[Construction of eukaryotic expression vector of human hCGbeta and establishment of stably transfected B16 cell line].
OBJECTIVE
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%.


CONCLUSIONS
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.
Zhang
Liang
L
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. zhangliangrpg@126.com
Wang
Yu
Y
Jiang
Le
L
Yan
Jin-qi
JQ
Wang
Yue
Y
Hu
Ming-ming
MM
Yu
Ji-yun
JY
D045744:Cell Line, Tumor / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D014162:Transfection
D018997:Chorionic Gonadotropin, beta Subunit, Human