重组减毒沙门菌ΔcrpSL1344(pET28-tPA)在BHK细胞中的表达及活性检测

Expression of t-PA Gene in BHK Cells delivered by attenuated Salmonella typhimurium and detection of its activity

为了探讨原核表达质粒中的真核基因在减毒沙门菌中表达的多肽是否能在真核细胞中加工修饰成具有生物活性的蛋白,试验根据人t-PA的编码序列设计1对引物,扩增t-PA目的基因片段,构建pET28-tPA原核表达质粒,将表达质粒电转化导入减毒鼠伤寒沙门菌ΔcrpSL1344中并转染BHK细胞,应用SDS-PAGE技术检测t-PA表达情况,ELISA法检测其表达水平,琼脂糖平板溶圈法检测纤溶活性。结果表明:重组菌ΔcrpSL1344(pET28-tPA)在BHK细胞中有66.0 ku的蛋白表达,转染96 h的表达量为108μg/L,细胞裂解液和转染表达质粒的细胞培养上清液均有促纤溶活性。说明携带t-PA原核表达质粒的减毒沙门菌具有较强的促纤溶活性。

To investigate the peptides which was expressed in attenuated Salmonella typhimurium from the eukaryotic genes in prokaryotic expression plasmid, could be processed and modified into biologically active protein. A pair of primers were designed according to the coding se- quence for human, then the t - PA target gene fragments was amplified. A prokaryotic expression plasmid pET28 - tPA was constructed and transformed into attenuated Salmonella typhimurium AcrpSL1344, then was transfected in BHK cells. SDS - PAGE was used to detect the ex- pression of t - PA gene, ELISA was used to detect the expression level, and the fibrin agarose plate assay was used to detect the fibrinolytic ac- tivity. The results showed that 66 ku protein was expressed in BHK cells delivered by recombinant germ AcrpSL1344 （ pET28 - tPA） . The ex- pression level was 108μg/L on the 96 h ,transfection. Both of cell lysates and cell culture superuatant had fibrinolytic activity. It is concluded that the fibrinolytic activity of the attenuated Salmonella typhimurium carrying t - PA gene in prokaryotic plasmid is higher than that of attenuated Salmonella typhimurium.