汉滩病毒M、S基因不同拼接方式在昆虫细胞中融合表达效果比较  

Comparison of Expression Results of Different Connection Manners of Hantaan Virus M and S Gene Segments in Insect Cells

作　　者：罗雯[1] 徐志凯[1] 张芳琳[1] 阎岩[1] 吴兴安[1] 刘勇[1] 白文涛[1] 王海涛[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《中国病毒学》2002年第3期226-229,F003,共5页Virologica Sinica

基　　金：国家自然科学基金资助 (3 0 0 70 686) ;国家教育部骨干教师计划资助

摘　　要：将汉滩病毒囊膜糖蛋白G1与核蛋白 (NP)部分片段以不同方式拼接 ,构建G1S0 .7或S0 .7G1嵌合基因 ,分别插入杆状病毒表达载体 pFBD ,转化DH10Bac致敏菌 ,获得含有嵌合基因的重组穿梭质粒Bacmid ,用其转染Sf9细胞 ,快速筛选出含有G1S0 .7或S0 .7G1嵌合基因的重组杆状病毒 ,在昆虫细胞中表达外源融合蛋白。利用间接免疫荧光、ELISA和免疫印迹对表达产物进行检测。结果表明 ,含G1S0 .7嵌合基因之重组杆状病毒可在昆虫细胞中表达出融合蛋白 ,该蛋白可被抗汉滩病毒核蛋白及糖蛋白G1特异性单抗所识别 ,其分子量约 97kD ;含S0 .7G1嵌合基因之重组杆状病毒在昆虫细胞中表达的融合蛋白 ,只能被抗汉滩病毒核蛋白特异性单抗所识别 ,其分子量约 4 3kD。上述结果提示 ,G1S0 .7嵌合基因可能在昆虫细胞中表达出完整的具有生物学活性的融合蛋白 ,S0 .7G1嵌和基因的昆虫细胞表达产物不完整 ,且生物学活性不如G1S0 .Hantaan virus glycoprotein G1 and nucleoprotein partial fragment were connected in different ways.The constructed chimeric gene G1S0.7 or S0.7G1 was inserted into baculovirus expression vector pFBD.The recombinant shuttle plasmids(Bacmids) containing chimeric genes were obtained in \%E.coli\% DH10Bac.Sf9 cells were transfected by the recombinant Bacmids,and then recombinant baculoviruses were selected and fusion proteins were expressed in insect cells.The expression was identified by ELISA,immunofluorescence and Western blot.It showes that,the recombinant baculovirus containing the chimeric gene G1S0.7 could express the fusion protein in insect cells.This protein,whose molecular weight was about 97kD,could be recognized by the hantaan virus nucleoprotein specific mAb and glycoprotein G1 specific mAb.The fusion protein,which was expressed by the recombinant baculoviurs containing the chimeric gene S0.7G1 in insect cells,could only be recognized by the hantaan virus nucleoprotein specific mAb.Its molecular weight was about 43kD.It suggests that the chimeric gene G1S0.7 can express biologically active integrated fusion protein in insect cells,while the product of \{S0.7G1\} isn't integrated and its biological activity isn't as good as the product of G1S0.7.

关 键 词：汉滩病毒 S基因 拼接方式 M基因 昆虫细胞 融合表达效果 比较 

分 类 号：Q786[生物学—分子生物学]