摘要
构建靶向乙肝病毒(HBV)X基因的真核表达载体pSOS-X-siRNA和pSOS-siRNA,转染肝癌细胞系HepG2和HepG2.2.15,筛选和验证高效siRNA。设计4个靶向X基因siRNA,将siRNA和HBx基因插入载体pSOS得重组质粒pSOS-X-siRNA;pSOS-X-siRNA经PacI酶切去除目的片段HBx后得pSOS-siRNA。将质粒pSOS-X-siRNA和pSOS-siRNA分别转HepG2和HepG2.2.15肝癌细胞株。荧光显微镜下观察HepG2细胞绿色荧光(GFP)减弱程度预估干扰效率。ELISA检测HepG2.2.15细胞上清HBsAg、HBeAg表达,Western blotting检测胞内蛋白HBsAg、HBcAg表达,Real time PCR检测胞内HBsmRNA、HBx mRNA的转录。转染4d后,siRNA4使HepG2细胞的GFP信号表达程度最低,为阴性对照的9%,预测其干扰效果最强。siRNA4使HepG2.2.15细胞转染后4d上清的HBsAg蛋白表达为对照的(13.92±1.14)%(P<0.05)、HBeAg为(21.69±4.92)%(P<0.05),胞内的HBsAg、HBcAg蛋白表达量灰度比值为0.175±0.025、0.0825±0.028,均为各处理组中最低(P<0.01),HBs mRNA和HBx mRNA分别降为对照的0.237±0.028(P<0.01)、0.110±0.022(P<0.01),差异有统计学意义,证实siRNA4为高效干扰位点。利用pSOS成功筛选并验证构建靶向HBV X基因的siRNA靶点。
The study was to construct the eukaryotic expression vector pSOS-X-siRNA and pSOS-siRNA targeting for Hepatitis B virus（HBV）X gene.After transfecting pSOS-X-siRNA and pSOS-siRNA into the hepatocellular carcinoma cell lines HepG2 and HepG2.2.15,respectively,we can screen and validate the HBx short interfering RNAs.To design and synthesis the specific four siRNAs for targeting HBx gene,the recombinant pSOS-X-siRNA was obtained by inserting the HBX gene and siRNA into pSOS.The pSOS-siRNA vector was produced through digesting by Pac I and removing the HBx gene of pSOS-X-siRNA system.The plasmids pSOS-X-siRNA were transfected into HepG2 and the plasmids pSOS-siRNA were transfected into HepG2.2.15,respectively.The knockdown efficiency of siRNA can be assessed through observing the fluorescence intensity in HepG2 cells under fluorescence microscope.ELISA detected the expression of HBsAg and HBeAg in supernatant,Western blotting detected the expression of HBsAg and HBcAg in intracellular,and Real Time PCR detected the expression of the HBs mRNA and HBx mRNA in HepG2.2.15 cells.After transfection for 4 days,the fluorescence intensity of siRNA4 transfected HepG2 cells was lowest and reduced more than 90%.Similarly,the siRNA4 tranfected HepG2.2.15 cells,the expression of HBsAg and HBeAg in supernatant were decreased to 13.92 1.14% and 21.69 4.92%.The levels of HBsAg and HBcAg in intracellular still were inhibited to（0.175±0.025）-fold and（0.0825±0.028）-fold,compared with the control.Furthermore,the levels of the HBs mRNA and HBx mRNA were noticeably reduced to（0.237±0.028）-fold,（0.110±0.022）-fold compared with control.siRNA4 have the most marked gene-silencing efficiencies.The system of screening and validating siRNA targeting for HBx mRNA was constructed successfully by use of the pSOS vector system.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第11期146-152,共7页
Biotechnology Bulletin
基金
国家自然科学基金(30771921)
