摘要
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、Western blot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一（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plRES—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。
AIM： To construct the eukaryotic expression vector of human hCGβ and stably transfect B16 cell line with it. METHODS： The full length of hCGβ cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector plRES-neo, added the restriction enzyme position and 6 x His tag. After identification of restriction digestion and PCR, The recombinant plasmid plRES-neo-hCGβ- （His）6 was obtained. Then transfected it into BI6 cells by lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, the transcription and expression of the hCGβ gene was identified by RT-PCR, Western blot and immunofluorescence assay. RESULTS. The eukaryotic expression vector plRES-neo-hCGβ-（ His）6 was successfully constructed. A stably transfected cell line was established and the expression rate of hCGβ gene was higher than 90%. CONCLUSION： The established cell line can highly express hCGβ stably, the expression of the target gene provide a solid experimental foundation for further studies on the function of the hCGβ, and which will contribute to the research of hCGβ gene in the tumor immunotherapy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第7期623-626,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助项目(2006AA02A237
2007AA02Z451)
国家自然科学基金资助项目(30772002)
