Application Of Antiapoptosis And Proliferation-arrested Strategies In Hybridoma Cells Large Scale Culture

The H18 hybridoma cell line producing monoclonal antibody Habl8 directed against Habl8G/Cdl47 molecule, is hepatoma associated antigen, was established in our laboratory. In order to enhance hybridoma cell production in bioreactor, we researched it from antiapoptosis and proliferation-arrested aspects, and integrate the above two factors, and culture hybridoma cell in 5L bioreactor.Part I Murine bcl-XL Clone and Expression in CHO cellsTo construct eukaryotic expression vector contenting murine bcl-XL and EGFP fusion gene and express it in CHO cells. METHODS: Murine bcl-XL was isolated by using Trizol reagent from murine brain tissue. RT-PCR was used to obtain murine bcl-XL cDNA, which was ligated into pGEMT vector and sequenced. In order to confirm cloned bcl-XL, which can be expressed fully, the recombinant expression vector pEGFP-bcl-XL was constructed by cloning bcl-XL cDNA intoeukaryotic expression vector pEGFP. After transfection into CHO cells through lipofectamine 2000? the expression of fusion gene was detected by fluoroscope. RESULTS: The obtained murine bcl-XL cDNA was consistent with that in GenBank. The eukaryotic expression vector pEGFP-bcl-XL was successfully constructed and expressed in CHO cells. CONCLUSION: The cloning and expression of murine bcl-XL gene are of significance in further use of bcl-XL gene in high density large scale cell culture.Part II Modification and identification of the antiapoptotic ability of H18 hybridoma cellsTo construct eukaryotic expression vector containing murine bcl-XL and stably express it in H18 hybridoma cells in order to enhance hybridoma cells antiapoptotic ability. PCR was used to obtain 710bp murine bcl-XL cDNA from pGEM-T-bcl-XL. Then the recombinant expression vector pEF-bcl-XL was constructed by cloning bcl-XL cDNA into eukaryotic expression vector pEF by PstI and Xhol double digestion. After transfection into H18 hybridoma cells through lipofectamine 2000? the stable expression cell line was screened by 800mg/L G418. The expression of bcl-XL gene was detected by western blotting. Flow cytometer was used to test the modified hybridoma cells ability to resist apoptosis induced by 0.4mmol/L Sodium Butyrate. The eukaryotic expression vector pEF-bcl-XL was successfully constructed and stably expressed in H18 hybridoma cells. Our data showed that stably transfected H18 cells expressed highlevels of Bcl-Xl Under the condition of 0.4mmol/L NaBu, the production of antibody was to be significantly increased by more than 3-fold in stably transfected H18, which resulted from suppressing the NaBu-induced apoptosis and allowing stably transfected H18 cells to grow at higher viability and extend culture longevity by > 3 days. The increased culture longevity by inhibition of NaBu-induced apoptosis by inducible expression of BcI-Xl combined with the enhanced secretion of antibody by NaBu contributed to the enhancement of final antibody concentration in the stably transfected H18 cells culture. The final antibody concentration of stably transfected H18 cells in the presence of NaBu was threefold higher than that of H18 cells culture in the absence of NaBu.' Together, our results showed that butyrate is of practical interest for production of antibody. NaBu-induced apoptosis of hybridoma cells could be inhibited by inducible expression of BcI-Xl. The expression of murine bcl-XL gene in hyridoma cells and the increasing antiapoptosis ability of hybridoma cells are of significance in further use of hybridoma cells in high density large scale cell culture.Part III NaBu Arrested Hybridoma Cells Proliferation Resulting in Enhanced Antibody ProductionNaBu, a well-known differentiation inducer in several myeloid cells, induces G1 phase arrest in many cell lines. In this study, we investigated the possibility of using NaBu to control H18.D4 hybridoma cells in the G1 phase of the cell cycle and analyzed whetherimprovement antibody production follow the Gl arrest using growth curve, ELISA and cell cycle assays. We showed that NaBu efficiently arrests H18.D4...