表达羊传染性脓疱病毒F1L基因的重组山羊痘病毒的构建与特性鉴定

Characterization of a recombinant Goatpox virus expressing Orfv F1L gene

摘要:

摘要:【目的】本文旨在构建可表达羊传染性脓疱病毒F1L 基因的重组山羊痘病毒，并对其生物学特性进行初步研究。【方法】将羊传染性脓疱病毒F1L基因与转移载体pUC-TK12连接，同时插入LacZ报告基因和一双向启动子;将重组转移载体转染已接种山羊痘病毒的BHK-21细胞，通过蓝白斑筛选、纯化，对重组病毒进行连续传代培养，应用PCＲ、间接免疫荧光和Western免疫印迹等方法进行鉴定，并对重组病毒进行不同温度、酸碱度、有机溶剂和紫外照射等处理，进行TCID50评价。rGpv-F1L免疫小鼠后，经酶联免疫吸附实验检测其特异性抗体水平。【结果】成功构建重组病毒转移载体pTL-F1L，将转移载体转染BHK-21细胞后，通过蚀斑纯化获得了可稳定遗传的rGpv-F1L。间接免疫荧光和Western免疫印迹检测发现其可稳定表达F1L蛋白。55℃30min或紫外照射1h即可灭活重组病毒，且其对强酸、强碱、有机溶剂等较敏感。接种重组山羊痘病毒的小鼠40d内机体可持续产生高水平的特异性抗体，与接种Gpv病毒组相比差异极显著(P＜0.01)。【结论】本文获得了可稳定表达羊传染性脓疱病毒F1L的重组山羊痘病毒疫苗候选株，其具有良好的抗原性和生物学活性，为同时预防羊传染性脓疱病和羊痘提供新途径。

Abstract:

Abstract:［Objective］In order to establish the vaccine against the contagious ecthyma，we constructed and characterized recombinant goatpox virus expressing F1L protein of Orf virus.［Methods］The F1L gene was amplified and cloned into the vector pUC-TK12 carrying the LacZ gene and a bidirectional promoter． With the help of lipidosome，the recombinant plasmid pTL-F1L was transfected into the BHK-21 cells，which had been infected by Gpv． The aim is to make the Gpv and pTL-F1L recombined randomly and get the recombinant virus，which was defined as rGpv-F1L. The rGpv-F1L was screened by blue plaque，and then the F1L recombination and translation were identified by PCR， indirect immunofluorescence and Western blot．By the means of TCID50，we evaluated the physicochemical properties of rGpv-F1L．Female mice were immunized with the rGpv-F1L，and the specific antibodies levels in serum were detected by ELISA.［Results］We obtained rGpv-F1L，which was stably expressing F1L protein．The results of biological characteristics showed the rGpv-F1L was sensitive to acids，alkalis，organic solvents and ultraviolet．The activity of specific antibodies significantly increased in mice infected by rGpv-F1L more than Gpv (P＜0.01).［Conclusion］In this research，we have successfully obtained the candidate vaccine，which is stably expressing F1L of Orf virus．Thereby the candidate vaccine with excellent antigenicity and biological activity provides new avenues for the prevention of contagious ecthyma and capripox．