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目的:探讨用重组PCR技术对人单核细胞趋化蛋白-1(HΜMCP-1)基因CDNA进行缺失突变,构建 N末端缺失7个氨基酸的编码序列HΜMCP-1突变体-HΜMCP-1(7ND)CDNA,以期实现7ND基因治疗抑制MCP-1 活性。方法:根据缺失前后的两段基因片段A和B分别设计两对引物即内引物与外引物,第一轮PCR反应通过每段各自的内外引物,分别获得加有互补末端的A+和B+DNA片段。然后进行第二轮PCR反应,以第一轮PCR产物为模板,加入两外引物,获得大量重组体AB基因片段,将PCR产物与T载体连接,进行酶切鉴定并测序证实成功进行了 HΜMCP-1的基因改造。为便于表达HΜMCP-1突变体,通过ECOR Ⅰ/HINDⅢ酶切,将目的基因克隆入PCDNA3．1真核表达载体中。结果:经酶切鉴定并测序,表明已成功地构建了HΜMCP-1CDNA突变体-7ND的真核细胞表达载体。结论:已成功进行了HΜMCP-1基因CDNA的缺失突变,获得了7ND CDNA的克隆,为进一步研究HΜMCP-1功能奠定了基础。
Objective: To investigate the deletion mutation of CDNA of human monocyte chemoattractant protein-1 (HMNPCP-1) gene by recombinant PCR and to construct the HMNCP-1 mutant HMPCP-1 mutant (7ND) with N-terminal deletion of 7 amino acids. CDNA, in order to achieve 7ND gene therapy to inhibit MCP-1 activity. METHODS: Two pairs of primers, inner and outer primers, were designed according to the two segments A and B before and after the deletion. The first round of PCR reaction was carried out by using the respective internal and external primers of each segment to obtain complementary A + and B + DNAs Fragment. Then the second round of PCR reaction was carried out. The first round of PCR product was used as a template, and two extra primers were added to obtain a large number of AB gene fragments. The PCR product was ligated with the T vector and identified by restriction enzyme digestion and sequencing. HIMMCP- 1 genetically modified. In order to facilitate the expression of HMMCP-1 mutant, the target gene was cloned into PCDNA3.1 eukaryotic expression vector by digestion with ECOR Ⅰ / HINDⅢ. Results: After restriction enzyme digestion and sequencing, the eukaryotic expression vector of HMNPCP-1CDNA mutant -7ND was successfully constructed. CONCLUSION: The deletion mutation of CDNA of HMMCP-1 gene has been successfully carried out and the clone of 7ND CDNA has been obtained, which lays the foundation for further study on the function of HMNPCP-1.