靶向EV71 VP1基因siRNA真核表达质粒的构建及筛选研究

发布时间：2018-07-10 02:25

  本文选题：VP1基因 + RNA干扰　； 参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的:构建和筛选靶向EV71病毒VP1基因的高效siRNA表达载体;将筛选出的高效siRNA重组到减毒沙门氏菌中,构建以减毒沙门氏菌为载体、靶向EV71病毒VP1基因的口服基因重组治疗性疫苗。为运用RNAi技术治疗EV71引起的手足口病提供理论基础和实验依据。 方法:采用DNA重组技术将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启动子、H1启动子和增强型绿色荧光蛋白(EGFP)的质粒pSEB-HUS载体中,构建重组质粒pSV,经限制性内切酶消化、PCR扩增及DNA序列测定等方法鉴定后,再将靶向EV71病毒的VP1基因的siRNA克隆到重组质粒pSV中,构建成重组质粒pSVsi1、pSVsi2、pSVsi3和pSVsi4,经DNA序列测定鉴定证实插入的siRNA序列正确后,转染A549细胞,用倒置荧光显微镜初步观察绿色荧光蛋白的表达情况;用荧光实时定量RT-PCR检测VP1基因mRNA转录水平,免疫细胞化学检测细胞中的VP1基因的表达水平,进而分析siRNA对VP1 mRMA转录和蛋白表达的抑制作用。筛选出抑制效率最高的siRNA,构建重组质粒pSsi1,并将其转化进减毒沙门氏菌中,进而探索以RNAi技术为基础、减毒沙门氏菌为载体的口服治疗性疫苗的可行性。 结果:经限制性内切酶、PCR、测序及Western Blot证实:成功构建可以表达EGFP的、靶向EV71病毒VP1基因的siRNA表达载体;从已构建的3个靶向VP1基因的siRNA表达载体中,筛选到可高效抑制VP1基因表达的pSVsi1,其VP1 mRNA和VP1蛋白的抑制率分别为87%和89%;成功构建si1的重组质粒pSsi1,并将其成功的转化进减毒沙门氏菌中。 结论:1.成功构建以pSEB-HUS为载体的重组siRNA表达质粒,为今后运用该质粒进行RNAi相关研究奠定了物质基础;2.筛选到对EV71病毒VP1基因的表达有一定抑制作用的siRNA,并将其重组质粒pSsi1成功转化进减毒沙门氏菌中,不仅为研究RNAi治疗由EV71引起的手足口病提供了物质基础,而且表明用RNA干扰技术抑制细胞内EV71病毒VP1基因的表达、进而治疗由EV71引起的手足口病是可行的。3.本研究设计的三个靶向EV71病毒VP1基因的序列,均对VP1的表达有大于50%的抑制率,说明RNA干扰对所靶向的mRNA的序列和部位依赖性不强,因此进一步预示了此项技术的可行性。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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