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Published on: December 28, 2015
[Construction of eukaryotic expression vector of human hCGbeta and establishment of stably transfected B16 cell line]
Liang Zhang1, Yu Wang, Le Jiang
1Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. zhangliangrpg@126.com
Aim:
To construct the eukaryotic expression vector of human hCGbeta and stably transfect B16 cell line with it.
Methods:
The full length of hCGbeta cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo, added the restriction enzyme position and 6xHis tag. After identification of restriction digestion and PCR, The recombinant plasmid pIRES-neo-hCGbeta-(His)6; was obtained. Then transfected it into B16 cells by lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, the transcription and expression of the hCGbeta gene was identified by RT-PCR, Western blot and immunofluorescence assay.
Results:
The eukaryotic expression vector pIRES-neo-hCGbeta-(His)6; was successfully constructed. A stably transfected cell line was established and the expression rate of hCGbeta gene was higher than 90%.
Conclusion:
The established cell line can highly express hCGbeta stably, the expression of the target gene provide a solid experimental foundation for further studies on the function of the hCGbeta, and which will contribute to the research of hCGbeta gene in the tumor immunotherapy.
