HBV表面抗原大蛋白与人胰腺CDK5RAP3蛋白相互作用的初步研究
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摘要
目的:为进一步研究HBV表面抗原大蛋白(LHBs)与宿主胰腺细胞的相互作用。方法:1.将酵母细胞表达载体pGBKT7-LHBs转化AH109酵母菌株,与含有胰腺细胞文库质粒的Y187酵母菌株进行配合,行酵母双杂交,筛选人胰腺中LHBs结合蛋白基因。2.应用逆转录聚合酶链反应(Reverse transcription PCR,RT-PCR),扩增人胰腺CDK5RAP3基因。3.构建真核表达质粒pcDNA-3.1 -myc-his-CDK5RAP3,通过真核表达系统,应用免疫共沉淀技术及Western blotting方法证实LHBs与人胰腺CDK5RAP3蛋白在细胞内的相互作用关系。4.构建真核表达质粒pACT-CDK5RAP3,通过真核表达系统,应用哺乳动物双杂交技术证实LHBs与人胰腺CDK5RAP3蛋白在细胞内的相互作用关系。5.构建CDK5RAP3的红色荧光表达载体pDsRed1-N1-CDK5RAP3,将其与绿色荧光表达载体pEGFP-LHBs分别转染Capan2细胞,模拟该蛋白在细胞中的表达,在激光共聚焦显微镜下观察LHBs及CDK5RAP3蛋白的亚细胞定位。结果:1.成功筛选出人胰腺细胞中11种与LHBs存在相互作用的已知功能蛋白,其中包括羧肽酶B1、胰凝乳蛋白C、人类胰蛋白酶原2、线粒体蛋白基因、组织相容性复合体13、乌头酸酶1、蛋白酶2、辅脂肪酶以及核糖体蛋白L10a等已知功能蛋白。2.成功扩增人胰腺CDK5RAP3基因。3.成功构建CDK5RAP3基因的真核表达载体pcDNA3.1-myc-his-CDK5RAP3,同pcDNA3.1(-)LHBs行免疫共沉淀,证实LHBs与人胰腺CDK5RAP3蛋白在细胞内存在相互作用关系。4.成功构建CDK5RAP3基因的真核表达载体pACT-CDK5RAP3,同pBIND-LHBs等共转染Capan 2细胞,行哺乳动物双杂交,证实LHBs与人胰腺CDK5RAP3蛋白在细胞内存在相互作用关系。5.成功构建CDK5RAP3的红色荧光表达载体,并在Capan2细胞中得到表达。激光共聚焦显微镜观察到LHBs及CDK5RAP3蛋白均主要定位于细胞浆中。结论:本实验成功筛选人胰腺中LHBs结合蛋白基因,并且证实LHBs与人胰腺CDK5RAP3蛋白在细胞内存在相互作用,为下一步研究LHBs的作用机制奠定基础。
Objective:To further study the interaction between HBV large protein and the host pancreatic cell.Methods:1.We transformed yeast cell expression vector pGBKT7-LHBs into yeast strain AH109 and performed yeast two hybrid by mating AH109 containing LHBs gene with Y187 containing plasmids of human pancreas cDNA library and screened of proteins binding to HBV large protein from human pancreas cDNA library.2.Homo sapiens CDK5RAP3 gene was amplified with reverse transcription polymerase chain reaction(RT-PCR). 3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using co-immunoprecipitation and Western blotting.4.We constructed eukaryotic expression vector pACT-CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using mammalian two-hybrid experiment.5. We constructed eukaryotic expression vector pDsRed1-N1-CDK5RAP3 and transfected into Capan2 cell lines by using lipofectamine,then observe the location by confocal fluorescence microscope. Results:1.We found eleven proteins which interact with HBV large protein,including Homo sapiens pancreatic lipase、Homo sapiens carboxypeptidase B1、Homo sapiens protease, serine, 2 (PRSS2)、Homo sapiens histocompatibility (minor) 13 (HM13)、Homo sapiens aconitase1、Homo sapiens elastase 2A (ELA2A)、Homo sapiens ribosomal protein L10a (RPL10A)and so on.2. Homo sapiens pancreatic CDK5RAP3 gene was obtained .3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3,which was cotransfected into Capan 2 cell line with pcDNA3.1(-)LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens colipase protein with Co-immunopercipitation.4.We constructed eukaryotic expression vector pACT-CDK5RAP3,which was cotransfected into Capan 2 cell line with pBIND- LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens CDK5RAP3 protein with mammalian two-hybrid.5.To investigate the subcellular localization of LHBs and CDK5RAP3, the plasmids expressing LHBs and CDK5RAP3 fusion protein with pEGFP-C1 and pDsRed1-N1 were constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. After 48 hours, the expression was detected by confocal fluorescence microscope. Conclusion:We found eleven proteins which could bind to HBV large protein and confirmed the interaction between HBV large protein and homo sapiens CDK5RAP3 protein in intra-cellular , which laid the foundation for further studying the mechanism of HBV large protein.
