【作者】 钱莺娟；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 根据已发表文献及DNAstar软件分析，选取GA株马立克氏病病毒（MDV）BamHI-I₃区糖蛋白B 250-460位氨基酸为目的片段，进行串联表达。根据GenBank发表的序列，Primer5.0软件的辅助下分别设计两对引物Primer（U₁）、Primer（U₂）和Primer（D₁）、Primer（D₂），并在上游片段5’端加上ATG GCG、下游片段5’端设计一个长9个氨基酸的linker（GSLAGALGL）和下游片段3’端加上TAA，使上游片段和下游片段串联后形成一个完整的阅读框。用PCR扩增出上游片段gB（U）和下游片段gB（L／D）。将PCR产物分别克隆入pBluescriptSKⅡ+，构建成pSKgB（U）和pSKgB（L／D）。从pSKgB（U）以XbaⅠ+BamHⅠ酶切出gB（U），克隆入pSKgB（L／D），构建成pSKgB（U／L／D）。然后从pSKgB（U／L／D）中以XbaⅠ+PstⅠ酶切出串联片段gB（U／L／D），克隆入pEFgpt12S，构建成pEFMDgB（U／L／D）重组鸡痘病毒转移质粒栽体。 确定转染时亲本毒的用量及筛选重组病毒选择性培养基中霉酚酸的浓度后，用磷酸钙共沉淀法，将鸡痘病毒转移质粒载体pEFMDgB（U／L／D）与鸡痘病毒FPV 282E4株在次代CEF上进行同源重组，将目的基因导入FPV ORF1区。用霉酚酸法连续筛选10代获得重组病毒rFPV-MDgB（U／L／D）。用间接免疫荧光试验（IFA）鉴定，可见阳性重组病毒表达产物产生特异性荧光，表明重组病毒成功表达了串联目的片段且串联表达产物具有免疫原性：对亲本毒和重组毒的TCID₅₀试验测定结果是两者的TCID₅₀值基本一致，表明外源基因插入鸡痘病毒ORF1后不影响病毒的复制和生物学特性。 将筛选到的重组鸡痘病毒rFPV-MDgB（U／L／D）继续在2％MEM中5代，测定重组病毒分别在选择性培养基和普通培养基中的病毒滴度，并与亲本毒的滴度相比较，结果基本一致，说明重组病毒已得到了纯化且比较稳定。用rFPV-MDgB（U／L／D）+CVI988（商品苗）和CVI988（商品苗）分别免疫1日龄SPF鸡，8日龄时用RB1B株超强毒MDV攻毒，隔离饲养9周，在此期间，剖检死亡鸡，检查记录MD的病变情况，并每周每组取2-3只鸡采血制备血清和每只鸡采翅部羽毛6根，饲养9周后全部扑杀存活鸡，统计记录MD的发病情况。疫苗对RB1B株MDV在羽毛囊中复制的影响的AGP测定结果为两试验组疫苗免疫均可抑制RB1B株超强毒MDV羽毛囊排毒；攻毒后血液中MDV抗体水串联表达GA沐MDv gB主要表位荃因重组鸡痘病毒的构建及其免没保护作用平的AGP检测结果是在疫苗免疫后的第三周开始试验鸡体内MOV抗体水平逐步稳定上升;疚苗免疚效果的结果是两种疫苗免疚后均能保护试验鸡抵抗RBIB株超强毒MOv的感染，与攻毒对照组的差异显著，但两试验组的差异不显著.更多还原

【Abstract】 According to the data concerned and with the aids of DNAstar software, the 250-460 amino acids of the glycoprotein B of BamHI-I3 domain of Marek’s disease virus GA strain was selected as target fragment for tandem expression. With the help of Primer5.0 software two pairs of primers, Primer(U1) and Primer(U2), Primer(D1) and Primer(D2), were designed according to the sequence published in the GenBank. And an ATG GCG was added to the 5’ tern of Primer(U1), a TAA was added to the 3’ tern of Primer(D2) and a linker of nine amino acids was designeo at the 5’ tern of Primer(D1) .Thus a complete ORF formed when upstream fragment gB(U) linkered with downstream fragment gB(L/D).The gB(U) and gB(L/D) were amplified respectively based on pairs of Primer(U1) and Primer(U2) , Primer(D1) and Primer(D2) by polymerase chain reaction (PCR). PCR products, gB(U) and gB(L/D) were individually cloned into pBluescriptSKII+ plasmid, pSKgB(U) and pSKgB(L/D) were constructed. Then the pSKgB(U) was digested with XbaI+BamHI and g(U) was cloned into pSKgB(L/D) , accordingly pSKgB(U/L/D) were constructed. Finally pSKgB(U/L/D) was digested with XbaI+PstI, and then gB(U/L/D) were cloned into pEFgpt12S, pEFMDgB(U/L/D) recombinant fowlpox virus transfer vector were constructed.The quantity of parental FPV used in transfection and the optimal titer of Mycophenolic Acid (MPA) in selectable culture were determined. The recombinant fowlpox virus transfer plasmid vector pEFMDgB(U/L/D) was cotransfected with fowlpox virus 282E4 strain at CEF by calcium phosphate-DNA coprecipitation method and the target linked fragment was inserted into ORF1 domain of FPV. Then the recombinant virus was selected with selectable culture containing MPA and was purified after 10 passages. Finally, Us specificity was identified positive on its infected CEF through Indirect Immunofluorescence Assay (IFA), this result shows that the recombinant fowlpox virus has successfully expressed the foreign genesand the tandem espressed products have antigenicity; Comparing the TCID50 of the recombinant fowlpox virus (rFPV-MDgB(U/L/D) ) with that of parental FPV, there was no obvious differences between them. The result shows that insertion of foreign genes into ORF1 domain of FPV doesn’t affect the propagation and biological characteristics of this virus.The selected recombinant fowlpox virus tandem espressing the linked fragment gB(U/L/D) was cultivated for 5 passages in normal culture (2% MEM) and then the tilers of the recombinant in the selectable culture (2% MXHAT) and normal culture were tested and compared to the titer of parental FPV in normal culture. Three values were same on the whole, which show that the recombinant was purified and its replication was stable relatively. One-day old SPF chickens were vaccinated with rFPV-MDgB(U/L/D) + CVI988 (commercial vaccine) bivalent vaccine and CVI988(commercial vaccine) respectively. All chickens, experimental groups and the control group, were challenged with RB1B strain very virulent MDV at 7 days post vaccination and housed in isolation for 9 weeks. During this course, 2-3 chickens of every group were sampled 6 feathers of wings and blood to prepare serum and if chickens died they were examined for gross lesions of MD. At the end of the ninth week, all the survivors were killed and were examined for lymphoid tumors and other lesions of typical of MDV. The result of AGP assay of effect of vaccines on level of challenge virus shedding from feather follicles of chickens shows that both vaccines can inhibit replication of RB1B strain MDV in feather follicles of chickens. And AGP test of antibodies in blood from chickens challenged with RB1B strain shows that antibodies in both experimental groups gradually raised 2 weeks post vaccination. Finally, the morbidity of experimental groups was significant lower than the control group, but the morbidity of these two experimental groups has no significant differences. This result demonstrates that both vaccines can protect chickens from infected by RB1B strain MDV.更多还原