国际病毒学杂志
國際病毒學雜誌
국제병독학잡지
INTERNATIONAL JOURNAL OF VIROLOGY
2014年
3期
100-104
,共5页
干扰素调节因子3%HBV%启动子活性
榦擾素調節因子3%HBV%啟動子活性
간우소조절인자3%HBV%계동자활성
Interferon regulatory factor 3%HBV%Promoter activation
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.
目的 構建榦擾素調節因子3(IRF-3) rs2304204野生型及突變型重組質粒併比較二者啟動子活性.為進一步研究IRF-3對HBV感染的影響打下基礎.方法 以人全血DNA為模闆,擴增含rs2304204位點的IRF-3啟動子區1355bp目的序列,插入pGL3-Basic載體中,構建rs2304204野生型的重組質粒(wIRF-3).以wIRF-3為模闆,利用基因定點突變試劑盒構建rs2304204突變型重組質粒(mIRF-3).wIRF-3、mIRF-3和pGL3-Basic質粒分彆轉染HEK293細胞,採用雙熒光素酶報告基因檢測試劑盒檢測熒光素酶活性,併計算熒光素酶相對活性單位(RLU).結果 成功構建IRF-3rs2304204野生型及突變型重組質粒,且wIRF-3質粒轉染組RLU明顯高于mIRF-3質粒轉染組(P＜0.005).結論 野生型重組質粒的啟動子活性明顯高于突變型重組質粒,啟動子活性的不同可能進而影響機體抗HBV感染的臨床結跼.
목적 구건간우소조절인자3(IRF-3) rs2304204야생형급돌변형중조질립병비교이자계동자활성.위진일보연구IRF-3대HBV감염적영향타하기출.방법 이인전혈DNA위모판,확증함rs2304204위점적IRF-3계동자구1355bp목적서렬,삽입pGL3-Basic재체중,구건rs2304204야생형적중조질립(wIRF-3).이wIRF-3위모판,이용기인정점돌변시제합구건rs2304204돌변형중조질립(mIRF-3).wIRF-3、mIRF-3화pGL3-Basic질립분별전염HEK293세포,채용쌍형광소매보고기인검측시제합검측형광소매활성,병계산형광소매상대활성단위(RLU).결과 성공구건IRF-3rs2304204야생형급돌변형중조질립,차wIRF-3질립전염조RLU명현고우mIRF-3질립전염조(P＜0.005).결론 야생형중조질립적계동자활성명현고우돌변형중조질립,계동자활성적불동가능진이영향궤체항HBV감염적림상결국.
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 recombinant 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 mIRF-3,RLU of wIRF-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.
