用PCR突变技术克隆HIV env125肽基因

Insertional mutagenesis and cloning of HIV env 125 peptide gene by PCR

作者在计算机辅助设计下,利用DNA合成仪合成HIV env 125-1,HIV env 125-2两个寡核苷酸引物及HIV env 101-1寡核苷酸探针,以PCP技术扩增env 125肽基因。扩增产物经电泳和Southern杂交鉴定后克隆入pUC19中,筛选、鉴定后进行序列分析。结果表明,上述方法获得的env 125肽基因5’端插入了EcoRI酶切位点和起始码ATG,3’端插入了终止码TAG和HindⅢ酶切位点,且序列及读框正确。

One oligonudeotide probe HIV env 101-1 and two aligonucleotide primers, HTV env 125-1 and HIV env 125-2 , were designed with the aid of a computer and synthesized by a DNA synthesizer. The env 125 peptide gene was amplified by PCR. After its being identified by electrophoresis and Southern blot, the production of PCR was cloned into plasmid pUC19. The recombinant penv125, identified with X-gal selection and restriction mapping was sequenced. The result showed that the cloned env 125 peptide gene contained inserted FcoRI site and ATG in 5' end, Hind Ⅲ site and TAG in 3' end The sequence and reading frame arc proved to be correct