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目的构建人Tec酪氨酸蛋白激酶真核表达载体;研究Tec参与肝细胞再生的可能信号途径。方法经PCR扩增、酶切、连接等基因重组技术将人Tec插入pCDNA3.1(hismycTec)真核表达载体中,并经酶切、PCR、测序鉴定;用脂质体法将构建的真核表达载体瞬时转染Hela细胞,用Western方法检测Tec蛋白是否表达。用荧光素酶报告基因系统,检测Tec是否参与HGF刺激信号途径的活化。结果构建的Tec真核表达载体使Hela细胞高表达Tec蛋白;并发现Tec在HGF介导下,对Elk信号分子活化的报告质粒中荧光素酶的表达有明显增强作用。结论构建了Tec真核表达载体,并能使HGF介导的MAPK途径活化,提示Tec可能参与HGF介导肝细胞增殖的信号调控。
Objective To construct eukaryotic expression vector of human Tec tyrosine protein kinase and to study the possible signaling pathways by which Tec participates in hepatocyte regeneration. Methods Human Tec was inserted into pCDNA3.1 (hismycTec) eukaryotic expression vector by PCR amplification, restriction enzyme digestion and ligation, and identified by restriction enzyme digestion, PCR and sequencing. The constructed eukaryotic The Hela cells were transiently transfected with the expression vector and the expression of Tec protein was detected by Western blot. Using the luciferase reporter gene system, it is tested whether Tec is involved in the activation of the HGF stimulation signaling pathway. Results The constructed Tec eukaryotic expression vector enhanced the expression of Tec protein in Hela cells. The results showed that Tec enhanced the expression of luciferase in the reporter plasmid activated by Elk signaling through HGF. Conclusion The eukaryotic expression vector of Tec was constructed and the HGF-mediated MAPK pathway was activated. It suggests that Tec might be involved in the signal regulation of HGF-mediated hepatocyte proliferation.