表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性

【目的】鸭瘟和小鹅瘟是番鸭和鹅的两种重要传染病，鸭瘟最主要的防治措施是定期接种鸭瘟病毒减毒活疫苗。根据2012年国际病毒分类委员会（ICTV）的报告，DEV被归为疱疹病毒科的α疱疹病毒亚科马立克氏病毒属。疱疹病毒如伪狂犬病毒、马立克氏病毒、火鸡疱疹病毒等已广泛用于病毒活载体的研究，而近几年也有关于鸭瘟病毒（DEV）作为疫苗活载体的报道。为了为免疫防控鸭瘟和小鹅瘟提供新手段，本研究拟在鸭瘟病毒疫苗株感染性克隆的基础上，构建表达小鹅瘟病毒（GPV）主要免疫原蛋白VP2的重组病毒rDEV-VP2，并研究其生物学特性，进而探讨重组病毒rDEV-VP2作为防治DEV和GPV的二联重组活载体疫苗的可能性。【方法】将密码子优化的GPV VP2基因通过常规基因克隆的方法插入转移载体pEP-BGH-end，构建含有GPV VP2表达框pCMV-VP2-BGH-pA的重组表达质粒。在鸭瘟病毒（DEV）疫苗株细菌人工染色体克隆pDEV-EF1的基础上，通过“Red E/T”两步重组法将GPV VP2基因表达框插入到DEV US7和US8基因之间构建了突变体克隆pDEV-VP2。利用磷酸钙法转染鸡胚成纤维细胞（CEFs）拯救获得重组病毒rDEV-VP2和删除Bac质粒序列的rDEV-VP2-Cre，并对重组病毒细胞体外生长曲线、蚀斑大小和VP2蛋白表达情况进行测定。将rDEV-VP2接种番鸭，在不同时间采集血清，采用间接ELISA法检测血清中GPV VP2抗体产生情况。【结果】间接免疫荧光检测和Western blotting分析表明，外源蛋白VP2在CEFs细胞成功表达。病毒生长曲线和蚀斑大小测定结果显示，rDEV-VP2在CEFs细胞上的增殖滴度与亲本株相比无显著差异，表明外源基因VP2的插入不影响rDEV重组病毒的增殖。动物试验结果表明，7日龄雏番鸭接种rDEV-VP2可以诱导产生针对GPV VP2的抗体，免疫后3周抗体阳性率为50%（4/8）。【结论】本实验将小鹅瘟病毒的主要免疫原基因VP2插入到DEV疫苗株基因组的US7和US8基因间构建了表达该免疫原性基因的重组鸭瘟病毒细菌人工染色体，继而在鸡胚成纤维细胞（CEFs）上拯救获得了重组病毒rDEV-VP2，病毒细胞生长特性与亲本株基本一致，且能诱导鸭体产生GPV VP2特异性的抗体。该研究为研制DEV-GPV二联重组活载体疫苗奠定了基础。

Construction and Characterization of a Recombinant Duck Enteritis Virus Expressing VP2 Gene of Goose Parvovirus

【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek''s disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection.