【摘要】：目的構(gòu)建共沉默Birc5和Hspa5的雙干擾siRNA質(zhì)粒,為進(jìn)一步以Birc5和Hspa5作為腫瘤生物治療靶點(diǎn)研究提供基礎(chǔ)。方法使用Ambion Target Finder設(shè)計Birc5和Hspa5mRNA的干擾序列;挑取酶切鑒定正確的質(zhì)粒轉(zhuǎn)化菌液Birc5-siRNA和Hspa5-siRNA測序鑒定;采用分子克隆技術(shù)構(gòu)建特異性沉默Birc5和Hspa5的雙干擾siRNA質(zhì)粒,命名為pgsiRNA-Birc5+Hspa5,酶切鑒定;將pgsiRNA-Birc5+Hspa5轉(zhuǎn)染HepG2細(xì)胞48h后采用Real-time PCR檢測Birc5和Hspa5的mRNA表達(dá)水平。結(jié)果經(jīng)酶切鑒定正確的質(zhì)粒轉(zhuǎn)化菌液Birc5-siRNA和Hspa5-siRNA測序結(jié)果顯示均為插入正確的克隆質(zhì)粒;經(jīng)酶切鑒定分析顯示pgsiRNA-Birc5+Hspa5符合酶切鑒定結(jié)果;轉(zhuǎn)染pgsiRNA-Birc5+Hspa5 48h后HepG2細(xì)胞Birc5和Hspa5mRNA表達(dá)均顯著下降,未轉(zhuǎn)染組Birc5和Hspa5mRNA的相對表達(dá)量均為1.0,pgsiRNA-Birc5+Hspa5組Birc5為0.15(P0.05),Hspa5為0.37(P0.05),表明雙干擾載體構(gòu)建成功。結(jié)論成功構(gòu)建了Birc5和Hspa5雙干擾siRNA質(zhì)粒,為進(jìn)一步同時靶向沉默腫瘤Birc5和Hspa5基因研究提供了工具和基礎(chǔ)。
[Abstract]:Objective To construct the co-silenced bi-interfering siRNA plasmids of Birc5 and Hspa5, and to provide the basis for further research on bi-c5 and Hspa5 as targets for tumor biotherapy. Subcloning technique was used to construct the bi-interfering siRNA plasmids with specific silencing of Birc5 and Hspa5, named pgsiRNA-Birc5+Hspa5, which were identified by enzyme digestion. The expression of Birc5 and Hspa5 in HepG2 cells was detected by Real-time PCR 48 hours after pgsiRNA-Birc5+Hspa5 was transfected into HepG2 cells. The results showed that all the cloned plasmids were inserted correctly. The results of enzyme digestion analysis showed that pgsiRNA-Birc5+Hspa5 accorded with the results of enzyme digestion identification. The expression of Birc5 and Hspa5mRNA in HepG2 cells decreased significantly after transfection of pgsiRNA-Birc5+Hspa5 for 48h. The relative expression of Birc5 and Hspa5mRNA in non-transfected group was 1.0, that in pgsiRNA-Birc5+H5 group was 0.15 (P 0.05), and that in Hspa5+H5 group was 0.15 (P 0.05). Spa 5 was 0.37 (P 0.05), indicating that the construction of the double interference vector was successful. Conclusion The double interference siRNA plasmids of Birc 5 and Hspa 5 were successfully constructed, which provided a tool and basis for further study of simultaneous targeted silencing of tumor genes Birc 5 and Hspa 5.
【作者單位】： 武漢科技大學(xué)醫(yī)學(xué)院病原生物與免疫學(xué)系;杭州醫(yī)學(xué)院病原生物與免疫學(xué)教研室;華中科技大學(xué)同濟(jì)醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院免疫學(xué)系;
【基金】：浙江醫(yī)學(xué)高等專科學(xué)校人才引進(jìn)項目(No.2015B08) 湖北省教育廳科學(xué)研究計劃重點(diǎn)項目(No.D20131103)
【分類號】：Q78;R73
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本文編號：2196039