干扰素调节因子3 rs2304204野生型及突变型重组质粒的构建及活性分析

目的构建干扰素调节因子3（IRF-3）rs2304204野生型及突变型重组质粒并比较二者启动子活性。为进一步研究IRF-3对HBV感染的影响打下基础。方法以人全血DNA为模板，扩增含rs2304204位点的IRF-3启动子区1355bp目的序列，插入pGL3-Basic载体中，构建rs2304204野生型的重组质粒（wIRF-3）。以wIRF-3为模板，利用基因定点突变试剂盒构建rs2304204突变型重组质粒（mIRF-3）。wIRF．3、mIRF-3和pGL3-Basic质粒分别转染HEK293细胞，采用双荧光素酶报告基因检测试剂盒检测荧光素酶活性，并计算荧光素酶相对活性单位（RLU）。结果成功构建IRF-3rs2304204野生型及突变型重组质粒，且wIRF-3质粒转染组RLU明显高于mIRF-3质粒转染组（P〈0．005）。结论野生型重组质粒的启动子活性明显高于突变型重组质粒，启动子活性的不同可能进而影响机体抗HBV感染的临床结局。

The recombinant plasmid of human interferon regulatory factor 3 and its promoter activation

Objective To construct rs2304204 wild type and mutant type plasmids in promoter of interferon regulatory factor 3 （IRF-3） , and compare their promoter activation. To study the relationship between IRF-3 and HBV infection. Methods Amplifying promoter region of IRF3 containing rs2304204 from full sequence of human blood DNA, and wild type recombinant plasmid （wIRF-3） was constructed by inserting the amplified sequence into pGL3-Basic vector. Agarose gel electrophoresis and gene sequencing were used to identify the recombinant plasmid. The mutant type recombinant plasmid （mIRF-3） was constructed from wIRF-3 using StarMut Site-directed Mutagenesis Kit. Gene sequencing were used to identify the recom- binant plasmid. Transfection of HEK293 with wIRF-3 ,mIRF-3 and pGL3-Basic plasmids was performed to induce luciferase gene expression and calculate the relative luciferase activity unit （RLU）. Results Wild type and mutant type recombinant plasmids were constructed successfully. Compared with mlRF-3, RLU of wlRF-3 was significantly higher than that of mIRF-3. Conclusions The promoter activation of wild type was significantly higher than mutant type, and the promoter activation may influence the outcome of HBV infection.