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目的:构建人hCGβ真核表达载体,稳定转染入小鼠黑色素瘤B16细胞,建立稳定表达人hCGβ的小鼠黑色素瘤细胞系。方法:采用PCR方法扩增出人hCGβ全长基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×His标签,得到重组表达质粒pIRES-neo-hCGβ-(His)6。利用阳离子脂质体介导法将其稳定转染入小鼠黑色素瘤B16细胞,经G418加压筛选出阳性克隆。RT-PCR、Westernblot及免疫荧光检测人hCGβ在B16细胞中的表达。结果:经限制性内切酶鉴定及序列分析,pIRES-neo-hCGβ-(His)6重组体构建正确,最终建立的表达人hCGβ基因的B16细胞系阳性率高于90%。结论:成功构建了真核表达载体pIRES-neo-hCGβ-(His)6,建立的稳定转染人hCGβ小鼠黑色素瘤细胞系能够高效表达hCGβ基因。该稳定转染细胞系的建立为进一步研究人hCGβ抗肿瘤基因疫苗的功能提供了良好的实验基础,为hCGβ在肿瘤免疫治疗中的应用奠定了研究基础。
OBJECTIVE: To construct a human hCGβ eukaryotic expression vector and stably transfected into mouse melanoma B16 cells to establish mouse melanoma cell line stably expressing human hCGβ. Methods: The cDNA coding region of human hCGβ gene was amplified by PCR and inserted into the eukaryotic expression vector pIRES-neo by DNA recombination. The restriction enzyme site and 6 × His tag were added to obtain Recombinant expression plasmid pIRES-neo-hCGβ- (His) 6. The positive cells were stably transfected into mouse melanoma B16 cells by cationic liposome-mediated method and the positive clones were screened by G418 pressure. The expression of human hCGβ in B16 cells was detected by RT-PCR, Western blot and immunofluorescence. Results: The recombinant plasmid pIRES-neo-hCGβ- (His) 6 was constructed correctly and finally the positive rate of B16 cell line expressing human hCGβ gene was higher than 90% by restriction enzyme analysis and sequence analysis. CONCLUSION: The eukaryotic expression vector pIRES-neo-hCGβ- (His) 6 was successfully constructed. The established hCGβ mouse melanoma cell line stably transfected with hCGβ could efficiently express hCGβ gene. The establishment of the stable transfected cell line provides a good experimental basis for further study on the function of human hCGβ anti-tumor gene vaccine and lays a foundation for the application of hCGβ in tumor immunotherapy.