Modification of the antiapoptotic ability of H18 hybridoma cells.
- Author:
Xian-Hui WANG
1
;
Jing XU
;
Yang ZHANG
;
Li MI
;
Zhi-Nan CHEN
Author Information
1. Cell Engineering Research Centre, The Fourth Military Medical University, Xi'an 710033, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies;
immunology;
metabolism;
Apoptosis;
drug effects;
genetics;
Blotting, Western;
Butyrates;
pharmacology;
Cell Line, Tumor;
Enzyme-Linked Immunosorbent Assay;
Flow Cytometry;
Humans;
Hybridomas;
immunology;
metabolism;
pathology;
Mice;
Proto-Oncogene Proteins c-bcl-2;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2003;19(6):705-708
- CountryChina
- Language:Chinese
-
Abstract:
To 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 Pst I and Xho I 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 high levels 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 Bcl-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 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-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.
