TIA-1对乙型肝炎病毒表达和复制的影响

发布时间：2018-01-01 11:12

本文关键词：TIA-1对乙型肝炎病毒表达和复制的影响　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

更多相关文章： IA-1蛋白 HPRE RNA 抑制性结合蛋白

【摘要】： 乙肝病毒感染是全球性的公共卫生问题,在我国危害尤其严重,HBV转录后调节涉及多个环节,HBV转录后调节基序(HBV post-transcriptional regulatory element ,HPRE)的作用是其中重要一环。HPRE最早发现是在1993年,是HBV基因组上的一个片段(1151nt-1684nt),现有的研究认为这段序列在转录后水平发挥着顺式调节作用,抑制转录体的剪接同时促进转录体由细胞核向细胞浆的转运。虽然HPRE的作用机制至今还不是完全清楚,但比较一致的认为是HPRE依赖与细胞蛋白的结合来发挥它的作用,因此验证HPRE结合蛋白就可以帮助明确HPRE在HBV RNA输出中发挥的作用。我室研究人员,利用噬菌体表面展示技术进行筛选新的与HPRE-RNA结合的蛋白分子,发现了一种新的蛋白,经鉴定为TIA-1(cytotoxic granule-associated RNA binding protein 1)蛋白。为了进一步分析TIA-1对HPRE的功能影响,开展了本研究。目的:表达纯化大量TIA-1-GST蛋白及HPRE-Biontin-RNA,验证两者之间的结合作用,并且纯化TIA-1各亚片断的GST融合蛋白,鉴定TIA-1与HPRE作用部位。利用检测系统pDM138及pDM138-HPRE进一步验证TIA-1对HPRE是否发挥作用。最后转染HepG2.2.15细胞检测TIA-1对HBV的功能有何影响。方法:1用XhoI酶切pGEM-HPRE质粒DNA,得一线性模板,利用T7RNA聚合酶,以体外转录的方法合成HPRE-Biotin RNA;在BL21细菌里大量诱导表达及纯化TIA-1-GST融合蛋白及GST蛋白。2 M-280 Streptavidin磁珠特异性结合Biotin,用磁力座纯化混合物,将纯化后的洗脱液做Western Blot分析,用GST单抗检测有无蛋白存在; Glutathione Sepharose 4B纯化柱特异性结合GST蛋白,将纯化后的洗脱液做RT-PCR,检测有无HPRE的RNA存在。3将蛋白TIA-1的真核表达质粒pcDNA3-FLAG-TIA-1和pDM138及pDM138-HPRE分组,按照一定比例转染细胞HepG2,通过ELISA检测报告基因CAT的表达验证蛋白TIA-1与HPRE RNA的相互作用。4将TIA-1蛋白的真核表达质粒pcDNA3-FLAG-TIA-1按照一定比例转染HepG2.2.15细胞,通过ELISA检测S抗原及e抗原的表达量和统计学分析验证蛋白TIA-1对HBV的影响。结果: 1 Western Blot用GST抗体检测,样品TIA-1-GST+HPRE-Biotin RNA有蛋白带存在,而阴性对照GST+HPRE-Biotin RNA没有条带。RT-PCR电泳结果显示样品TIA-1-GST+HPRE-Biotin RNA有HPRE RNA ,而阴性对照GST+HPRE-Biotin RNA没有。2报告基因CAT表达量经统计学处理后显示:转染质粒pDM138-HPRE的细胞能增加CAT表达,同时转染质粒pDM138-HPRE和pcDNA3-FLAG-TIA-1之后,CAT的表达受到明显抑制。3检测S抗原及e抗原的表达量,统计学处理后显示:与正常的HepG2.2.15细胞相比,转染pcDNA3-TIA-1质粒的HepG2.2.15细胞,其e抗原的表达量有明显的增高,而S抗原表达量有明显的降低。结论:TIA-1与HPRE可以发生结合作用,为HPRE的抑制性结合蛋白,能调节HBV S抗原和e抗原的表达。
[Abstract]:Hepatitis B virus infection is a global public health problem, particularly in China, HBV post transcriptional regulation involving multiple links, HBV post transcriptional regulatory motifs (HBV post-transcriptional regulatory element, HPRE) is one of the important role of a ring.HPRE was first discovered in 1993, is a fragment of HBV genome (the 1151nt-1684nt), some researchers believe that this sequence at the post transcriptional level plays a cis regulatory role, inhibiting transcription splicing of transcripts and promote transport from the nucleus to the cytoplasm. Although the mechanism of HPRE is still not completely clear, but that is more consistent with HPRE dependent cell protein to play the role of it, so the validation of the HPRE binding protein can help clear HPRE play in the HBV RNA output function.
My room for researchers, using phage display technique for screening new HPRE-RNA combined with the protein molecule, the discovery of a new protein, was identified as TIA-1 (cytotoxic granule-associated RNA binding protein 1) protein. In order to further analyze the effect of TIA-1 on the function of HPRE, carry out this study.
Objective: to express and purify TIA-1-GST protein and HPRE-Biontin-RNA, combined with the effect of verification between the two, and the purified TIA-1 sub fragment of GST fusion protein and identification of TIA-1 and HPRE sites of action. The system uses pDM138 and pDM138-HPRE to validate the TIA-1 on whether HPRE play a role. What is the effect of the last HepG2.2.15 cells transfected with detection of TIA-1 function of HBV.
Methods: 1 XhoI pGEM-HPRE was digested with DNA, a linear template, using T7RNA polymerase, by the method of in vitro transcription synthesis of HPRE-Biotin RNA in BL21; a large number of bacteria induced expression and purification of TIA-1-GST fusion protein and GST protein.2 M-280 Streptavidin beads binding specificity of Biotin, purified mixture with a magnetic base, the eluent Western Blot analysis of purified GST monoclonal antibody, a Glutathione Sepharose 4B protein; purification column specific binding to GST protein, purified eluate RT-PCR, detection without HPRE RNA.3 eukaryotic expression plasmid pcDNA3-FLAG-TIA-1 and pDM138 and pDM138-HPRE group protein TIA-1, according to a certain proportion of transfected cells HepG2 through the interaction of.4, ELISA to detect the expression of reporter gene CAT TIA-1 and HPRE RNA to validate the protein TIA-1 protein eukaryotic expression plasmid according to pcDNA3-FLAG-TIA-1 HepG2.2.15 cells were transfected in a certain proportion. The expression of S antigen and e antigen was detected by ELISA and the effect of protein TIA-1 on HBV was verified.
Results: 1 Western Blot GST antibody detection, sample TIA-1-GST+HPRE-Biotin RNA protein band, while the negative control GST+HPRE-Biotin RNA no.RT-PCR band electrophoresis results showed that the sample TIA-1-GST+HPRE-Biotin RNA HPRE RNA, GST+HPRE-Biotin RNA.2 and negative control did not report the expression level of CAT gene was revealed: transfection of plasmid pDM138-HPRE cells can increase the expression of CAT at the same time, pDM138-HPRE and pcDNA3-FLAG-TIA-1 after transfection, the expression of CAT was inhibited by.3 to detect the expression of S antigen and e antigen, statistical analysis showed that: compared with the normal HepG2.2.15 cells, the pcDNA3-TIA-1 plasmid was transfected into HepG2.2.15 cells, the expression of e antigen was increased, and the expression of S antigen was obvious reduced.
Conclusion: TIA-1 and HPRE can bind to the binding protein of HPRE, and can regulate the expression of HBV S antigen and e antigen.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R512.6;R373

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