本文選題：VP1基因 + RNA干擾　； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:構(gòu)建和篩選靶向EV71病毒VP1基因的高效siRNA表達(dá)載體;將篩選出的高效siRNA重組到減毒沙門氏菌中,構(gòu)建以減毒沙門氏菌為載體、靶向EV71病毒VP1基因的口服基因重組治療性疫苗。為運用RNAi技術(shù)治療EV71引起的手足口病提供理論基礎(chǔ)和實驗依據(jù)。 方法:采用DNA重組技術(shù)將EV71病毒的VP1基因序列和靶向EV71病毒的VP1基因的siRNA克隆到含U6啟動子、H1啟動子和增強型綠色熒光蛋白(EGFP)的質(zhì)粒pSEB-HUS載體中,構(gòu)建重組質(zhì)粒pSV,經(jīng)限制性內(nèi)切酶消化、PCR擴增及DNA序列測定等方法鑒定后,再將靶向EV71病毒的VP1基因的siRNA克隆到重組質(zhì)粒pSV中,構(gòu)建成重組質(zhì)粒pSVsi1、pSVsi2、pSVsi3和pSVsi4,經(jīng)DNA序列測定鑒定證實插入的siRNA序列正確后,轉(zhuǎn)染A549細(xì)胞,用倒置熒光顯微鏡初步觀察綠色熒光蛋白的表達(dá)情況;用熒光實時定量RT-PCR檢測VP1基因mRNA轉(zhuǎn)錄水平,免疫細(xì)胞化學(xué)檢測細(xì)胞中的VP1基因的表達(dá)水平,進而分析siRNA對VP1 mRMA轉(zhuǎn)錄和蛋白表達(dá)的抑制作用。篩選出抑制效率最高的siRNA,構(gòu)建重組質(zhì)粒pSsi1,并將其轉(zhuǎn)化進減毒沙門氏菌中,進而探索以RNAi技術(shù)為基礎(chǔ)、減毒沙門氏菌為載體的口服治療性疫苗的可行性。 結(jié)果:經(jīng)限制性內(nèi)切酶、PCR、測序及Western Blot證實:成功構(gòu)建可以表達(dá)EGFP的、靶向EV71病毒VP1基因的siRNA表達(dá)載體;從已構(gòu)建的3個靶向VP1基因的siRNA表達(dá)載體中,篩選到可高效抑制VP1基因表達(dá)的pSVsi1,其VP1 mRNA和VP1蛋白的抑制率分別為87%和89%;成功構(gòu)建si1的重組質(zhì)粒pSsi1,并將其成功的轉(zhuǎn)化進減毒沙門氏菌中。 結(jié)論:1.成功構(gòu)建以pSEB-HUS為載體的重組siRNA表達(dá)質(zhì)粒,為今后運用該質(zhì)粒進行RNAi相關(guān)研究奠定了物質(zhì)基礎(chǔ);2.篩選到對EV71病毒VP1基因的表達(dá)有一定抑制作用的siRNA,并將其重組質(zhì)粒pSsi1成功轉(zhuǎn)化進減毒沙門氏菌中,不僅為研究RNAi治療由EV71引起的手足口病提供了物質(zhì)基礎(chǔ),而且表明用RNA干擾技術(shù)抑制細(xì)胞內(nèi)EV71病毒VP1基因的表達(dá)、進而治療由EV71引起的手足口病是可行的。3.本研究設(shè)計的三個靶向EV71病毒VP1基因的序列,均對VP1的表達(dá)有大于50%的抑制率,說明RNA干擾對所靶向的mRNA的序列和部位依賴性不強,因此進一步預(yù)示了此項技術(shù)的可行性。
[Abstract]:Objective: to construct and screen the highly efficient siRNA expression vector targeting VP1 gene of EV71 virus, and to construct the vector of attenuated Salmonella by recombination of the siRNA into attenuated Salmonella. Oral Recombinant Therapeutic Vaccine targeting VP1 Gene of EV 71 virus. To provide theoretical and experimental basis for the treatment of hand, foot and mouth disease caused by EV71 by RNAi technique. Methods: VP1 gene sequence of EV71 virus and siRNA of VP1 gene targeting EV71 virus were cloned into plasmid pSEB-HUS containing U6 promoter H1 promoter and enhanced green fluorescent protein (EGFP) by DNA recombination technique. The recombinant plasmid pSVwas constructed. After PCR amplification with restriction endonuclease digestion and DNA sequencing, the siRNA targeting VP1 gene of EV71 virus was cloned into the recombinant plasmid PSV. The recombinant plasmids pSVsi1, pSVsi2, pSVsi3 and pSVsi4 were constructed. After DNA sequencing, the inserted siRNA sequence was confirmed to be correct, then transfected into A549 cells, and the expression of green fluorescent protein was preliminarily observed by inverted fluorescence microscope. The mRNA transcription level of VP1 gene and the expression level of VP1 gene were detected by fluorescence real-time quantitative RT-PCR and immunocytochemistry, and the inhibitory effect of siRNA on VP1 mRMA transcription and protein expression was analyzed. The siRNAs with the highest inhibition efficiency were selected and the recombinant plasmid pSsi1 was constructed and transformed into attenuated Salmonella. The feasibility of oral therapeutic vaccine with attenuated Salmonella based on RNAi technology was explored. Results: the siRNA expression vector targeting VP1 gene of EV71 virus was successfully constructed by restriction endonuclease PCR, sequencing and Western blot analysis, and the siRNA expression vector targeting VP1 gene of EV71 virus was constructed from three siRNA expression vectors targeting VP1 gene of EV71 virus. The inhibition rates of VP1 mRNA and VP1 protein were 87% and 89%, respectively. The recombinant plasmid pSsi1 of si1 was successfully constructed and transformed into attenuated Salmonella. Conclusion 1. The recombinant siRNA expression plasmid using pSEB-HUS as vector was successfully constructed, which laid a material foundation for RNAi related research in the future. SiRNAs which could inhibit the expression of VP1 gene of EV71 virus were screened, and the recombinant plasmid pSsi1 was successfully transformed into attenuated Salmonella, which not only provided a material basis for the study of RNAi treatment of HFMD caused by EV71. It is suggested that it is feasible to inhibit the expression of VP1 gene of EV71 virus by RNA interference, and then to treat HFMD caused by EV71. The sequence of VP1 gene of EV71 virus was designed in this study, which showed that RNA interference had not strong dependence on the sequence and site of targeted mRNA, so the feasibility of this technique was predicted further. 3. The sequence of VP1 gene targeting EV71 virus was inhibited by more than 50% inhibition rate of VP1 gene expression, which indicated that RNA interference was not strongly dependent on the sequence and site of targeted mRNA.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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