摘要
目的 用基因工程方法表达人的热休克类似蛋白 73 ( HSC73 )并提纯 HSC73蛋白 ,为研究该蛋白提供条件。方法 用人 HSC73基因构建原核细胞表达载体 p ETHSC73 ,在大肠杆菌 BL 2 1中用异丙基硫代 -β-D半乳糖苷 ( IPTG)诱导表达 ,ATP亲和层析法提取 HSC73。经 SDS-PAGE、3 .a.3抗体 Western blot作 ECL检测。结果 人HSC73基因构建的载体 p ETHSC73能很好地在大肠杆菌表达出分子量为 73 k D的蛋白 ,蛋白提取方法能有效提纯表达的蛋白 ,用 3 .a.3抗体作 Western blot检测证实该蛋白具有人 HSC73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报道的一致。结论 人 2 .1kb的 HSC73基因片段构建的高表达载体能够很好表达人 HSC73 ,用 ATP亲和层析法可以有效提纯有活性的人
Objective To provide a human heat shock cognate protein 73 (HSC73),which could be expressed by gene recombination and purified with a purpose for its further study.Methods 2.1 kb human HSC73 gene was inserted to plasmid vector pET3c to construct a recombinant plasmid,pETHSC73,which then transformed to E. coli strain BL21.BL21 expressed human HSC73 under inducement of IPTG.The protein was extracted and purified by affinity chromatography.Western blotting was used to check the expressed protein. Results The recombinant plasmid vector pETHSC73 could express high level HSC73,a molecular weight of 73 kD protein,in E. coli strain BL21.Western blotting showed the protein possessing the antigen specificity of human HSC73 protein.The measure of amino acid ratio and N terminal sequence of the protein coincided with the relational reports.Chromatography could effectively purify the protein.Conclusion HSC73 gene recombinant plasmid vector pETHSC73 is a high expression vector.HSC73 protein with biologic activity can be obtained effectively by affinity chromatography.
基金
铁道部科技基金资助项目 !( J96 Z0 89)
