Objective:To clone the human B7-H3 (CD276) gene's complete coding sequence and express the recombinant B7-H3 protein in Pichia pastoris,and purify the expressed product.Methods:B7-H3 gene's complete coding sequence was cloned by RT-PCR from A549 human lung cancer cells and 6×His tag gene was inserted into it's 3'end.The PCR product was inserted into the pPIC9K vector to construct an expression plasmid,which was named as pPIC9K/B7-H3.The pPIC9K/B7-H3 plasmid was subsequently transformed into Pichia pastoris strain GS115,and clones were screened by different concentrations of G418 on the YPD plate and were identified by PCR method.Then the positive clones were induced by methanol,the recombinant protein was purified by Ni-NTA Agarose,analyzed by SDS-PAGE and Western Blot.Results:The DNA fragment about 1 500bp was amplified by RT-PCR.The recombinant plasmid pPIC9K/B7-H3 was constructed successfully and the reading frame was correct.SDS-PAGE result showed that the expressed product of GS115/pPIC9K/B7-H3 had a specific band of 70kD.Western Blot showed that the purified protein bound to both mouse anti-6×His antibody and anti-B7-H3 antibody.Conclusion:The human B7-H3 gene's complete coding sequence was successfully cloned and expressed in Pichia pastoris,which laid a solid foundation for further preparation of study on the B7-H3's function and it's regulation mechanism during tumorigenesis and development.%目的:克隆人B7-H3(CD276)基因的完全编码区序列,在毕赤酵母中表达B7-H3重组蛋白并纯化.方法:应用RT-PCR从人肺癌A549细胞中克隆B7-H3基因的完全编码区序列并在3'端加入6×His标签基因,克隆于毕赤酵母表达载体pPIC9K中,构建表达质粒pPIC9K/B7-H3,以该质粒转染酵母菌株GS115,在含有不同浓度遗传霉素(G418)的YPD平板上进行筛选,然后挑取单克隆进行PCR鉴定,甲醇诱导表达鉴定结果为阳性的酵母重组子,表达产物通过离子交换柱纯化后经SDS-PAGE电泳和Western Blot进行检测.结果:应用RT-PCR从人肺癌A549细胞中获得约1 500bp、与目的基因大小相符的条带,且测序正确;构建的酵母表达质粒pPIC9K/B7-H3表达框正确无误;阳性酵母重组子表达产物经6×His标签纯化后,SDS-PAGE电泳显示在70kD处有与预期大小相符的蛋白条带,Western Blot显示该蛋白可分别与抗6×His单克隆抗体及抗人B7-H3抗体呈特异性反应.结论:成功克隆了人B7-H3基因的完全编码区序列,在毕赤酵母中表达并进一步纯化,为B7-H3在肿瘤发生、发展中的作用及机制研究奠定了基础.
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