本文關(guān)鍵詞：TIA-1對乙型肝炎病毒表達(dá)和復(fù)制的影響　出處：《重慶醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： IA-1蛋白 HPRE RNA 抑制性結(jié)合蛋白

【摘要】： 乙肝病毒感染是全球性的公共衛(wèi)生問題,在我國危害尤其嚴(yán)重,HBV轉(zhuǎn)錄后調(diào)節(jié)涉及多個(gè)環(huán)節(jié),HBV轉(zhuǎn)錄后調(diào)節(jié)基序(HBV post-transcriptional regulatory element ,HPRE)的作用是其中重要一環(huán)。HPRE最早發(fā)現(xiàn)是在1993年,是HBV基因組上的一個(gè)片段(1151nt-1684nt),現(xiàn)有的研究認(rèn)為這段序列在轉(zhuǎn)錄后水平發(fā)揮著順式調(diào)節(jié)作用,抑制轉(zhuǎn)錄體的剪接同時(shí)促進(jìn)轉(zhuǎn)錄體由細(xì)胞核向細(xì)胞漿的轉(zhuǎn)運(yùn)。雖然HPRE的作用機(jī)制至今還不是完全清楚,但比較一致的認(rèn)為是HPRE依賴與細(xì)胞蛋白的結(jié)合來發(fā)揮它的作用,因此驗(yàn)證HPRE結(jié)合蛋白就可以幫助明確HPRE在HBV RNA輸出中發(fā)揮的作用。 我室研究人員,利用噬菌體表面展示技術(shù)進(jìn)行篩選新的與HPRE-RNA結(jié)合的蛋白分子,發(fā)現(xiàn)了一種新的蛋白,經(jīng)鑒定為TIA-1(cytotoxic granule-associated RNA binding protein 1)蛋白。為了進(jìn)一步分析TIA-1對HPRE的功能影響,開展了本研究。 目的:表達(dá)純化大量TIA-1-GST蛋白及HPRE-Biontin-RNA,驗(yàn)證兩者之間的結(jié)合作用,并且純化TIA-1各亞片斷的GST融合蛋白,鑒定TIA-1與HPRE作用部位。利用檢測系統(tǒng)pDM138及pDM138-HPRE進(jìn)一步驗(yàn)證TIA-1對HPRE是否發(fā)揮作用。最后轉(zhuǎn)染HepG2.2.15細(xì)胞檢測TIA-1對HBV的功能有何影響。 方法:1用XhoI酶切pGEM-HPRE質(zhì)粒DNA,得一線性模板,利用T7RNA聚合酶,以體外轉(zhuǎn)錄的方法合成HPRE-Biotin RNA;在BL21細(xì)菌里大量誘導(dǎo)表達(dá)及純化TIA-1-GST融合蛋白及GST蛋白。2 M-280 Streptavidin磁珠特異性結(jié)合Biotin,用磁力座純化混合物,將純化后的洗脫液做Western Blot分析,用GST單抗檢測有無蛋白存在; Glutathione Sepharose 4B純化柱特異性結(jié)合GST蛋白,將純化后的洗脫液做RT-PCR,檢測有無HPRE的RNA存在。3將蛋白TIA-1的真核表達(dá)質(zhì)粒pcDNA3-FLAG-TIA-1和pDM138及pDM138-HPRE分組,按照一定比例轉(zhuǎn)染細(xì)胞HepG2,通過ELISA檢測報(bào)告基因CAT的表達(dá)驗(yàn)證蛋白TIA-1與HPRE RNA的相互作用。4將TIA-1蛋白的真核表達(dá)質(zhì)粒pcDNA3-FLAG-TIA-1按照一定比例轉(zhuǎn)染HepG2.2.15細(xì)胞,通過ELISA檢測S抗原及e抗原的表達(dá)量和統(tǒng)計(jì)學(xué)分析驗(yàn)證蛋白TIA-1對HBV的影響。 結(jié)果: 1 Western Blot用GST抗體檢測,樣品TIA-1-GST+HPRE-Biotin RNA有蛋白帶存在,而陰性對照GST+HPRE-Biotin RNA沒有條帶。RT-PCR電泳結(jié)果顯示樣品TIA-1-GST+HPRE-Biotin RNA有HPRE RNA ,而陰性對照GST+HPRE-Biotin RNA沒有。2報(bào)告基因CAT表達(dá)量經(jīng)統(tǒng)計(jì)學(xué)處理后顯示:轉(zhuǎn)染質(zhì)粒pDM138-HPRE的細(xì)胞能增加CAT表達(dá),同時(shí)轉(zhuǎn)染質(zhì)粒pDM138-HPRE和pcDNA3-FLAG-TIA-1之后,CAT的表達(dá)受到明顯抑制。3檢測S抗原及e抗原的表達(dá)量,統(tǒng)計(jì)學(xué)處理后顯示:與正常的HepG2.2.15細(xì)胞相比,轉(zhuǎn)染pcDNA3-TIA-1質(zhì)粒的HepG2.2.15細(xì)胞,其e抗原的表達(dá)量有明顯的增高,而S抗原表達(dá)量有明顯的降低。 結(jié)論:TIA-1與HPRE可以發(fā)生結(jié)合作用,為HPRE的抑制性結(jié)合蛋白,能調(diào)節(jié)HBV S抗原和e抗原的表達(dá)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R512.6;R373

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