本文選題：丙型肝炎病毒非結(jié)構(gòu)蛋白4A + 鈣離子信號(hào)調(diào)節(jié)親環(huán)素配體��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年博士論文

【摘要】： 目的：探討丙型肝炎病毒非結(jié)構(gòu)蛋白4A(HCV NS4A)與鈣離子信號(hào)調(diào)節(jié)親環(huán)素配體(CAML)的相互作用、作用位點(diǎn)及相互作用后對(duì)細(xì)胞基因表達(dá)的影響。 方法：應(yīng)用酵母雙雜交技術(shù)篩選人白細(xì)胞文庫(kù)中與HCV NS4A相互作用的蛋白。對(duì)克隆重復(fù)率較高的CAML基因進(jìn)行克隆，并構(gòu)建其酵母表達(dá)載體，在酵母細(xì)胞中與HCV NS4A進(jìn)行回交驗(yàn)證之后，構(gòu)建HCV NS4A(pCMV／Myc-NS4A)及CAML(pcDNA3.1／His-A-CAML)的真核表達(dá)載體，共轉(zhuǎn)染293細(xì)胞，，進(jìn)行免疫共沉淀驗(yàn)證實(shí)驗(yàn)。構(gòu)建CAML不同剪接體及缺失突變體的酵母表達(dá)載體，轉(zhuǎn)染入酵母Y187，分別在酵母細(xì)胞中與轉(zhuǎn)染了HCV NS4A酵母表達(dá)載體(pGBKT7-NS4A)的AH109進(jìn)行配合，尋找HCV NS4A與CAML相互作用的具體位點(diǎn)。將HCV NS4A真核表達(dá)載體轉(zhuǎn)染入穩(wěn)定轉(zhuǎn)染CAML的293細(xì)胞，利用基因芯片技術(shù)檢測(cè)二者相互作用后對(duì)細(xì)胞基因表達(dá)的影響。 結(jié)果：篩選出在鋪有X-α-gal的四缺培養(yǎng)基上能夠生長(zhǎng)并變成藍(lán)色的真陽(yáng)性菌落45個(gè)，其中包括29個(gè)CAML。對(duì)CAML基因成功克隆，在酵母細(xì)胞中回交驗(yàn)證了HCV NS4A與CAML的相互作用。構(gòu)建了HCV NS4A及CAML的真核細(xì)胞表達(dá)載體，并利用免疫共沉淀技術(shù)在293細(xì)胞中驗(yàn)證了兩者的相互作用。包含CAML蛋白氨基末端1～57位氨基酸的CAML不同缺失突變體及剪接體的酵母表達(dá)載體轉(zhuǎn)染入酵母Y187后，均可以與轉(zhuǎn)染pGBKT7-NS4A的AH109成功配合�；蛐酒Y選結(jié)果提示轉(zhuǎn)染HCV NS4A后的穩(wěn)定轉(zhuǎn)染了CAML基因的293細(xì)胞中共有48種基因的表達(dá)水平上調(diào)，36種基因的表達(dá)水平下調(diào)，其中與Rho蛋白信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因6種。 結(jié)論：在293細(xì)胞中HCV NS4A與CAML存在相互作用，其相互結(jié)合的位點(diǎn)位于CAML蛋白親水的氨基末端1～57位氨基酸。二者相互作用可以使細(xì)胞內(nèi)的一系列基因表達(dá)發(fā)生改變，其中包括與細(xì)胞凋亡相關(guān)的Rho蛋白信號(hào)轉(zhuǎn)導(dǎo)相關(guān)基因。HCV NS4A可能通過(guò)與CAML相互作用對(duì)Rho GTPases信號(hào)通路活性產(chǎn)生影響，賦予細(xì)胞對(duì)凋亡的抵抗，并可能由此而參與丙型肝炎病毒感染慢性化的機(jī)制。
[Abstract]:Aim: to investigate the interaction of hepatitis C virus nonstructural protein (4A(HCV NS4A) with calcium signal regulated cyclophile ligand (CAMLL), the interaction sites and the effect of interaction on cell gene expression. Methods: yeast two-hybrid technique was used to screen proteins interacting with HCV NS4A in human leukocyte library. The CAML gene with high repetition rate was cloned, and its yeast expression vector was constructed. After backcrossing with HCV NS4A in yeast cells, the eukaryotic expression vectors of HCV NS4ApCMV / Myc-NS4A) and CAML-pcDNA3.1 / His-A-CAMLS were constructed and cotransfected into 293 cells. The immune coprecipitation test was carried out. The yeast expression vectors of different splicing and deletion mutants of CAML were constructed and transfected into yeast Y187.The yeast expression vector pGBKT7-NS4A was transfected into yeast cells respectively to find the specific site of interaction between HCV NS4A and CAML. The eukaryotic expression vector of HCV NS4A was transfected into 293 cells stably transfected with CAML. Results: 45 true-positive colonies, including 29 CAMLs, could grow and turn blue on X- 偽 -gal medium. The CAML gene was cloned successfully and the interaction between HCV NS4A and CAML was verified by backcross in yeast cells. The eukaryotic expression vectors of HCV NS4A and CAML were constructed, and the interaction between them was verified by immunoprecipitation technique in 293 cells. The yeast expression vector containing amino terminal amino acid of CAML protein at position 1 and 57 amino acids of CAML with different deletion mutants and splicing vectors were transfected into yeast Y187, which could be successfully combined with AH109 transfected with pGBKT7-NS4A. The results of gene chip screening showed that the expression level of 48 genes up-regulated and down-regulated the expression of 36 genes in 293 cells with stable transfection of CAML gene after HCV NS4A transfection, including 6 genes related to signal transduction of Rho protein. Conclusion: there is interaction between HCV NS4A and CAML in 293 cells, and the binding site is located at the amino terminal of 1 ~ 57 amino acid at the hydrophilic end of CAML protein. The interaction of the two changes a series of genes expression in cells, including apoptosis related Rho protein signal transduction related gene. HCV NS4A may affect the activity of Rho GTPases signal pathway by interacting with CAML. Endow cells with resistance to apoptosis, which may be involved in the mechanism of chronic hepatitis C virus infection.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 宋培新;慢性HBV感染者肝組織中HBV cccDNA定量檢測(cè)方法的建立[D];南京醫(yī)科大學(xué);2006年

2 劉佩;不同物種液泡型Na～+/H～+逆向運(yùn)轉(zhuǎn)體基因的克隆及鹽、干旱脅迫下的功能比較[D];山東農(nóng)業(yè)大學(xué);2007年

本文編號(hào)：1843906