發(fā)布時(shí)間：2018-05-22 11:40

  本文選題：丙型肝炎病毒 + 核心蛋白�。� 參考：《安徽理工大學(xué)》2008年碩士論文

【摘要】： 目的:本實(shí)驗(yàn)構(gòu)建丙型肝炎核心蛋白(HCV core)原核表達(dá)載體,在大腸埃希菌中進(jìn)行表達(dá)、并純化融合蛋白;制備抗core蛋白單克隆抗體并進(jìn)行鑒定。研究HCVcore在肝源性細(xì)胞系中與載脂蛋白AⅠ(apoAⅠ)、轉(zhuǎn)位蛋白(Translin)相互作用,為丙型肝炎病毒引起的脂肪肝和肝癌的分子機(jī)制提供理論基礎(chǔ)。 方法:構(gòu)建原核表達(dá)載體pET-32a(+)HCV-core,轉(zhuǎn)化大腸埃希菌BL21中,以IPTG誘導(dǎo)獲得core融合蛋白。利用Ni~+親和柱對(duì)表達(dá)蛋白進(jìn)行純化及上柱復(fù)性。用純化蛋白免疫BALB/c小鼠,取小鼠脾細(xì)胞與骨髓瘤細(xì)胞融合,獲得抗HCV-core蛋白的單克隆抗體。利用Western blot、ELISA法和免疫組化對(duì)單克隆抗體進(jìn)行特異性和靈敏度分析及鑒定。構(gòu)建真核表達(dá)質(zhì)粒:pDNA3.1-myc-his-apoAⅠ、pDNA3.1-myc-his-Translin、pDNA3.1(-)core、pact-apoAⅠ、pact-Translin和pBIND-core,共轉(zhuǎn)染到HepG2細(xì)胞中。用免疫共沉淀(CoIP)和哺乳動(dòng)物雙雜交的方法,Westren blot分析免疫共沉淀雜交帶,用Dual Luciferase Reporter AssaySystem檢測(cè)熒光素酶強(qiáng)度。 結(jié)果:1:HCV核心蛋白基因PCR產(chǎn)物及連接到pET32a(+)中,經(jīng)雙酶切和DNA測(cè)序鑒定,成功構(gòu)建pET-32a(+)-core,HCV-core融合蛋白表達(dá)成功。通過Ni~+親和柱對(duì)表達(dá)蛋白進(jìn)行純化,獲得core蛋白(42KD)。用純化蛋白常規(guī)免疫BALB/c小鼠,并鼠尾靜脈取血。用ELISA檢測(cè)免疫后小鼠血清出現(xiàn)抗core抗體,并且血清抗體滴度成逐漸增高趨勢(shì)。Western blot證明血清抗體能檢測(cè)到HCV肝硬化患者肝組織中core蛋白,進(jìn)一步證明我們制備的core融合蛋白與天然存在的core蛋白結(jié)構(gòu)基本一致。用脾細(xì)胞與骨髓瘤細(xì)胞融合,細(xì)胞融合率59.2%。用ELISA和Westren blot篩選單克隆抗體株陽(yáng)性率6.3%。有限稀釋再次篩選共獲得穩(wěn)定分泌抗體的雜交瘤細(xì)胞株。用ELISA測(cè)定其效價(jià),上清效價(jià)最高的細(xì)胞株是4E10。以4E10細(xì)胞上清作為一抗,Western blot檢測(cè)純化抗原可檢測(cè)到8ng的抗原;免疫組織化學(xué)發(fā)現(xiàn):在HCV肝癌組織中,HCVcore主要位于肝細(xì)胞胞漿中,呈灶性或彌漫性分布。 2:共轉(zhuǎn)染pDNA3.1-myc-his apoAⅠ和pDNA3.1(-)HCVcore進(jìn)入HepG2細(xì)胞中,經(jīng)免疫共沉后Western blot結(jié)果顯示:單獨(dú)core蛋白的雜交帶相對(duì)分子量大概19kDa,共沉淀帶相對(duì)分子量大約50kDa。所構(gòu)建的真核載體能在HepG2細(xì)胞中表達(dá),并且HCVcore和apoA1二種蛋白能相互結(jié)合。而且也用pDNA3.1-myc-hisapoAⅠ和pDNA3.1(-)HCVcore單獨(dú)轉(zhuǎn)染入HepG2細(xì)胞中,每種質(zhì)粒都能在細(xì)胞中過表達(dá),且不與HepG2細(xì)胞內(nèi)其它蛋白結(jié)合。pACT-apoAⅠ和pBIND-core共轉(zhuǎn)染時(shí),相對(duì)熒光素酶活性值較pACT和pBIND空載體轉(zhuǎn)染組以及pACT-apoA1和pBIND空載體、pACT空載體和pBIND-core轉(zhuǎn)染組明顯升高(12～16倍)。表明HCVcore和apoAⅠ在體內(nèi)能相互結(jié)合,且單獨(dú)轉(zhuǎn)染后沒有自身激活作用。 3:共轉(zhuǎn)染pDNA3.1-myc-his Translin和pDNA3.1(-)HCVcore進(jìn)入HepG2細(xì)胞中,經(jīng)免疫共沉后Western blot結(jié)果顯示:單獨(dú)core蛋白的雜交帶相對(duì)分子量大概19kDa,共沉淀帶只出現(xiàn)Translin蛋白雜交帶相對(duì)分子量大約21.8kDa。pACT-Translin和pBIND-core共轉(zhuǎn)染時(shí),相對(duì)熒光素酶活性值較其他組明顯升高(6～8倍)。表明HCVcore和Translin在體內(nèi)能相互結(jié)合。 結(jié)論:成功表達(dá)、純化HCV-core基因融合蛋白,獲得高特異性、高效價(jià)鼠抗HCV-core單克隆抗體,為研究core基因的生物學(xué)功能提供了新的手段。HCV-core可與apoAⅠ、Translin在體內(nèi)相互作用,為慢性丙型肝炎致肝脂肪變和肝癌的機(jī)制的研究提供理論依據(jù)。
[Abstract]:Objective: to construct the prokaryotic expression vector of hepatitis C core protein (HCV core), to express in Escherichia coli and to purify the fusion protein; to prepare monoclonal antibodies against core protein and to identify it. The interaction of HCVcore in the hepatogenic cell line with apolipoprotein A I (apoA I) and transposition protein (Translin) was used as a hepatitis C virus. The molecular mechanism of virus induced fatty liver and liver cancer provides a theoretical basis.
Methods: the prokaryotic expression vector, pET-32a (+) HCV-core, was transformed into the Escherichia coli BL21, and the core fusion protein was induced by IPTG. The expression protein was purified by the Ni~+ affinity column. The purified protein was used to immunize the BALB/c mice and the murine splenocytes were fused with the myeloma cells to obtain the monoclonal antibody against the HCV-core protein. The specificity and sensitivity of the monoclonal antibodies were analyzed and identified by Western blot, ELISA and immunohistochemistry. The eukaryotic expression plasmids were constructed: pDNA3.1-myc-his-apoA I, pDNA3.1-myc-his-Translin, pDNA3.1 (-) core, pact-apoA I, pact-Translin and pBIND-core, and were transferred to HepG2 cells. Immunoprecipitation (CoIP) and mammal double were used. The method of hybridization was used to analyze the co immunoprecipitation hybrids with Westren blot, and Dual Luciferase Reporter AssaySystem was used to detect the intensity of luciferase.
