重组人Grn的克隆与表达

Cloning and Expression of Human Grn Gene

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。

Objects To construct a prokaryotic expression vector containing human Grn gene and observe its expression. Methods The gene Grn was amplifified by PCR from THP-1 c DNA. The cloned gene was digested by restrictive endonuclease and subcloned into eulcaryotic experssion vecter p GEX-4T-2. Grn had been cloned into p GEX-4T-2, and the recombined plasmid was transduced into TOP10. The positive cloning was identified by PCR and sequencing. The recombinant plasmid was transformed into competent cell of DE3. The expression of GRN was analyzed by Western blot. Results It was verified by PCR and nucleotide sequencing that the constructed Grn eukaryotic vector was correct. After p GEX-4T-2 was transfected into DE3, a GST fusion protein molecule of 91 KD was found in supernatant of lysis of bactoria by Western blot. Conclusion A prokaryotic expression plasmid containing human grn gene is successfully constructed, and it can express out objective protein, which has laid a concrete fundation for future study on grn.