稳定高效表达人血栓调节蛋白的CHO细胞株的建立

目的:构建稳定高效表达人血栓调节蛋白(human thrombomodulin,hTM)的CHO细胞株,以便于大量生产纯化hTM蛋白。方法:利用脂质体li-pofectamine 2000将包含hTM基因全长序列的重组表达质粒pThr402转染CHO细胞。转染后用G418的选择性培养液筛选、挑取抗性克隆。采用流式细胞术和Western blotting检测hTM在CHO细胞膜表面的表达情况,筛选建立稳定高效表达hTM的CHO细胞株并对其稳定性进行观察。结果:重组表达质粒pThr402转染CHO细胞后,流式细胞术证实hTM稳定高效表达于随机挑选的5个抗性克隆细胞株的细胞膜表面,但表达量有差异。Western blotting分析检测显示hTM表达量较高的CHO-TM1、CHO-TM4与CHO-TM5细胞株的细胞裂解液出现了与预期值相符的约105000的特异性条带。CHO-TM5已经经过冻存复苏前后各20次传代,且无论有无G418选择压力存在,hTM表达水平无明显差异。结论:成功构建了稳定高效表达hTM的CHO细胞株,可望获得大量蛋白,为进一步研究hTM的生物学功能以及制备和筛选抗hTM的单抗开辟新的途径。

Establishment of stable CHO cell line expressing high-level human thrombomodulin

AIM: To establish a cell line in Chinese hamster ovary cells(CHO) that is able to express human thrombomodulin(hTM) stably at high level for large-scale production of the recombinant hTM protein.METHODS: The full-length hTM cDNA expressing plasmid pThr402 was transfected into CHO cells by lipofectamine 2000 reagent.The drug resistant cell clones,which were confirmed to have stable high level expression of hTM on membrane by flow cytometry and western blotting assays,were obtained under the pressure of G418 selection.Moreover,the stability of cell line with the hTM highest expression in the presence or absence of G418 of cryopreservation was screened by flow cytometry.RESULTS: The recombinant plasmid pThr402 was transfected into CHO cells with the help of lipofectamine 2000 reagent by G418 selection successfully,and 5 drug resistant cell clones were selected randomly to culture for further investigation.Flow cytometry demonstrated that all of the 5 drug resistant cell clones randomly selected were positive cell lines expressing hTM on membrane,but there was difference between individual cell lines of the hTM protein production.Meanwhile,Western blotting assays indicated the protein molecular weight expressed on CHO-TM1,CHO-TM4 and CHO-TM5 cell membrane under reduction condition to be approximately 105000,which was accord with theoretical value of hTM protein molecular weight.On the other hand,the CHO-TM5 cell line in the presence or absence of G418 were cultured for twenty generations before or after cryopreservation,the mean fluorescence intensity of hTM protein was examined by flow cytometry.It was found that not only the duration but also the absence of G418 could not decrease hTM protein production.CONCLUSION: The stable CHO cell lines expressing high-lever human thrombomodulin on membrane were obtained successfully and will provide a base for further clinic diagnose and therapy application.