人热休克蛋白73的基因重组、表达和纯化

Gene Recombination, Expression and Purification of Human Heat Shock Protein 73

目的 用基因重组的方法表达人热休克蛋白７３，并纯化表达产物，用于进一步分析。方法 用人 热休克类似蛋白ＨＳＰ７３基因构建原核细胞表达载体ｐＥＴＨＳＰ７３，在大肠杆菌ＢＬ２１中用半乳糖苷（ＩＰＴＧ）诱导表达，用 电泳法和 ＡＴＰ亲和层析法提取 ＨＳＰ７３。经ＳＤＳ－ＰＡＧＥ、用３ ａ３抗体 Ｗｅｓｔｅｒｎ ｂｌｏｔ作 ＥＣＬ检测。结果人 ＨＳＰ７３基因 构建的载体ｐＥＴＨＳＰ７３能很好地在大肠杆菌表达出相对分子质量为７３ ０００的蛋白。两种蛋白提取方法均能有效提纯表达的蛋白，该蛋白具有人ＨＳＰ７３抗原特性。所得蛋白质氨基酸测定和Ｎ端测序的结果与有关的报道一致。结 论 ＨＳＰ７３基因重组、表达和纯化方法的建立，为研究ＨＳＰ７３的结构、功能与临床应用提供了必要条件。

Objective To expresse human heat shock protein 73(HSP73) by gene recombination and purify the expressed product for further analysis. Methods A recombinant plasmid, pETHSP73, was constructed using HSP73 gene and transformed to E. coli strain BL21,then HSP73 was expressed under the inducement of IPTG, purified by electrophoresis and affinity chromatography, and detected by SDS-PAGE, Western-blot with 3 a 3 McAb and ECL. Results The HSP73 with a molecular weight of 73 000 was highly expressed in pETHSP73,and both electrophoresis and affinity chromatography could be used for the effective purification of expressed HSP73. Western blot proved that the expressed product has the antigenicity of human HSP73. The results of amino acid detection and T-terminal sequencing were consistent with relevant reports. Conclusions A series of methods for the gene recombination,expression and purification of HSP73 were established. The study will provide necessary conidtions for the study on the structure,function and clinical application of HSP73.