation and clonal expansion for γδ T cells versus β T cells.     

Comparison of biological activities of natural and recombinant chicken interferon-gamma

In recent years, chicken interferon-gamma (ChIFN-γ) has been identified and cloned from a chicken T cell line. In this study, recombinant ChIFN-γ produced in the baculovirus and prokaryotic (Escherichia coli) expression systems were characterized and their activity was compared to that of naturally ChIFN-γ produced by mitogen-activated splenic T cells. The baculovirus-derived ChIFN-γ protein (Bac-ChIFN-γ) proved to have physiochemical properties and biological activities similar to those of natural ChIFN-γ. Indeed, Bac-ChIFN-γ was able to inhibit the replication of cytolytic viruses in chicken embryo fibroblasts and to activate macrophages, as was determined by nitric oxide production. Levels ranging between 100 and 300 μg/ml of BacChIFN-γ could be obtained in the supernatants of infected insect cells. On the other hand, yields of the E. coli produced ChIFN-γ rarely exceeded 100 μg/ml after purification steps and although it was also able to activate the HD11 macrophage cell line in a specific manner, no anti-viral activity could be demonstrated. Therefore, the baculovirus expression system is an appropriate system for the high-level expression of biologically active ChIFN-γ and will allow further studies of the immunomodulatory and therapeutic effects of this cytokine in vivo.     

口蹄疫病毒3D蛋白促进TLR3介导的Ⅰ型干扰素产生

口蹄疫是由口蹄疫病毒（FMDV）引起的急性、热性、高度接触性传染病。口蹄疫病毒感染宿主引起一系列严重的炎症反应，而TLR3通路是介导细胞炎性反应的主要途径之一。为研究口蹄疫病毒蛋白对TLR3通路的影响，本研究首先用双荧光素酶报告系统筛选影响TLR3通路的FMDV蛋白；接着用Q-PCR试验验证筛出来的候选蛋白对TLR3通路下游基因表达水平的影响；并用免疫共沉淀试验验证与候选蛋白有相互作用的TLR3通路蛋白；最后用Western blot试验检测候选蛋白对TLR3通路下游分子磷酸化水平的影响。双荧光素酶报告系统结果显示，口蹄疫病毒3D蛋白促进TLR3通路介导的Ⅰ型干扰素的产生并呈剂量依赖性，Q-PCR试验表明，3D能够促进TLR3通路下游基因表达水平；免疫共沉淀试验表明，FMDV 3D与TLR3有相互作用；Western blot试验进一步显示，过表达3D能够促进TLR3下游分子的磷酸化水平。综上，口蹄疫病毒3D蛋白能促进TLR3介导的Ⅰ型干扰素的产生，从而调控天然免疫反应。     

布鲁氏菌侵染过程中has-miR-5196-3p对炎症小体的调控作用

试验旨在研究miRNA分子has-miR-5196-3p在布鲁氏菌侵染THP1细胞过程中对NOD样受体蛋白3炎症小体（NLRP3）的调控作用。运用TargetScan、MiRDB等生物信息学预测网站预测has-miR-5196-3p和NLRP3-3'UTR的结合位点,构建其野生型及突变型双荧光素酶报告载体,分别与has-miR-5196-3p mimics及阴性对照共转染293T细胞,通过双荧光素酶报告系统检测各组相对荧光素酶活性的变化,验证has-miR-5196-3p和NLRP3-3'UTR的靶向关系;布鲁氏菌侵染THP1细胞,实时荧光定量PCR检测NLRP3、半胱氨酰天冬氨酸特异性蛋白酶（caspase1）及has-miR-5196-3p mRNA的表达情况,Western blotting检测NLRP3和caspase1蛋白表达水平;将has-miR-5196-3p mimics、has-miR-5196-3p inhibitor及阴性对照转染至THP1细胞,实时荧光定量PCR及Western blotting检测NLRP3和半胱氨酰天冬氨酸特异性蛋白酶（caspase1）的表达水平。结果显示,试验成功构建含有NLRP3-3'UTR的野生型及突变型双荧光素酶报告载体;has-miR-5196-3p mimics极显著降低pmirGLO-NLRP3-3'UTR-wt的相对荧光素酶活性（P<0.01）;布鲁氏菌侵染THP1细胞后,NLRP3炎症小体被激活,而has-miR-5196-3p无明显变化（P>0.05）;has-miR-5196-3p mimics能显著抑制NLRP3的表达（P<0.05）,而has-miR-5196-3p inhibitor则相反。结果表明,miRNA分子has-miR-5196-3p可与NLRP3-3'UTR结合并抑制其表达,布鲁氏菌侵染THP1细胞过程中通过has-miR-5196-3p靶向负调节NLRP3炎症小体的表达及炎症的发生。     

