摘要
作者设计并合成了一对突变引物PGO1和PGO2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、BamHI和ATG及TAA序列,以便于HlV-1gag基因序列的定向克隆和表达。用PCO1和PGO2作引物,采用PCR方法从HIV-1基因组DNA中扩增出一个长504bp的DNA片段,用EcoRl和BamHI双酶切位点将此片段定向克隆入pUC19质粒。将克隆基因插入M13mp18进行DNA序列分析,结果表明,该基因序列及读框完全正确,且在其5′末端突变出EcoRI位点和ATG起始码,3′末端突变出TAA终业码和BamHI位点,从而为该基因的表达研究奠定了基础。
In the present paper. the primers PG01 and PG02 were synthesized in which the EcoRI and BamHI recognition sites as well as the initiation and termination codons were designed for convenience of subsequent cloning and expression. Micrograms of target gene sequence was produced by 30 rounds of amplification with pJG 423 as a template. The amplified gene fragment was digested with both EeoRI and BamHI and ligated to pUC19 predigested with the same restriction endonucleases. The recombinant plasmid pCY52 was identified and the cloned P20 gene was inserted into phage M13mpl8 for sequence analysis. It was revealed that the sequence of the cloned P20 gene (471bp) was identical to, its template. And the site mutations at both the 5′ and 3′ ends were also successful.
出处
《中国病毒学》
CSCD
1992年第3期265-271,共7页
Virologica Sinica
关键词
聚合酶链反应
克隆
免疫缺陷病毒
Human immunodeficiency virus Polymerase chain reaetion Cloning Sequence analyvsis Mutagenesis Restriction enzyme
