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目的:用重组PCR技术对人单核细胞趋化蛋白鄄1(hμMCP鄄1)基因cDNA进行缺失突变,构建其突变体—7NDcDNA。方法:根据hμMCP鄄1基因缺失突变前后的两段基因分别设计两对含有酶切位点的引物,以pBluescript鄄hμMCP鄄1为模板,进行重组PCR反应,将PCR产物与T载体连接进行TA克隆,酶切鉴定并测序确定其长度为342bp,继之将目的基因克隆入pcDNA3.1真核表达载体。结果:成功构建hμMCP鄄1cDNA突变体—7ND的真核细胞表达载体,测序结果表明,该突变体N端第2至8位氨基酸缺失。结论:重组PCR技术是十分有效、可靠的基因缺失突变方法。
OBJECTIVE: To study the mutation of hMCP-1 gene cDNA by recombinant PCR and to construct its mutant-7ND cDNA. METHODS: Two pairs of primers containing restriction enzyme sites were designed according to the two genes before and after the deletion mutation of hμMCP-1 gene. PCR was carried out by using pBluescript-hμMCP-1 as a template. The PCR product was ligated with the T vector for TA cloning , Identified by restriction enzyme digestion and sequenced to determine the length of 342bp, followed by cloning the target gene into pcDNA3.1 eukaryotic expression vector. Results: The eukaryotic expression vector of hMCP-1 cDNA mutant-7ND was successfully constructed. The sequencing results showed that the amino acids 2 to 8 of the N-terminal of the mutant were deleted. Conclusion: Recombinant PCR is a very effective and reliable method for gene deletion mutation.