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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 of full-length Syk (L) gene and establish hep-2 laryngeal cell line stably expressing Syk (L). Methods The gene of SyK (L) was inserted into pMD18-simpleT vector by chemical synthesis and PCR. After verified by sequencing, it was digested with XhoI and Hind III and ligated with T4 DNA ligase to construct pIRES2-EGFP vector The recombinant plasmid pIRES2-EGFP-SyK (L) was constructed and transfected into laryngeal carcinoma Hep-2 cells by lipofectamine 2000. The stable transfected Hep-2 cells were established by G418 screening, The mRNA expression of SyK (L) was detected by real-time fluorescence quantitative PCR. Results The eukaryotic expression vector pIRES2-EGFP-SyK (L) was constructed successfully. Hep-2 cell line stably transfected with pIRES2-EGFPSyK (L) was established. Real-time PCR results showed that pIRES2- The expression level of SyK (L) mRNA in Hep-2 cell line was significantly higher than that in empty vector pIRES2-EGFP transfected cell line and empty transfected cell line, the difference was statistically significant (P <0.01). Conclusion The construction of pIRES2-EGFP-SyK (L) eukaryotic expression vector and the establishment of a stable and highly expressed SyK (L) Hep-2 cell line laryngeal cancer lay the foundation for further study of the effect of SyK (L) on the biological function of laryngeal carcinoma cells Good experimental foundation.