A组轮状病毒我国地方株VP6基因的序列测定及其在杆状病毒系统中的表达  

NUCLEOTIDE SEQUENCE OF THE VP6 GENE OF GROUP A ROTAVIRUS FIELD STRAIN AND THE EXPRESSION IN BACULOVIRUS EXPRESSION SYSTEM

作　　者：李国华[1] 钱渊[1] 何湘君[1] 霍云雯[1] 

机构地区：[1]首都儿科研究所北京市感染与免疫中心实验室,北京100020

出　　处：《病毒学报》1999年第3期231-237,共7页Chinese Journal of Virology

基　　金：国家自然科学基金;卫生部科研基金;北京市自然科学基金

摘　　要：通过反转录－ 聚合酶链反应（ Ｒ Ｔ－ Ｐ Ｃ Ｒ） 获得了轮状病毒地方株 Ｔ１１４ Ｖ Ｐ６ 全基因的ｃ Ｄ Ｎ Ａ 片段，将其克隆入转移载体质粒ｐ Ｖ Ｌ１３９３ 中，构建成重组质粒ｐ Ｖ Ｌ１３９３ － Ｖ Ｐ６ 。对克隆的 Ｖ Ｐ６ 基因进行序列测定，并用它和杆状病毒（ Ａｃ Ｍ Ｎ Ｐ Ｖ） 野毒株 Ｄ Ｎ Ａ 共转染 Ｓｆ９ 细胞，筛选纯化得到含 Ｖ Ｐ６ 基因插入片段的重组杆状病毒，并进行了表达重组蛋白 Ｖ Ｐ６ 的检测。测序结果显示 Ｖ Ｐ６ 基因全长１ ３５６ｂｐ ，序列分析显示与 Ｗａ 株非常接近，提示 Ｔ１１４ 为亚组Ⅱ病毒株。用高价免疫血清经 Ｗｅｓｔｅｒｎ ｂｌｏｔ 检测表达产物，结果显示，重组病毒感染细胞裂解液样品中可见大小约４５ｋ Ｄ 的特异条带；亚组Ⅱ特异性单抗检测到大小约１２０ｋ Ｄ 的条带，提示重组蛋白 Ｖ Ｐ６ 获得了表达，具有正常的抗原反应性和天然 Ｖ Ｐ６ 的三体结构。A cDNA fragment of the full-length VP6 gene from rotavirus strain T114 was obtained by RT-PCR and cloned into the transfer vector pVL1393 to construct recombinant plasmid pVL1393-VP6. The nucleotide sequence of VP6 gene fragment in pVL1393-VP6 was determined. The pVL1393-VP6was then cotransfected Sf9(Spodoptera frugiperda) cells with the AcMNPV DNA to produce the recombinant virus which included VP6 gene and the expression of recombinant protein VP6 was detected. The T114 VP6 gene was 1,356bp in length and the sequencce was very close to strain Wa, it suggested that strain T114 belonged to subgroup Ⅱ. The expression of VP6 was detected by Western blotting with hyperimmune serum against strain Wa. The monomer of VP6 (about 45kD) was found in cell lysates of Sf9 cells infected with recombinant virus and a band of molecular weight about 120kD was detected with the monoclonal antibody which is specific for subgroup Ⅱ epitopes of VP6. The results demonstrated that VP6 was expressed in baculovirus system and the immunoreactivity and conformational properties were similar to authentic VP6(trimeric).

关 键 词：VP6 轮状病毒 杆状病毒 表达 

分 类 号：R373.2[医药卫生—病原生物学]