禽流感病毒PA基因的原核表达载体的构建及鉴定

以重组质粒pcDNA-PA为模板扩增PA全基因,并构建原核表达载体pGEX-6p-1-PA。根据GenBank公开发表的禽流感病毒PA基因的序列设计引物,用PCR方法从重组质粒pcDNA-PA中扩增禽流感病毒的PA基因,将该基因定向插入到原核表达载体pGEX-6p-1中,然后将连接产物转化宿主菌DH5α,挑取阳性克隆进行PCR和BamHⅠ、NotⅠ双酶切鉴定并测序。测序结果表明,禽流感病毒PA基因全长2151 bp,编码717个氨基酸,原核表达载体pGEX-6p-1-PA已构建成功。从而为禽流感蛋白PA的表达及其多克隆抗体的制备奠定了基础。

Construction of prokaryotic expression vector of PA gene of avian influenza virus and identification of the expression vector

The complete PA gene of Avian Influenza Virus(AIV) was amplified with recombinant plasmid pcDNA-PA as template,and prokaryotic expression vector pGEX-6p-1-PA was constructed.According to the relevant nucleotide sequence from GenBank,a pair of specific primers were designed,and the complete PA gene of AIV was amplified with recombinant plasmid pcDNA-PA as template by PCR method.The PCR fragment was inserted into pGEX-6p-1.The ligated product was transformed into E.coli DH5α competent cells.The positive clones were identified by PCR, digestion with BamH I and Not I,and sequence.Sequence analysis demonstrated that PA gene contained one open reading frame of 2 151 bp,which encoded a polypeptide of 717 amino acids,therefore the prokaryotic expression vector pGEX-6p-1-PA was constructed successfully.This established a foundation for expression of AIV PA protein and preparation of its polyclonal antibody.