【作者】 陈志琳；

【导师】 崔治中； 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2002， 博士

【摘要】 30年来抗马立克氏病(MD)疫苗有了很大的发展，抗MD疫苗有下列几种：①单价疫苗，包括血清Ⅲ(HVT)，血清Ⅱ型(SB-1)及致弱的血清Ⅰ型(CV1988)MDV疫苗。②多价苗，包括二价苗(血清Ⅰ型+血清Ⅱ型，血清Ⅰ型+血清Ⅲ型和血清Ⅱ型+血清Ⅲ型)及三价苗。伴随着抗MD疫苗的发展，马立克氏病病毒(MDV)本身也在不断突变、演化，毒力不断增强，常规的单价苗、多价苗已难以控制毒力不断增强的MDV，在过去的10年中，科学家们通过对MDV分子生物学的研究，力图研制出比现在常规疫苗效果更好、使用更方便的抗MD基因工程疫苗。MDV分子生物学的研究进展，MDV全基因组测序工作的完成，及MDV一些重要基因的鉴定、定位及结构与功能方面的研究成果，促进了马立克氏病基因工程疫苗的研制和开发。MDVgB糖蛋白是MDV主要保护性抗原，rFPV-gB及rHVT-gB已显示良好的保护性免疫，但仅一个靶抗原，其免疫保护性仍然有局限性，因此人们也在寻找除gB之外，其他与保护性有关的抗原成分作为基因工程目的基因，对疱疹病毒囊膜糖蛋白的研究发现，gD、gB、gI、gC、gE和gG具有免疫保护作用，而把多个靶抗原联合使用时，具有协同免疫保护作用。糖蛋白gI是疱疹病毒中重要的囊膜糖蛋白，它可与其他囊膜糖蛋白组成双价或多价疫苗，提高单一囊膜糖蛋白的免疫保护效力。此外，有研究报告表明gI的同源编码产物在细胞间传播、毒力介导、FC受体活性等方面都起一定的作用。以MDVgI作为靶抗原，开展保护性免疫研究，国内外未见报道。本研究以MDV gI为研究对象，以鸡痘病毒(FPV)中国株为载体，构建表达MDV gI基因的重组鸡痘病毒(rFPV)，在构建成功表达MDV gI的rFPV的基础上，进一步分析rFPV-gI扬州大学博士学位论文的稳定性及鸡对rFPv一gI的抗体反应性，然后研究它的免疫保护效力，为研制rFPV一MDv一gl基因工程疫苗，进一步开发多价MDV基因工程重组疫苗奠定基础。 根据MDV 648A株病毒基因组gl基因序列，我们设计了两个引物，用PCR扩增出MDVgl基因。通过琼脂糖凝胶电泳，获得一条1 060 bp DNA条带，与MDvgl基因的大小是一致。在此基础上，用Qiagen公司胶回收试剂盒来纯化PCR产物。用Smal及Xhol来酶切PCR产物。用Smal和Sall酶切鸡痘病毒转移质粒载体pFGI 175一1。连接，转化大肠杆菌DHS。，转化菌经筛选后，得到一个含gl基因的阳性克隆，进一步纯化此阳性克隆DNA。用全自动测序仪测定插入片段DNA序列，证明插入片段DNA序列与MDv·648A一gI序列完全一致。此含MDVgi基因的重组转移质粒命名为pFGgill75一1。通过脂质体方法，将pFGglll75一1转染已感染FPv野毒(wt)的鸡胚成纤维细胞(cEF)，孵育3天后，通过蓝斑筛选法，筛选纯化四次后得到纯化的:FPv，用PCR扩增rFPV DNA，证实此rFpV含MDV 91基因。用FPv(wt)及rFPv感染96孔板上CEF，用pBS洗涤，丙酮固定，然后加抗gI单克隆抗体37℃孵育30分钟，PBS充分洗涤后，与抗鼠IgG荧光标记抗体作用，结果显示，rFPv感染的cEF有很强的荧光，而FPv(wt)感染的CEF没有荧光。说明MDvgl得到表达，此表达MDVgl基因rFpV命名为rFpV一MDV 64891。 用rFpv一MDv64sgl感染eEF，传代20次后，此rFPv仍然能够在CEF上稳定复制。用lo4PFurFPv一MDv64sgl及FPv(wt)分别接种l日龄sFP鸡，饲养于带正压过滤空气隔离器中，接种前及接种后20天采血收集血清，用间接免疫荧光法(IFA)分析血清与感染MDv的cEF反应情况。结果表明接种FPv一MDv648gl后鸡血清中含有抗gI抗体。 用104或lo6PFurFPv一Mnv“591接种l日龄sPF鸡和蛋鸡，在接种后10天后用MDV RBIB攻毒，隔离饲养60天，在此期间，解剖死亡鸡，检查记录MD大体病变，饲养第60天扑杀存活鸡，统计记录MD大体病变。接种1了PFu rFPv的伊莎蛋鸡的保护指数为22.97，rFPv一gl免疫接种组MDv发病率及死亡率低于FPv(wt)接种组，但差异不显著。接种1护PFU rFPv的白来航蛋鸡的保护指数为27.40，rFpV一91免疫接种组MDv发病率显著低于FPv(讯)接种组(P<0.05)，陈志琳:表达马立克氏病病毒gl基因重组鸡痘病毒的构建及其免疫保护作用rFPv一gI免疫接种组MDv死亡率低于FPv(wt)接种组，但差异不显著。接种100PFu的sPF鸡保护指数为23.53，rFPv一gI免疫接种组MDv发病率及死亡率均显著低于FPV(wt)接种组(P<0.05)。 此结果表明表达MDVgl基因的rFPv有一定程度的抗超强毒株免疫保护力。本研究是国内外首次报导构建表达MDvgi基因重组FPv，首次证实rFPV一MDVgi具有一定程度的抗超强毒株免疫保护力。更多还原

