人NPTX2基因原核表达载体构建及重组蛋白的表达纯化

Construction of prokaryotic expression vector of human neuronal pentraxin 2 gene,and expression and purification of its recombinant protein

目的在大肠埃希菌中表达人神经元正五聚蛋白2(NPTX2),并对该重组蛋白进行纯化、鉴定。方法双酶切pReceiv-er-M15 NPTX2质粒获得人NPTX2全长cDNA,经NotI和EcoRI双酶切后,连接至谷胱甘肽-S-转移酶(GST)融合表达载体pGEX-4T-1中,经限制性酶切、PCR和测序验证正确后,转化至大肠埃希菌BL21(DE3),经IPTG诱导重组蛋白表达,采用Western blotting鉴定表达产物,亲和层析法纯化GST-NPTX2蛋白。结果经酶切与测序鉴定证实原核重组载体pGEX4T-1-NPTX2构建成功,表达产物相对分子量为70kD左右,与预期值相符,Western blotting证实该蛋白具有免疫反应特异性,亲和层析法获得了纯化的GST-NPTX2蛋白。结论构建了pGEX-4T-NPTX2原核表达载体,并在大肠埃希菌BL21中诱导表达了GST-NPTX2融合蛋白。亲和纯化获得较高纯度的GST融合蛋白,为下一步继续研究NPTX2的生物学功能奠定了基础。

Objective To construct the prokaryotie expression vector of human neuronal pentraxin 2 （NPTX2） gene, induce the expression of the recombinant fusion protein in E. coil BL21, and purify the recombinant fusion protein. Methods The pReceiver-M15 plasmid containing full-length cDNA of NPTX2 was digested by restrictive enzyme EcoR Ⅰ and Not Ⅰ, and then cloned into the plasmid pOEX- 4T-1 which contained the glutathione-s-transferase （GST）. After restriction enzyme digestion identification, polymerase chain reaction （PCR） amplification and sequencing, the pGEX-4T-NPTX2 plasmid containing the correct target DNA fragments was obtained and transformed into E. coli BL21 （DE3）. By restriction enzyme digestion and sequencing, the positive transformed clones were identified. The ex pression of GST-NPTX2 fusion protein was induced with IPTG, and the products were analyzed by SDS-PAGE and Western blotting. The proteins were purified through affinity chromatography. Results The results of digestion and sequencing indicated that the recombinant prokaryotie vector pGEX-4T-NPTX2 was successfully constructed. After transformation with pGEX 4T-NPTX2 and induction with IPTG, the recombinant target protein of about 70kD was obtained by SDS-PAGE analysis, which was consistent with our anticipation. Western blotting suggested that the new band was purified recombinant GST-NPTX2 protein and possessed good immunogenicity. Conclusions The prokaryotic expression vector of human NPTX2 gene has been successfully constructed. The GST-NPTX2 fusion protein could be expressed in prokaryotie expression system of E. coli. Purified recombinant proteins are obtained through affinity chromatography, which is helpful for further study.