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目的 :构建人Grn基因原核表达载体并观察其表达。方法 :从人单核细胞系THP-1细胞c DNA中扩增出Grn片段,通过酶切与连接,将Grn片段构建到p GEX-4T-2质粒载体上,再转化入感受态细胞TOP10,菌落PCR鉴定阳性克隆并测序分析,测序正确的重组质粒再转化入感受态细胞DE3中表达蛋白,用Western blot鉴定结果。结果 :构建的p GEX-4T-2表达载体作PCR和双酶切鉴定,证实其中有目的片段完整插入,插入片段测序结果与Grn序列设计完全一致,将其转化入DE3中,Western blot结果表明在细菌裂解上清有分子质量为91KD的融合蛋白。结论 :重组人Grn的克隆与表达成功,为进一步研究Grn的功能奠定了基础。
Objective: To construct prokaryotic expression vector of human Grn gene and observe its expression. METHODS: The Grn fragment was amplified from c DNA of human monocytic cell line THP-1. The Grn fragment was constructed by digestion and ligation into pGEX-4T-2 plasmid vector and then transformed into competent cells TOP10. The positive clones were identified by colony PCR and sequenced. The correct recombinant plasmids were transformed into the expressed protein of competent cells DE3, and the results were identified by Western blot. Results: The constructed pGEX-4T-2 expression vector was verified by PCR and double enzyme digestion. The complete insert of the target fragment was confirmed. The sequence of the inserted fragment was exactly the same as that of the Grn sequence, and was transformed into DE3. The results of Western blot The bacterial lysate supernatant has a molecular mass of 91KD fusion protein. Conclusion: The successful cloning and expression of recombinant human Grn laid the foundation for further study on the function of Grn.