Results: the PCR product of 1:HCV core protein gene and its connection to pET32a (+) were identified by double enzyme cutting and DNA sequencing. The pET-32a (+) -core was successfully constructed and the expression of HCV-core fusion protein was successfully expressed. The expression protein was purified by Ni~+ affinity column and core protein (42KD) was obtained. The purified protein was routinely immune to BALB/c mice and the rat tail vein was taken blood. After immunization, the serum anti core antibody was detected and the serum antibody titer increased gradually in.Western blot. The serum antibody showed that the serum antibody could detect the core protein in the liver tissues of the patients with HCV liver cirrhosis, and further demonstrated that the core fusion protein prepared by us was basically consistent with the natural existing core protein structure. The fusion of splenocyte and myeloma cells was the same. The cell fusion rate 59.2%. was selected by ELISA and Westren blot to screen the hybridoma cell line of the monoclonal antibody positive rate 6.3%. to obtain the stable secretory antibody again. The titer was measured by ELISA. The highest titer of the supernatant was 4E10. with the 4E10 cell supernatant as one anti, Western blot detection of the purified antigen could detect 8ng. Immunohistochemistry revealed that in HCV hepatoma tissues, HCVcore was mainly located in the cytoplasm of hepatocytes, with a focal or diffuse distribution.
2: co transfected pDNA3.1-myc-his apoA I and pDNA3.1 (-) HCVcore into HepG2 cells. The results of Western blot after immunization showed that the relative molecular weight of the single core protein hybrid band was about 19kDa, and the eukaryotic carrying capacity of the coprecipitation zone was expressed in the HepG2 cells by the relative molecular weight of approximately 50kDa.. PDNA3.1-myc-hisapoA I and pDNA3.1 (-) HCVcore were also transfected into HepG2 cells separately, each plasmid could be overexpressed in the cell, and the relative luciferase activity value was compared with pACT and pBIND no-load transfection groups and pACT-apoA1 and pBI when the other proteins in the HepG2 cells were co transfected with.PACT-apoA I and pBIND-core. ND no-load, pACT no-load and pBIND-core transfection group were significantly increased (12~16 times). It showed that HCVcore and apoA I could be combined in the body, and there was no self activation after transfection.
3: cotransfected pDNA3.1-myc-his Translin and pDNA3.1 (-) HCVcore into HepG2 cells. The result of Western blot after immunization showed that the relative molecular weight of the single core protein hybrid band was about 19kDa, and the relative molecular weight of the Translin protein hybridization band was only about 21.8kDa.pACT-Translin and co transfection. The activity of enzyme was significantly higher than that of other groups (6~8 times), indicating that HCVcore and Translin could bind to each other in vivo.
Conclusion: successfully expressed, purified HCV-core gene fusion protein and obtained high specificity and high efficiency mouse anti HCV-core monoclonal antibody. It provides a new means to study the biological function of core gene,.HCV-core can interact with apoA I, Translin in the body, and provides a theory for the study of the mechanism of hepatic steatosis and liver cancer induced by chronic hepatitis C inflammation. Basis.
【學(xué)位授予單位】：安徽理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 劉崇柏;非甲非乙肝炎(NANB)的研究進(jìn)展[J];病毒學(xué)報(bào);1990年02期

