By using the site - directed mutagenesis technique, the mutative C and A chains of human proinsulin gene were joined through PCR, and the human trypsin C and A mutant genes were constructed. "The PCR products B and CA fragments were directly cloned into T vector by using T - A method, the middle recombinant pHB and pHCA were constructed, and their sequencing result was completely consistent with the published sequence in Genbank. After the double digestions of pHB and pHCA by the restrictive endo-nucleases BamH I and Sse8387 I, the retrieved and purified target fragments were directedly connected to construct the recombinant plasmid pHBCA, which was verified to be successful by enzymatic digestion analysis.%采用定点突变技术,将组成人胰岛素原基因的C、A两条肽链进行了PCR突变连接,构建了人胰岛素C、A突变基因.采用T-A法将PCR产物B、CA克隆到T载体,分别构建了中间体pHB和pHCA,其测序结果与GenBank公布的一致.应用Bam-H Ⅰ和Sse8387 Ⅰ对pHB和pHCA进行双酶切,然后将回收纯化的片段进行定向连接,构建了重组质粒pHBCA,酶切分析证实重组质粒pHBCA构建成功.
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