Objective:To further study the interaction between HBV large protein and the host pancreatic cell.Methods:1.We transformed yeast cell expression vector pGBKT7-LHBs into yeast strain AH109 and performed yeast two hybrid by mating AH109 containing LHBs gene with Y187 containing plasmids of human pancreas cDNA library and screened of proteins binding to HBV large protein from human pancreas cDNA library.2.Homo sapiens CDK5RAP3 gene was amplified with reverse transcription polymerase chain reaction(RT-PCR). 3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using co-immunoprecipitation and Western blotting.4.We constructed eukaryotic expression vector pACT-CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using mammalian two-hybrid experiment.5. We constructed eukaryotic expression vector pDsRed1-N1-CDK5RAP3 and transfected into Capan2 cell lines by using lipofectamine,then observe the location by confocal fluorescence microscope. Results:1.We found eleven proteins which interact with HBV large protein,including Homo sapiens pancreatic lipase、Homo sapiens carboxypeptidase B1、Homo sapiens protease, serine, 2 (PRSS2)、Homo sapiens histocompatibility (minor) 13 (HM13)、Homo sapiens aconitase1、Homo sapiens elastase 2A (ELA2A)、Homo sapiens ribosomal protein L10a (RPL10A)and so on.2. Homo sapiens pancreatic CDK5RAP3 gene was obtained .3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3,which was cotransfected into Capan 2 cell line with pcDNA3.1(-)LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens colipase protein with Co-immunopercipitation.4.We constructed eukaryotic expression vector pACT-CDK5RAP3,which was cotransfected into Capan 2 cell line with pBIND- LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens CDK5RAP3 protein with mammalian two-hybrid.5.To investigate the subcellular localization of LHBs and CDK5RAP3, the plasmids expressing LHBs and CDK5RAP3 fusion protein with pEGFP-C1 and pDsRed1-N1 were constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. After 48 hours, the expression was detected by confocal fluorescence microscope. Conclusion:We found eleven proteins which could bind to HBV large protein and confirmed the interaction between HBV large protein and homo sapiens CDK5RAP3 protein in intra-cellular , which laid the foundation for further studying the mechanism of HBV large protein.
引文
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[3]成军现代肝炎病毒分子生物学第二版科学出版社,2009年:699
[4]张艳玲,张宏,梁秀彬,侯平,王海燕.应用酵母双杂交系统筛选新基因A ngRem104相关蛋白。北京大学学报(医学版)。2003,35 (4):369-372
[5]陈永健,周永列,夏骏,等.血清乙型肝炎病毒表面抗原大蛋白检测的临床意义与评价.中华实验和临床病毒学杂志.2007,9:241-243
[6]韩学尧专论:2型糖尿病的遗传学研究突破将糖尿病的病因学研究引入新的时代.中国糖尿病杂志2008年第16卷第8期
[7] Zeggini E, Weedon MN,lingren CM,et al.Relication of genome-wide association signals in UK samples reveals risk loci for type2.diabetes.Science,2007, 316; 1336-1341
[8] Wu Y,Li YH.Relationship between hepatitis C virus infection and type 2 diabetes mellitus.Zhongguo Ganran Kongzhi Zazhi,2008,7:188-191.
