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.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
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)
关键词
鹅源副黏病毒
F基因
定点突变
表达
goose paramyxovirus F gene site-specific mutagenesis expression
