鹅源副黏病毒NA-1株F基因定点突变及其在Bac-to-Bac系统中的表达  

Site-Specific Mutagenesis of F Gene of Goose Paramyxovirus and Its Expression in Bac-to-Bac System

作　　者：杜眉[1] 丁壮[1] 徐明[1] 宋子运[1] 毕玉海[1] 尹仁福[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《吉林农业大学学报》2008年第3期348-351,355,共5页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(30571375)

摘　　要：根据已发表的鹅源副黏病毒(GPMV)NA-1株F基因序列设计2对引物,引物中含有突变位点,分3次扩增F基因片段,从而得到目的基因片段,再利用酶切位点将基因片段与转座载体pFastBac 1连接,获得重组质粒,命名为pFastBac 1-F′,从而使改造后的F′基因编码的氨基酸裂解位点为112Gly-Arg-Gln-Gly-Arg-Leu117。并将pFastBac 1-F′进行测序鉴定后转化到DH10Bac宿主菌,将目的基因定向插入到Bacmid质粒中,经筛选获得的重组Bacmid F′转染Sf 9昆虫细胞,并进行表达检测。结果,表达产物经SDS-PAGE和Western-blot检测获得蛋白特异条带,相当分子量约63 kD。According to nucleotide sequence of goose paramyxovirus（GPMV）NA-1 strain,two pairs of primers were designed and synthesized.A new F gene fragment containing the mutational sites of GPMV NA-1 was amplified by PCR,and the reconstructed F gene was linked into the transposon vector pFastBacⅠto be pFastBac-F′.The F′gene has a typical feature of attenuated viruses whose multiple basic amino acids at the deduced cleavage site of the fusion protein is 112 Arg-Arg-Gln-Gly-Arg-Leu117.pFastBac-F was transformed into E.coli DH10Bac which contains bacmid and helper plasmid,and then transposition was carried out,and the recombinant bacmid was constructed in it.The recombinant bacmid was transfected into Sf 9 cells to generate recombinant baculovirus expressing F′recombination protein.Results of SDS-PAGE and Western-blot showed that the molecular weight of the expressed F′recombinant protein was about 63 kD,and it could be specifically recognized by polyclonal antibody against GPMV.

关 键 词：鹅源副黏病毒 F基因 定点突变 表达 

分 类 号：S852.65[农业科学—基础兽医学]