用PCR插入突变法扩增和克隆HIV-env282肽基因

AMPLIFICATION AND CLONING OF HIV-ENV282 PEPTIDE GENE BY PCR INSERTIONAL MUTAGENESIS

本文报道借助于微机分析设计并合成了2个引物,通过聚合酶链反应插入突变法扩增出含有4个功能区(T肽区,与NLK部分同源区,显性中和位点及与CD4结合区)的HIV-env282肽基因,其5′端连有EcoR Ⅰ酶切位点和翻译起始码,3′端与终止码和BamH Ⅰ酶切位点相连。通过基因重组将其插入到大肠杆菌表达载体pBV220内并转化至宿主菌DH5α中,经电泳初筛、酶切分析和PCR鉴定,确定了一株含目的基因的重组菌,称之为pXW1。

The HIV-env282 peptide gene fragment (nt 6310-7155) coding for four functional regions (T peptide,part of homologous region of neuroleukin, neutralization dominant and CD4 binding region) were generated using the PCR from plasmid pAE332 containing HIV env gene. The primers with start and stop codons and restriction enzyme sites were designed and synthesized. PCR product were cloned into plasmid pBV220 and characterized by restriction enzyme mapping and PCR analysis. A recombinant plasmid containing HIV-env 282 peptide gene were obtained and named pXWl.