發(fā)布時間：2018-05-04 17:22

  本文選題：丙型肝炎病毒非結構蛋白4A + 鈣離子信號調節(jié)親環(huán)素配體��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2007年博士論文

【摘要】： 目的：探討丙型肝炎病毒非結構蛋白4A(HCV NS4A)與鈣離子信號調節(jié)親環(huán)素配體(CAML)的相互作用、作用位點及相互作用后對細胞基因表達的影響。 方法：應用酵母雙雜交技術篩選人白細胞文庫中與HCV NS4A相互作用的蛋白。對克隆重復率較高的CAML基因進行克隆，并構建其酵母表達載體，在酵母細胞中與HCV NS4A進行回交驗證之后，構建HCV NS4A(pCMV／Myc-NS4A)及CAML(pcDNA3.1／His-A-CAML)的真核表達載體，共轉染293細胞，，進行免疫共沉淀驗證實驗。構建CAML不同剪接體及缺失突變體的酵母表達載體，轉染入酵母Y187，分別在酵母細胞中與轉染了HCV NS4A酵母表達載體(pGBKT7-NS4A)的AH109進行配合，尋找HCV NS4A與CAML相互作用的具體位點。將HCV NS4A真核表達載體轉染入穩(wěn)定轉染CAML的293細胞，利用基因芯片技術檢測二者相互作用后對細胞基因表達的影響。 結果：篩選出在鋪有X-α-gal的四缺培養(yǎng)基上能夠生長并變成藍色的真陽性菌落45個，其中包括29個CAML。對CAML基因成功克隆，在酵母細胞中回交驗證了HCV NS4A與CAML的相互作用。構建了HCV NS4A及CAML的真核細胞表達載體，并利用免疫共沉淀技術在293細胞中驗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載體轉染入酵母Y187后，均可以與轉染pGBKT7-NS4A的AH109成功配合�；蛐酒Y選結果提示轉染HCV NS4A后的穩(wěn)定轉染了CAML基因的293細胞中共有48種基因的表達水平上調，36種基因的表達水平下調，其中與Rho蛋白信號轉導相關基因6種。 結論：在293細胞中HCV NS4A與CAML存在相互作用，其相互結合的位點位于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胞內的一系列基因表達發(fā)生改變，其中包括與細胞凋亡相關的Rho蛋白信號轉導相關基因。HCV NS4A可能通過與CAML相互作用對Rho GTPases信號通路活性產生影響，賦予細胞對凋亡的抵抗，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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R373

【共引文獻】

相關碩士學位論文 前2條

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

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

本文編號：1843906