人性激素结合球蛋白的真核表达、纯化鉴定及质控品应用分析

Eukaryotic expression,purification and identification of human sex hormone binding globulin and application analysis of quality control products

目的构建人性激素结合球蛋白(SHBG)的重组真核表达载体,获得高纯度的人SHBG重组蛋白。方法根据人SHBG基因序列,利用Primer Premier 6.0设计引物,扩增获得人SHBG基因;构建人SHBG-pCMV3H重组真核表达载体;将其转染至HEK-293F细胞,提取人SHBG重组蛋白,并用ConA柱亲和层析法纯化;采用人SHBG检测试剂盒对其蛋白浓度进行鉴定。结果人SHBG基因产物长度与预期一致,约为1200 bp;测序比对分析结果显示人SHBG基因成功插入pCMV3H载体;电泳结果显示在相对分子质量约为42×10^(3)处有一明显条带,与预期蛋白质位置一致;人SHBG检测试剂盒鉴定结果显示人SHBG重组蛋白作为质控品与西门子、安图生物、罗氏一致性良好;含钙离子缓冲液中人SHBG重组蛋白稳定性优于不含钙离子缓冲液。结论真核表达人SHBG重组蛋白能够作为SHBG检测试剂的质控品,且各厂家检测一致性良好;钙离子有利于提升人SHBG蛋白的稳定性。

Objective To construct a recombinant eukaryotic expression vector of human sex hormone binding globulin(SHBG)and obtain high-purity recombinant human SHBG protein.Methods According to the sequence of human SHBG gene,Primer Premier 6.0 was used to design primers and amplify human SHBG gene.The recombinant eukaryotic expression vector of human SHBG-pCMV3H was constructed,the recombinant human SHBG protein was extracted and purified by ConA column affinity chromatography.The protein concentration was identified by human SHBG detection kit.Results The length of human SHBG gene product was consistent with the expectation,about 1200 bp.Sequencing analysis showed that human SHBG gene was successfully inserted into pCMV3H vector.Electrophoresis showed that there was a clear band at the relative molecular weight of about 42×10^(3),which was consistent with the expected protein position.The identification results of the human SHBG detection kit showed that the recombinant human SHBG protein as a quality control product was in good agreement with Siemens,Antu Biotech,and Roche.The stability of SHBG recombinant protein in buffer containing calcium ion was better than that in buffer without calcium ion.Conclusion The eukaryotic expression of human SHBG protein can be applied to the quality control products of SHBG detection reagents,and the detection consistency of each manufacturer is good.Calcium ion help to improve the stability of human SHBG protein.