MHC-Ⅰ类链相关基因A真核表达载体的构建及稳定转染舌鳞癌细胞的实验研究

Construction of eukaryotic expression vector of major histocompatibility complex class Ⅰ-related chain A and establishment of its stable transfected Tca8113-Tb cell line

目的构建人类MHC-Ⅰ类链相关基因A(MICA)的真核表达载体,转染人舌鳞癌脑高转移Tca8113-Tb细胞,建立稳定过表达MICA基因的口腔鳞癌细胞系。方法采用PCR技术扩增pCMV-SPORT6-MICA中编码MICA基因的cDNA序列,重组至有绿色荧光蛋白标记的真核表达载体pEGFP-N1,构建最终的表达载体pEGFP-N1-MICA,脂质体法转染Tca8113-Tb细胞,G418筛选,荧光显微镜下观察绿色荧光蛋白的表达,有限稀释法建立稳定过表达MICA基因的Tca8113-Tb细胞系,RT-PCR、real time PCR和免疫细胞化学检测MICA在该细胞中的表达。结果通过PCR技术获取了MICA基因并成功克隆入载体,测序鉴定该序列与GenBank中的序列相同。转染的细胞可见绿色荧光蛋白表达,RT-PCR、real time PCR及免疫细胞化学检测到目的基因MICA在转染细胞中为过表达。结论 pEGFP-N1-MICA真核表达载体的成功构建与稳定转染Tca8113-Tb细胞系的建立,为进一步研究该基因的功能奠定了良好的实验基础。

Objective To construct the eukaryotic expression vector,encoding major histocompatibility complex class Ⅰ-related chain A gene（MICA）,for the further research of transfecting Tca8113-Tb cell line（a metastatic cell line of brain metastasis from human tongue cancer Tca8113 cells in nude mouse）,and to establish a stable MICA overex-pression oral squamous cell line.Methods cDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR,and subcloned into eukaryotic expression vector pEGFP-N1 marked with green fluorescent protein（GFP）.The recom-binant plasmid was sequenced and transfected into Tca8113-Tb cell line by lipofectamineTM 2000.After screen culture by G418,stable tranfected Tca8113-Tb cell line was established using definite dilution method.The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR,real time PCR and immunocytochemistry.Results The MICA gene was amplified by PCR and then cloned into the vector,whose sequence was identical to that in the GenBank.The transfected cells showed the expression of GFP.And the overexpression of MICA gene in transfected cells was detected by RT-PCR,real time PCR and immunocy-tochemistry.Conclusion The recombinant eukaryotic expression vector pEGFP-N1-MICA has been constructed suc-cessfully and stably expressed in Tca8113-Tb cell line,providing a foundation for further studies on the function of MICA in vitro.