摘要
利用PCR从pGEM T bcl XL 质粒中获得bcl XL 基因 ,构建真核表达载体pEF bcl XL,脂质体法转染杂交瘤细胞 ,G4 18筛选稳定表达株 ,Westernblotting检测目的蛋白表达 ,流式细胞仪检测Bcl XL 提高杂交瘤抗正丁酸钠诱导凋亡的功能。将构建的编码鼠bcl XL 基因的真核表达载体pEF bcl XL,转染H18细胞后 ,获得稳定的表达株细胞 ;稳定表达Bcl XL 的细胞具有抗正丁酸钠诱导凋亡的功能。鼠bcl XL 基因在杂交瘤细胞中稳定表达 ,提高了杂交瘤抗凋亡的能力 ,对高密度大规模培养杂交瘤细胞具有重要意义。
To construct eukaryotic expression vector containing murine bcl-X L and stably express it in H18 hybridoma cells in order to enhance hybridoma cells antiapoptotic ability. PCR was used to obtain 710bp murine bcl-X L cDNA from pGEM-T-bcl-X L. Then the recombinant expression vector pEF-bcl-X L was constructed by cloning bcl-X L cDNA into eukaryotic expression vector pEF by Pst Ⅰ and XhoⅠ double digestion. After transfection into H18 hybridoma cells through lipofectamine 2000 TM, the stable expression cell line was screened by 800mg/L G418 The expression of bcl-X L 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-X L was successfully constructed and stably expressed in H18 hybridoma cells. Our data showed that stably transfected H18 cells expressed high levels of Bcl-X L. 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 Bcl-X L 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 three-fold 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 Bcl-X L. The expression of murine bcl-X L gene in hyridoma cells and the increasing antiapoptosis ability of hybridoma cells are of significance in furt
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第6期705-708,共4页
Chinese Journal of Biotechnology
基金
国家 8 63计划生物工程主题项目 (No .2 0 0 2AA2 170 11)
国家重大科技专项 (No.2 0 0 2AA2Z3 441)~~
