Construction of prokaryotic expression vector of PA gene of avian influenza virus and identification of the expression vector
2008
Zhao Cuijun | Huang Huiling | Du Juchao
Chinese. 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Gen―Bank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DH5a，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2151bp，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。[著者文摘]
Show more [+] Less [-]English. 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 con- structed. 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 ligat- ed product was transformed into E. coli DHSa competent cells. The positive clones were identified by PCR, digestion with BarnH 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.
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