J亚群禽白血病病毒跨膜蛋白gp37基因克隆与表达

Cloning and Expression of gp37 Gene of Avian Leukosis Virus Subgroup J

禽白血病病毒(ALV)跨膜蛋白(TM)在病毒-细胞融合过程中起关键作用,其蛋白内部存在的许多高度保守序列有可能成为抗病毒的重要靶点。对TM结构和功能的深入研究将为抗禽白血病病毒乃至其它反转录病毒相关制剂的研制提供科学的理论基础。本研究通过PCR从本实验室分离的ALV-J山东汶上毒株获得表达TM的gp37基因,克隆连接pMD18-T载体并测序,从已构建的质粒pMD18-T-gp37中酶切回收gp37基因,构建重组转移载体pFastBacHTb-gp37。利用昆虫杆状病毒表达系统对gp37基因进行表达,通过间接免疫荧光和Western blot检测gp37基因表达产物,间接免疫荧光显示,重组杆状病毒感染的sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的sf9细胞蛋白显示出约21kD的阳性条带。结果表明,gp37基因在sf9细胞内表达成功。

The transmembrane protein（TM） encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs.Several highly conserved regions in TM are important targets for antivirus studies.Studies on structure and function of TM will provide basic information for anti-retrovirus,especially for avian leukosis virus.In the study,gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701.The gp37 gene was cloned into pMD18-T vector,and was sequenced.Then,pFastBacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system.The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot.The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot.It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.