pIRES2-EGFP-SyK(L)载体构建及稳定转染喉癌Hep-2细胞株的建立  

Construction of eukaryotic expression vector pIRES2-EGFP-SyK(L) and establishment of stably-transfected laryngocarcinoma Hep-2 cell line

作　　者：李志海[1] 蔡志毅[1] 陶宝鸿[1] 金巧智[1] 

机构地区：[1]台州市立医院耳鼻咽喉科,浙江台州318000

出　　处：《中国卫生检验杂志》2013年第13期2718-2721,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的构建Syk(L)全长结构基因真核表达载体,建立稳定高表达Syk(L)的喉癌hep-2细胞系。方法化学合成结合PCR拼接SyK(L)基因,插入pMD18-simpleT载体中,测序验证正确后经XhoI和Hind III双酶切,经T4 DNA连接酶,将其与同样双酶切处理pIRES2-EGFP载体连接,构成新的重组质粒pIRES2-EGFP-SyK(L),测序验证正确后,以脂质体法转染喉癌Hep-2细胞,通过G418筛选,建立稳定转染的Hep-2细胞株,用实时荧光定量PCR检测SyK(L)的mRNA表达。结果成功构建了pIRES2-EGFP-SyK(L)真核表达载体;建立了稳定转染pIRES2-EGFPSyK(L)的Hep-2细胞株;实时荧光定量PCR结果表明pIRES2-EGFP-SyK(L)转染Hep-2细胞株中SyK(L)mRNA表达水平明显高于空载体pIRES2-EGFP转染细胞株及空转染细胞株,差别有统计学意义(P<0.01)。结论 pIRES2-EGFP-SyK(L)真核表达载体的构建及稳定高表达SyK(L)喉癌Hep-2细胞株的建立,为进一步研究SyK(L)对喉癌细胞生物功能的影响奠定了良好的实验基础。Objective To construct eukaryotic expression vector containing Syk（L） and establish stably transfected Hep-2 cell line with high expression of SyK（L）.Methods The synthesized DNA encoding SyK（L） was inserted into vector pMD18-simpleT after being identified by nucleotide sequencing.After being digested by XhoI and HindIII,the purified SyK（L） gene fragment was subcloned into the eukaryotic expression vector pIRES2-EGFP.After analysis by restrictive digestion reaction,the recombinant plasmid was transfected into Hep-2 cells with lipofectamine method and G418 screening,and the expression of SyK（L） mRNA in resistant clones was detected directly by real-time quantita-tive PCR with Taqmn technique.Results Recombinant plasmid pIRES2-EGFP-SyK（L） was successfully constructed as indicated by restriction endonuclease analysis.The stably transfected Hep-2 cell line highly expressing Syk（L） was successfully constructed.The expression of Syk（L） mRNA was significantly higher in stable transfectants with the Syk（L） vector than those with empty vector or untransfected cells（P 0.01）.Conclusion The construction of the eukaryotic expression vector pIRES2-EGFP-SyK（L） and the establishment of stable transfected hep-2 cell line have provided solid experiment foundation for further studies on the function Syk（L）.

关 键 词：脾酪氨酸激酶 真核表达载体 HEP-2细胞 基因表达 

分 类 号：R739.65[医药卫生—肿瘤]