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.pdfVIP

吉林农业大学学报 2008，30(3)：348～351，355 http：／／xuebao．flau．edu．Clt Journal of in Agricultural E-mail：flndxb@vip．sina．con 鹅源副黏病毒NA一1株 F基因定点突变 及其在Bac．to．Bac系统中的表达 杜 眉，丁 壮 ，徐 明，宋子运，毕玉海，尹仁福 (吉林大学畜牧兽医学院，长春 130062) 摘 要：根据已发表的鹅源副黏病毒(GPMV)NA一1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 基因编码的氨基酸裂解位点为”0Gly—Arg-Gln-Gly．Arg-I_eu“ 。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Sf9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。 关键词：鹅源副黏病毒；F基因；定点突变；表达 中图分类号：$852．65 文献标识码：A 文章编号：1000-5684(2008)03．0348．04 Site-Specific Mutagenesis of F Gene of Goose Paramyxovirus and Its Expression in Bac·-to·-Bac System DU Mei，DING Zhuang，XU Ming，SONG Zi—yun，BI Yu—hai，YIN Ren—fu (College ofAnimal Science and Veterinary Medicine，Jilin University，Changchun 130062，China) Abstract：According to nucleotide sequence of goose paramyxovims(GPMV)NA一1 strain，two pairs of pfime~were designed and synthesized．A new F gene fragment containing the mutational sites of GPMV NA一1 was amplified by PCR，and the reconstructed F gene Was linked into the transposon vector pFastBac I to 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 1 1 2 Arg-Arg-Gln—Gly—Arg-Leu ¨．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 trans— fected into Sf 9 cells to generate recombinant baculovims expressing F recomb ination protein．Results of SDS—PAGE and We