SHARP-2特异的RNA干扰腺病毒载体构建

发布时间：2018-06-27 00:37

本文选题：RNA干扰 + 小发夹RNA　；参考：《浙江大学》2008年硕士论文

【摘要】： 背景: 大鼠SHARP-2蛋白是归属重要转录因子basic helix-loop-helix(bHLH)蛋白质家族,在小鼠和人的相应的蛋白质为Stra13和Dec1。SHARP-2转录因子在细胞增殖,分化,肿瘤形成的起了重要的作用。我们的前期研究表明TGF-β可促进SHARP-2的表达,而SHARP-2转录因子又可促进IL-2的分泌。以及在Stra13敲除小鼠中INF-γ和IL-2的表到下降,由此推测SHARP-2基因在器官移植中具有重要的作用。我们借助于RNA干扰技术,通过靶向抑制SHARP-2的表达,来研究SHARP-2在器官移植中的作用。RNA干扰主要是通过dsRNA被核酸酶切割成21-23 bp的siRNA双链,进而由RNA诱导的沉默复合体(RNAinduced silencing complex,Risc)分离并结合,后者在siRNA反义链的介导下识别并靶向切割同源性mRNA而阻止基因的翻译过程。通过构建shRNA表达载体,在细胞内表达shRNA,通过Dicer酶的作用下形成siRNA分子,达到对目的基因的沉默。目前腺病毒载体作为RNA干扰载体已有长足的发展,并能够在哺乳动物细胞中达到目的基因的长时程抑制。课题组获得国家自然基金资助,本课题作为基础,构建了特异性SHARP-2基因RNA干扰的腺病毒载体系统,为SHARP-2基因在器官移植中的作用研究奠定基础。目的: 构建转录SHARP-2基因小发夹RNA(SHARP-2-shRNA)的腺病毒(Rad-hSHARP)。方法: 一、设计能转录SHARP-2-shRNA模板的两对DNA序列,退火后克隆至PDC316-EGFP-U6质粒,构建重组质粒PDC316-SHARP-shRNA。二、将PDC316-SHARP-shRNA与腺病毒骨架质粒pBHGloxdelEl3cre共转染293细胞,进行腺病毒载体(Rad-hSHARP)的构建。三、提取重组腺病毒的DNA,PCR鉴定正确者即为正确序列插入的重组腺病毒Rad-hSHARP。四、在NRK细胞中对重组腺病毒干扰效果的鉴定。结果: 一、测序鉴定表明PDC316-SHARP-shRNA构建成功。二、穿梭载体和骨架质粒共转染293细胞成功构建重组腺病毒颗粒,滴度较高。三、PCR测序鉴定表明重组腺病毒(Rad-hSHARP)包含目的基因序列的插入。四、RT-PCR表明重组腺病毒在NRK细胞中有干扰效果。结论: 成功构建转录SHARP-2基因小发夹RNA(SHARP-2-shRNA)的腺病毒(Rad-hSHARP),为利用特异性沉默SHARP-2基因在器官移植中的作用研究奠定基础。
[Abstract]:Background: rat SHARP-2 protein belongs to an important transcription factor basic helix-loop-helix (bHLH) protein family. The corresponding proteins in mice and humans are Stra13 and Dec1. SHARP-2 transcription factors play an important role in cell proliferation differentiation and tumor formation. Our previous studies have shown that TGF- 尾 can promote the expression of SHARP-2, and SHARP-2 transcription factor can promote the secretion of IL-2. And the apparent decrease of INF- 纬 and IL-2 in Stra13 knockout mice, which suggested that SHARP-2 gene plays an important role in organ transplantation. We study the role of SHARP-2 in organ transplantation by targeting the inhibition of SHARP-2 expression by RNA interference. RNA interference is mainly mediated by dsRNA cleavage into 21-23 BP siRNA double strands by dsRNA. Then RNA-induced silencing complexRisc (RNA-induced silencing complexRisc) was isolated and conjugated, which was mediated by siRNA antisense chains to recognize and target homologous mRNAs to block gene translation. By constructing shRNA expression vector and expressing shRNAs in cells, siRNA molecules were formed by Dicer enzyme to silence the target gene. At present, adenovirus vectors as RNA interference vectors have been greatly developed, and can achieve long-term inhibition of the target gene in mammalian cells. The project was supported by the National Fund for Nature. Based on this project, the adenovirus vector system of specific SHARP-2 gene RNA interference was constructed, which laid a foundation for the study of the role of SHARP-2 gene in organ transplantation. Aim: to construct the adenovirus (Rad-hSHARP) of SHARP-2 gene small hairpin RNA (SHARP-2-shRNA). Methods: first, two pairs of DNA sequences were designed and cloned into PDC316-EGFP-U6 plasmid after annealing, and the recombinant plasmid PDC316-SHARP-shRNA was constructed. Secondly, PDC316-SHARP-shRNA and adenovirus skeleton plasmid pBHGloxdelEl3cre were co-transfected into 293 cells to construct the adenovirus vector (Rad-hSHARP). Thirdly, the recombinant adenovirus Rad-hSHARPwas identified by DNA polymerase chain reaction (DNA-PCR) of the recombinant adenovirus inserted with the correct sequence. 4. Identification of interference effect of recombinant adenovirus in NRK cells. Results: first, sequencing showed that PDC316-SHARP-shRNA was successfully constructed. Secondly, the recombinant adenovirus particles were successfully constructed by co-transfection of shuttle vector and skeleton plasmid into 293 cells, and the titer of recombinant adenovirus particles was higher. PCR sequencing showed that the recombinant adenovirus (Rad-hSHARP) contained the insertion of the target gene sequence. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the recombinant adenovirus had interference effect in NRK cells. Conclusion: the successful construction of the small hairpin RNA of SHARP-2 (SHARP-2-shRNA) adenovirus (Rad-hSHARP) lays a foundation for the study of the role of specific silencing of SHARP-2 gene in organ transplantation.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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