表达鸡传染性法氏囊病病毒VP 2基因的重组火鸡疱疹病毒的构建及生物学特性分析

Construction of Recombinant Herpesvirus of Turkey Expressing Infectious Bursal Disease Virus VP2 Gene and Analysis of Its Biological Characteristics

鸡传染性法氏囊病(IBD)是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒(HVT)活载体疫苗,本研究构建了表达鸡传染性法氏囊病病毒(IBDV)保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体,获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下,连接于入门质粒pENTR,构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应,构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞(CEF),拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测,并绘制重组病毒体外生长曲线,分析其体外复制特性。结果表明,重组病毒rHVT-VP2能够稳定表达VP2蛋白,rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体,并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础,对IBD的防控具有重要意义。

Infectious bursal disease(IBD)is a highly lethal and immunosuppressive infectious disease that seriously harms the poultry industry.In order to develop a recombinant herpesvirus of turkey(HVT)vector vaccine against IBD,a recombinant HVT expressing the protective antigen VP2 gene of infectious bursal disease virus(IBDV)was constructed and its biological characteristics in vitro were analyzed.The VP2 gene of very virulent IBDV(vvIBDV)was amplified by RT-PCR and cloned into pCI vector to obtain the recombinant eukaryotic expression plasmid pCI-VP2.The expression frame of VP2 gene carrying CMV promoter was cut off by restriction endonuclease and ligated into entry plasmid pENTR to construct a recombinant entry plasmid pENTR-VP2.The recombinant expression fosmid H3-VP2 were constructed by LR recombination reaction between pENTR-VP2 entry plasmid and HVT recombinant fosmid H3-Kan/ccdB.The recombinant virus rHVT-VP2 was obtained by co-transfection of H3-VP2 with four other fosmids that overlap each other and cover the whole genome of HVT into chicken embryo fibroblasts(CEF).After continuously passaged in CEF for 20 generations,the recombinant virus was detected by PCR,indirect immunofluorescence,and Western blot.The growth curve of recombinant virus in vitro was determined and its replication characteristics in vitro were analyzed.The results showed that the recombinant virus rHVT-VP2 stably expressed VP2 protein,and the replication ability of rHVT-VP2 in CEF was not significantly different from that of the parent virus.The recombinant virus rHVT-VP2 induced virus neutralization antibodies against IBDV and provided 90%protection against vvIBDV-induced death in chickens.The construction of recombinant virus rHVT-VP2 lays a foundation for the development of recombinant HVT live vector vaccine against IBD and is of great significance for the prevention and control of this disease.