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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 express human heat shock protein 73 by gene recombination and to purify the expressed product for further analysis. Methods The prokaryotic expression vector pETHSP73 was constructed by human heat shock protein HSP73. The recombinant plasmid pETHSP73 was induced by IPTG in E. coli BL21. HSP73 was extracted by electrophoresis and ATP affinity chromatography. After SDS-PAGE, Western blot with 3 a3 antibody for ECL detection. Results The vector pETHSP73 constructed by human HSP73 gene expressed a good relative molecular mass of 73 000 in E. coli. Both protein extraction methods can effectively purify the expressed protein, which has the human HSP73 antigenic characteristic. The results of the amino acid determination and N-terminal sequencing of the obtained proteins are consistent with the related reports. Conclusion The establishment of HSP73 gene recombination, expression and purification methods provides the necessary conditions for studying the structure, function and clinical application of HSP73.