【Abstract】 For the past 30 years, the vaccines against Marek’s Disease (MD) have been developed effectively. At present monovalent vaccine and polyvalent vaccine are maily used against MD. The monovalent vaccines include serotype 3 (HVT), serotype 2 (SB-1) and attenuated serotype 1 ( CVI988). The polyvalent vaccines include bivalent vaccine (serotype 1 + serotype 2, serotype 1 + serotype 3 and serotype 2 + serotype 3) and trivalent vaccine. The forgoing succession of MD vaccines paralleled the continued evolution of field strains of serotype 1 Marek’s disease virus (MDV) to greater virulence. The conventional vaccines are more difficult to control very virulent and vv+ MDV. For the past 10 years, by research on molecular biology of MDV, scientists have tried to develop more effective and more convenient recombinant vaccines. The genomes of several MDVs were completely sequenced, some of important genes of MDV have been identified and localized, and the progresses of research on structure and function of these gene have been going well, all of these promoted the development of recombinant MD vaccines. MDV-gB is the main protective antigen of MDV. rFPV-gB and rHVT-gB have showed good protection. But one target antigen is difficult to induce complete protective immunity. Apart from gB, scientists are looking for other target antigens which can induce protection. In glycoproteins of herpesvirus, experiments suggested that gD, gB, gI, gC, gE, and gG could induce different levels of protection. When these glycoproteins were used in mixture, significant protective synergism was observed. Glycoprotein I is an important one among glycoproteins in herpesvirus, it can provide protective synergism when it was used with other glycoproteins. In addition, gI homologue of herpesviruses is required to mediate efficient cell-to-cell spread, involved in virulence, and functions as a receptor for the Fcdomain of immunoglobulin G. Up to now, there are no reports about MDV gI in protective experiments. In this program, we focussed on MDV gI. First we constructed a recombinant fowlpox virus (rFPV) expression gI of MDV, then explored immunogenecity and stability of the rFPV, finally investigated its immune protection.According to gI sequences of MDV 648A strain, two primers were designed, and MDV gI gene was amplified by PCR. PCR products were identified by agarose gel electrophoresis. A DNA band of 1060 bp was obtained, which was corresponded to that of MDV gI gene, and purified from gel with Qiagen gel extract kit. The PCR products were ligated into plasmid pFG1175-1 and transformed into E.coli DH5 α. After screening, one postive clone was got. The clone DNA was extracted by Qiagen midi plasmid kit and sequenced by automatic sequencer. The sequence of inserted DNA was exactly same as that of MDV-648A-gI gene. The transferring plasmid carrying gI gene was named pFGgI1175-l. Chick embryo fibrobast (CEF) cells infected FPV were transfected with pFGI1175-l using lipofectin reagent. After 3 days of incubation, the recombinant FPV were identified by blue plaque assays and purified by 4 passages of singer plaque. By PCR, the recombinant FPV was confirmed of containing gI gene of MDV. 96 well cultures of CEF cells infected with parental FPV and recombinant FPV were washed in PBS and fixed with acetone. The fixed cells were then incubated with MDV gI monoclonal antibody for 30 min at 37℃ and washed extensively in PBS. Bound antibodies were detected with fluorescein-conjugated anti-mouse IgG. The results showed that positve staining were observed only in cells infected with the recombinant FPV, but not in cells infected with parental FPV. These indicated that the recombinant FPV expressed gI gene of MDV in CEF. The recombinant FPV expressing gI gene of MDV was named rFPV-MDV648gI.The recombinant virus can replicate stably after 20 passages when it was used to infect CEF. Two groups of one-day-old specific pathogen free (SPF) chickens kept in isolators were injected subcutaneous with 104 PFU of the recombinant FPV and the parental FPV更多还原