2 叢勉爾,劉崇柏,孟宗達(dá),陳淑芬;我國(guó)非腸道傳播非甲非乙型肝炎(丙型)病毒NS3蛋白的部份cDNA克隆及序列分析[J];病毒學(xué)報(bào);1991年01期

3 金冬雁,李景源,楊永平,薛水星,吳長(zhǎng)龍,劉崇柏,李玉英,侯云德;丙型肝炎病毒核殼蛋白抗原的化學(xué)合成及其在血清學(xué)診斷中的應(yīng)用[J];病毒學(xué)報(bào);1992年04期

4 成軍,陳菊梅;丙型肝炎病毒核心蛋白結(jié)合蛋白的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).病毒學(xué)分冊(cè);2000年04期

5 劉妍,成軍,邵得志,王琳,鐘彥偉,夏小兵;丙型肝炎病毒核心蛋白反式激活SV40病毒早期啟動(dòng)子/增強(qiáng)子的研究[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);2001年03期

6 成軍;丙型病毒性肝炎與肝臟脂肪變[J];遼寧醫(yī)學(xué)雜志;2004年02期

7 楊少奇 ,胡建國(guó) ,郭新寧 ,蔣棟;慢性丙型肝炎與脂肪肝關(guān)系的研究[J];陜西醫(yī)學(xué)雜志;2003年03期

8 張健;成軍;李莉;劉愛兵;吳勤;李克;董菁;王琳;陸蔭英;;丙型肝炎病毒感染者血清載脂蛋白AⅠ及AⅡ水平的研究[J];世界華人消化雜志;2002年09期

9 成軍,李莉,張玲霞,陳菊梅;丙型肝炎病毒感染與脂類代謝的相關(guān)性[J];肝臟;2002年01期

，

本文編號(hào)：1921909