牛UCP3和MYH1基因启动子在C2C12和3T3-L1细胞中的启动活性分析

为阐明牛解偶联蛋白3（uncoupling protein 3,UCP3）和肌球蛋白重链1（myosin heavy chain 1,MYH1）基因启动子的核心区及其在小鼠C2C12和3T3-L1两种细胞株中的启动活性,本研究利用PCR、双荧光素酶和脂质体转染等方法进行关岭牛UCP3和MYH1基因启动子的扩增、重组载体构建和启动子活性分析。结果显示,试验成功构建了pGL3-Basic-UCP3-pro和pGL3-Basic-MYH1-pro重组真核表达载体,分别转染至小鼠C2C12和3T3-L1细胞后发现,UCP3、MYH1基因启动子的核心区分别为UCP3-P6（－385~+3 bp）和MYH1-P5（－255~+15 bp）区域;pGL3-Basic-MYH1-P2~pGL3-Basic-MYH1-P5在C2C12细胞中启动子活性均高于3T3-L1细胞,表明MYH1基因启动子活性在成肌细胞C2C12中较高。本研究阐明了UCP3和MYH1基因启动子的核心区及其启动子活性,为进一步研究UCP3和MYH1基因的转录调控机理提供了科学依据。     

载体介导的shRNA抑制猪瘟病毒在PK-15细胞中增殖特性的研究

利用生物学软件分析猪瘟病毒NS3基因,设计针对NS3基因不同位置的干扰序列,化学合成这些序列,然后退火连接为双链干扰片段,再定向克隆到干扰载体中,将酶切和序列测定鉴定正确的重组载体命名为pGene-NS3-1、pGene-NS3-2、pGene-NS3-3、pGene-NS3-Negative。重组干扰载体经纯化后转染PK-15细胞,并进行G418抗性筛选。克隆细胞扩大培养后,接种猪瘟病毒Shimen株,收集细胞分别进行实时定量PCR和ELISA光吸收度分析。Real-time PCR分析表明,与对照细胞比较,pGene-NS3-1、pGene-NS3-2、pGene-NS3-3转录产生的shRNA分子均在一定程度上沉默了病毒的基因,抑制率约为63%、46%、49%。ELISA检测结果表明,转染pGene-NS3-1、pGene-NS3-2、pGene-NS3-3重组载体的PK-15细胞均在不同程度上抑制了猪瘟病毒粒子的增殖。     

Cloning and Bioinformatics Analysis of MAP3K5 Gene CDS Sequence in Pig

In this study,we cloned MAP3K5 gene cDNA sequence,compared the sequence of MAP3K5 with MAP3K families in pig and other species,and studied their conservative structure domain.Obtained the full-length of MAP3K5 gene (5 452 bp) by RACE-PCR,predicted the open reading frame (ORF) and amino acid (AA) sequence,and obtained the information of 31 exons.The low similarity were found in mRNA and amino acid sequences of MAP3K families in pig (<50%).And MAP3K5 had the comparatively high similarity with MAP3K6 in pig.The comparative analyzed the mRNA and protein sequence of pig MAP3K5 gene with other species MAP3K5 gene,founded that the ten species of MAP3K5 mRNA and protein sequence had the high similarity (>78%),and pig MAP3K5 had the comparatively high similarity with Bos taurus,Ovis aries,Canis lupus and Homo sapiens.Function domain analyzed the high similarity protein with MAP3K5 in pig,this research found that pig MAP3K6 protein had the similar conserved function domain with pig MAP3K5.However,the other pig MAP3K families didn't have the similar conserved function domain with pig MAP3K5.The ten species of MAP3K5 protein had the similar conserved function domain.The results obtained the sequence and molecular structure of MAP3K5 gene,and provided an important foundation for further function research of MAP3K5 gene in pig.     

Bactrian camel (Camelus bactrianus) integrins αvβ3 and αvβ6 as FMDV receptors: Molecular cloning, sequence analysis and comparison with other species

Integrins are heterodimeric adhesion receptors that participate in a variety of cell–cell and cell–extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the αv integrin family of cellular receptors, αvβ3, αvβ6, αvβ1 and αvβ8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the αv integrins from a susceptible species as viral receptors, we have cloned Bactrian camel αv, β3 and β6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin αv, β3 and β6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel αv, β3 and β6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.     

Meiotic competence of mouse oocytes reconstructed by replacing the germinal vesicle with a male pronucleus and somatic nucleus

The present study examines the contribution of the nucleus to meiotic competence in mouse oocytes that were reconstructed using nuclear transfer. Three types of reconstructed oocytes were produced: MP‐GV, by transplanting the male pronucleus (MP) into germinal vesicle (GV) stage oocytes; 3T3‐GV, by transplanting the nucleus of a National Institute of Health (NIH) 3T3 cell into a GV stage oocyte; and 3T3‐MII, by transplanting the nucleus of an NIH 3T3 cell into a metaphase II (MII) stage oocyte. The fusion rates differed, but not significantly, in the MP‐GV, 3T3‐GV, and 3T3‐MII groups (77, 63, 56%, respectively). Then, meiotic competence was compared in MP‐GV, 3T3‐GV and non‐manipulated GV stage oocytes as a control. Nuclear envelope breakdown occurred in all the reconstructed oocytes, as well as the control ones. The percentage of first polar body extrusion differed between the MP‐GV (100%), 3T3‐GV (72%), and control (67%) groups. DNA staining with Hoechst 33342 revealed that in the MP‐GV‐group oocytes that had reached MII stage, the chromosomes were condensed and aligned in a regular array similar to the normal metaphase plate. By contrast, in 3T3‐GV group oocytes, the condensed chromosomes were irregularly scattered in the cytoplasm. These results suggest that the donor nucleus affects meiotic competence in reconstructed oocytes.