【作者】 姜世金；

【导师】 杨汉春；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2003， 博士

【摘要】 本研究将Ⅰ型MDV Md11株的pp24基因的完整ORF克隆入原核表达载体pGEX-6P-1中，重组质粒pGEX-pp24转化BL21宿主菌后，经IPTG诱导表达。诱导菌体裂解物经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)后，用山羊抗GST抗体进行Western-blotting试验，结果确证了GST-pp24融合蛋白的表达。将表达产物从凝胶中回收，免疫4周龄小白鼠，五次免疫后，采血分离血清。所得血清分别与GA、Md11和CVI988株MDV感染的鸡胚成纤维细胞(CEF)进行间接免疫荧光试验(IFA)，结果表明大肠杆菌表达的pp24基因至少保留了部分天然pp24蛋白的抗原性。 将Ⅰ型MDV Md11株的pp38基因和pp24基因的完整ORF分别克隆入真核表达载体pcDNA3.1/Zeo(+)中，重组质粒pcDNA3.1-pp38和pcDNA3.1-pp24在脂质体作用下分别转染CEF，在转染后24、48、72小时，用单抗H19和pGEX-pp24原核表达产物制备的抗血清分别对pcDNA3.1-pp38和pcDNA3.1-pp24转染的CEF进行IFA检测，结果检测到了pp38和pp24在CEF中的表达，该试验也使pp24基因在原核和真核系统中的表达得到了相互验证。 为研究pp38和pp24之间的关系，将Ⅰ型MDV Md11株的pp38基因和pp24基因的完整ORF克隆到真核双表达载体pBudCE4.1中，转染CEF后，通过IFA检测和用抗pp24多克隆血清进行Western-blotting试验检测到了pp38和pp24磷蛋白的共表达。 对包括Ⅰ型MDV疫苗毒、强毒、超强毒、特超强毒共7个国际参考株与7个中国分离野毒株的pp24基因和pp38基因分别进行了克隆测序，序列分析结果表明，Ⅰ型MDV的pp24基因和pp38基因均非常保守，比较的15个毒株的pp24基因的核苷酸和氨基酸之间的同源性分别高于98.9％和98.1％，同时比较的14个毒株的pp38基因的核苷酸和氨基酸之间的同源性分别高于99.4％和99.0％。除了pp38基因的第320位(A→G)和第326位(A→G)核苷酸发生变异，相应的第107位(Q→R)和第109位(E→G)氨基酸发生改变最终导致抗原位点发生变化外，两个基因中的其它核苷酸变异均未引起明显的抗原性改变。对pp24基因和pp38基因进行同源性比较分析的结果表明，绝大多数毒株二者的前195个核苷酸完全一致，不同毒株间的第81位核苷酸的差异(G/C)并不引起编码的氨基酸变化，仅仅与地域分布有关，这很可能是MDV在长期病毒衍化过程中形成的地域性遗传标志，而与病毒的分离年代及MDV的致病型等因素无关。 另外，本文还用pp38特异的PCR产物标记的核酸探针，对来自包括台湾在内的全国10个省37个鸡群共792只鸡的病理组织样品进行了检测，同时用REV SNV株全基因组cDNA克隆标记的探针检测REV，用CAV Cux-1株全基因组克隆标记的探针检测CAV。结果表明：发病鸡中这三种病毒的检出率均相当高，分别为84％(MDV)、62.8％(CAV)和58.8％(REV)：病鸡中存在非常严重的双重感染(32.6％)和三重感染(45.7％)；37个鸡群中的28个存在三重感染(75.6％)，5个鸡群存在MDV和CAV的共感染(13.5％)，3个鸡群存在MDV和REV的共感染(8.1％)，仅有1个鸡群未检测到这三种病毒；在地域分布上，除了广西未检测到CAV外，其余的9个省份均检测到了MDV、CAV和REV，显示这三种病毒在我国已广泛存在。更多还原

【Abstract】 Marek’ s Disease Virus(MDV) phosphoprotein 24(pp24) gene was amplified from Md11 strain by Polymerase Chain Reaction(PCR). Then we cloned it into the downstream of GST gene according to the right open reading frame (ORF) in pGEX-6P-l vector. The recombinant was transformed into E. co2i’BL21 strain for expression with the induce of IPTG. SDS-PAGE and Westen-blotting validated the expression of pp24. The expressed specific band was excised from the gel and injected into mice once two weeks for 5 times, then we collected the antiserum from the mice and used it for IFA with Chicken Embryonic Fibroblasts(CEF) infected by MDV Mdll, CVI988 and GA strains respectively. The positive straining was found in the MDV plaques, which shows the in vitro expressed protein of pp24 has some epitopes of MDV.We constructed pcDNAS. 1-pp38 and pcDNAS. 1-pp24 eukaryotic expressing plasmids, which contain pp38 gene and pp24 gene respectively. These two plasmids were transfected into CEF by lipofectamine reagent. 24, 48, 72 hours later after transfection, we testified the expression of pp38 with Mab H19, and pp24 with the antiserurn of pp24 expressed in E. coli. The tests justified the expression of pp24 in prokaryotic and eukaryotic expressing systems.In order to study the correlation of pp38 and pp24, we cloned pp38 gene and pp24 gene into pBudCE4. 1 vector which can express two different genes at the same time. After transfection, we also testified the co-expression of pp38 and pp24 by IFA and by Western-blotting with Anti-GST-pp24 sera.We cloned and sequenced the pp38 and pp24 gene amplified from 814, CVI988(mMDV), GA(vMDV), Md5(vvMDV), Md11(vvMDV), RBlB(vvMDV), 648A(vv+MDV), and other 7 isolated strains from China. Sequence analysis shows pp24 and pp38 gene are very conservative. The homogeneity of pp24 and pp38 is higher than 98. 9% and 99. 4% in nuclear acid and higher than 98. 1% and 99. 0% in amino acid among different strains.There are two mutations in pp38 gene at the sites of 320 (A-G) and 326(A-G). These mutations cause the changes of the amino acid at 107(Q-R) and 109(E-G). The rest mutations in pp38 and pp24 are at random. Sequence analysis also shows the first 195 nuclear acids of pp38 and pp24 are the same except for the 81 site(G/C), but this mutation does not cause the change of amino acid. We regard this as a genetic marker connecting with geography in the evolution of MDV but not related to isolated time and pathotype of different strain of MDV I .In order to investigate the epidemic of MDV, reticuloen-endotheliosis virus (REV) and chicken anemia virus(CAV), we used the special PCR amplified product of pp38 gene as a probe to detect MDV, the cDNA clone of whole genome of SNV strain as a probe to detect REV, the clone of whole genome of Cux-1 strain as a probe to detect CAV. The tissue samples of 792 diseased chickens from 37 flocks of 10 provinces (including Taiwan Province) in China are tested. The results indicate that the checkout ratios of the three viruses are quite high: 84%(MDV), 62. 8% (CAV) and 58. 8%(REV) ;all chickens have very serious dual infection (32.6%) and triple infection(45. 7%); 28 of 37 chicken flocks occur triple infection (75. 6%) ,5 ones occur dual infection of MDV and CAV(13. 5%),3 ones occur dual infection of MDV and REV(8. 1%), only one not occurs MDV, CAV or REV. Expect CAV is negative in the samples from Guangxi province, the other 9 provinces exist MDV, CAV and REV, it shows that the three viruses have existed widely in China.更多还原