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.