摘要
目的：将获得纯化的重组人睾丸前列腺素D合成酶(L-PGDS)用于基础和临床研究. 方法：在原核表达系统中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组质粒pGEX-2T/htL-PGDS的表达,用谷胱甘肽琼脂糖珠亲和并纯化重组融合蛋白GST/htL-PGDS,再用凝血酶裂解融合蛋白,从而获得纯化的重组L-PGDS.对表达纯化重组蛋白的质粒进行DNA测序,并对重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,鉴定其是否为所需目的蛋白. 结果：用IPTG诱导重组质粒pGEX-2T/htL-PGDS表达的最佳浓度为1 mmol/L,最佳时间为4 h.在此基础上获得的融合蛋白用凝血酶裂解后获得单一纯化的重组蛋白L-PGDS,最佳裂解时间为2 h.SDS-PAGE和DNA测序证实,所获的重组蛋白确系所需目的蛋白. 结论：用上述方法可获得纯化的人睾丸L-PGDS.
Objectives:To acquire purified recombinant human testis Lipocalin-type prostaglandin D synthetase (htL-PGDS) for basic and clinical s tudies. Methods:Recombinant pGEX-2T/htL-PGDS expressing fusio n GST/htL-PGDS protein was induced with isopropyl-B-D-l thiogalactoside(IPTG ), and the fusion GST/htL-PGDS protein in the supernatant were purified with g lutathione sepharose 4B in prokaryotic expression system. The resultant was enzy molysed by thrombin. After centrifugation, the supernatant were collected as pur ified recombinant L-PGDS. It was identified by sodium dodecyl sulfate polyacry lamide gel electrophoresis (SDS-PAGE) and DNA sequenced for recombinant pGEX -2T/htL-PGDS expressing the purified recombinant L-PGDS. Results :The optimal concentration of IPTG, which can induce the recombina nt pGEX -2T/ht L-PGDS to express fusion GST/htL-PGDS protein , is 1mmol/L, and the o ptimal induction period is 4h. Purified recombinant L-PGDS are acquired after fusion GST/htL-PGDS protein are enzymolysed by thrombin, and the optimal enzym olysis period is 2 h. It is the protein that we hope to acquire after identifyin g with SDS-PAGE and DNA sequencing. Conclusions:Purified recombi nant L-PGDS protein can be acquired successfully with above mentioned method.
出处
《医学研究生学报》
CAS
2001年第5期397-400,共4页
Journal of Medical Postgraduates
