【摘要】 目的构建人可溶性生长刺激表达基因2(sST2)蛋白的重组真核表达载体,获得高纯度的人sST2重组蛋白。方法根据人sST2基因序列设计引物,利用RT-PCR技术获得人sST2基因;构建人sST2-pcDNA3.1-his(-)重组真核表达载体;脂质体法转染COS7细胞,表达人sST2重组蛋白,并用镍柱亲和层析法纯化;用western blot和ELISA法对人sST2重组蛋白进行鉴定。结果 PCR扩增产物用琼脂糖凝胶电泳分析,结果显示产物长度与预期一致,约为1 000 bp;同源性比对分析结果显示,人sST2基因成功插入PGH-T载体;用BamHⅠ和HindⅢ对重组真核表达载体双酶切后凝胶电泳分析,结果显示产物电泳位置与预期一致;表达产物经SDS-PAGE电泳结果显示在相对分子质量(Mr)约为65 000处有一明显条带,与预期蛋白质位置一致;western blot鉴定结果显示人sST2重组蛋白有His标签,ELISA鉴定结果显示人sST2重组蛋白有与抗sST2抗体结合的特异性抗原表位。结论通过重组DNA技术成功构建人sST2基因重组真核表达载体,通过蛋白质纯化技术成功获得人sST2重组蛋白。更多还原

【Abstract】 Objective To construct a recombinant eukaryotic expression vector of human soluble growth stimulating expression gene 2(sST2) protein to obtain high-purity human sST2 recombinant protein. Methods The primers were designed based on the human sST2 gene sequence and human sST2 gene was obtained by RT-PCR technology. Human sST2-pcDNA3.1-his(-) recombinant eukaryotic expression vector was constructed. COS7 cells were transfected with liposome method to express human sST2 recombinant protein which was purified by nickel column affinity chromatography. The recombinant human sST2 protein was identified by western blot and ELISA. Results The PCR amplified products were analyzed by agarose gel electrophoresis, the results of which showed that the length of the products was consistent with the expectation, approximately 1 000 bp. The results of homology analysis showed that human sST2 was successfully inserted into the PGH-T vector. The results of gel electrophoresis analysis after double enzyme digestion of the nuclear expression vector showed that the electrophoresis position of the product was consistent with the expectation. SDS-PAGE electrophoresis results showed that there was a clear band at about(Mr) 65 000, which was consistent with the expected protein position. Western blot identification showed that the recombinant human sST2 protein had a His tag, and ELISA identification showed that the recombinant human sST2 protein had a specific antigenic epitope that bound to the anti-sST2 antibody. Conclusion Human sST2 recombinant eukaryotic expression vector was successfully constructed by recombinant DNA technology, and human sST2 recombinant protein was successfully obtained by protein purification technology.更多还原