丙型肝炎病毒NS4A与钙离子信号调节亲环素配体相互作用的研究

发布时间：2018-05-04 17:22

本文选题：丙型肝炎病毒非结构蛋白4A + 钙离子信号调节亲环素配体　；参考：《中国人民解放军军事医学科学院》2007年博士论文

【摘要】： 目的：探讨丙型肝炎病毒非结构蛋白4A(HCV NS4A)与钙离子信号调节亲环素配体(CAML)的相互作用、作用位点及相互作用后对细胞基因表达的影响。方法：应用酵母双杂交技术筛选人白细胞文库中与HCV NS4A相互作用的蛋白。对克隆重复率较高的CAML基因进行克隆，并构建其酵母表达载体，在酵母细胞中与HCV NS4A进行回交验证之后，构建HCV NS4A(pCMV／Myc-NS4A)及CAML(pcDNA3.1／His-A-CAML)的真核表达载体，共转染293细胞，，进行免疫共沉淀验证实验。构建CAML不同剪接体及缺失突变体的酵母表达载体，转染入酵母Y187，分别在酵母细胞中与转染了HCV NS4A酵母表达载体(pGBKT7-NS4A)的AH109进行配合，寻找HCV NS4A与CAML相互作用的具体位点。将HCV NS4A真核表达载体转染入稳定转染CAML的293细胞，利用基因芯片技术检测二者相互作用后对细胞基因表达的影响。结果：筛选出在铺有X-α-gal的四缺培养基上能够生长并变成蓝色的真阳性菌落45个，其中包括29个CAML。对CAML基因成功克隆，在酵母细胞中回交验证了HCV NS4A与CAML的相互作用。构建了HCV NS4A及CAML的真核细胞表达载体，并利用免疫共沉淀技术在293细胞中验证了两者的相互作用。包含CAML蛋白氨基末端1～57位氨基酸的CAML不同缺失突变体及剪接体的酵母表达载体转染入酵母Y187后，均可以与转染pGBKT7-NS4A的AH109成功配合。基因芯片筛选结果提示转染HCV NS4A后的稳定转染了CAML基因的293细胞中共有48种基因的表达水平上调，36种基因的表达水平下调，其中与Rho蛋白信号转导相关基因6种。结论：在293细胞中HCV NS4A与CAML存在相互作用，其相互结合的位点位于CAML蛋白亲水的氨基末端1～57位氨基酸。二者相互作用可以使细胞内的一系列基因表达发生改变，其中包括与细胞凋亡相关的Rho蛋白信号转导相关基因。HCV NS4A可能通过与CAML相互作用对Rho GTPases信号通路活性产生影响，赋予细胞对凋亡的抵抗，并可能由此而参与丙型肝炎病毒感染慢性化的机制。
[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- 伪 -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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373

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