[9] Wang L,Li K,Cheng J,et al.Interaction between hepatitis C virus core protein and apolipoprotein A1.Shijie Huaren Xiaohua Zazhi,2002,10:1018-1021.
[10]王琳,李克,成军,等.丙型肝炎病毒核心蛋白与载脂蛋白A1结合的研究.世界华人消化杂志,2002,10:1018-1021..
[11] Wyatt J,Baker H,Prasad P,et al.Steatosis and fibrosis in patients with chronic hepatitis C.J Clin Pathol,2004,57:402-406.
[12] Bahrami H,Daryani NE,Mirmomen S,et al. Clinical and histological features of nonalcoholic steatohepatitis in Iranian patients. BMC Gastroenterol 2003;3:27.
[13] Tsang SW,Ng WF,Wu BP,et al. Predictors of fibrosis in Asian patients with non-alcoholic steatohepatitis. J Gastroenterol Hepatol 2006;21:116-121.
[14] Bedogni G,Miglioli L,Masutti F,et al. Prevalence of and risk factors for nonalcoholic fatty liver disease:the Dionysos nutrition and liver study.Hepatology 2005;42:44-52.
[15] Reaven GM. Role of insulin resistance in human disease. Diabetes 2000;37:1595-1607.
[16] Ikeda Y,Suehiro T,Nakamura T,et al. Clinical significance of the insulin resistance index as assessed by homeostasis model assessment. Endocr.J 2001;48:81-86.
[17] Konrad T,Zeuzem S,Toffolo G,et al. Severity of HCV-induced liver damage alters glucose homeostasis in noncirrhotic patients with chronic HCV infection. Digestion 2000;62:52-59.
[18] Narita R,Abe S,Kihara Y,et al. Insulin resistance and insulin secretion in chronic hepatitis C virus infection. J Hepatol.2004 Jul;41(1):132-138.
[19] Mohammad Alizadeh AH,Fallahian F,Alavian SM,et al. Insulin resistance in chronic hepatitis B and C. Indian J Gastroenterol 2006;25:286-289.
[20] Hui JM, Sud A, Farrell GC,et al.Insulin resistance is associated with chronic hepatitis C and virus infection fibrosis progression. Gastroenterology 2003;125:1695-1704.
[21] Shintani Y,Fujie H,Miyoshi H,et al. Hepatitis C virus infection and diabetes:Direct involvement of the virus in the development of insulin resistance. Gastroenterology2004 Mar;126(3):840-848.
[22] Bonora E,Coscelli C,Orioli S,et al. Hyperinsulinemia of chronic active hepatitis: impaired insulin removal rather than pancreatic hypersection. Horm Metab Res 2000;16:111-114.
[23] Custro N,Carrocio A,Ganci A,et al. Glycemic homeostasis in chronic viral hepatitis and liver cirrhosis. Diabetes Metab 2001;27(4 pt 1):476-481.
[24]范小玲,徐道振,李瑾等.北京地区慢性肝炎合并糖尿病临床调查.中华传染病杂志.2001,(19),2:115-116.
[25]时德仁,东传凌,陆立等.肝硬化时糖代谢紊乱与肝细胞胰岛素受体及胰腺细胞HBV DNA表达的关系.中华实验和临床病毒学杂志2003,17(4):372-374.
[26]邵清,成军.丙型肝炎病毒与2型糖尿病关系的研究.世界华人消化杂志,2004;12(1):143-145.
[27] Shintani Y,Fujie H,Miyoshi H,et al. Hepatitis C virus infection and diabetes:Direct involvement of the virus in the development of insulin resistance. Gastroenterology. 2004 Mar: 126(3):840-848.
[28] Rubbia-Brandt L,Quadri R,Abid K,et al. Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3. J Hepatol 2000;33:106-115.
[29]唐星火,卢东红.乙型肝炎相关疾病HBV-DNA载量与细胞免疫功能的研究.广西医科大学学报2007,24(04).
[30]罗国辉,杨冀衡,陆法元等.乙型肝炎病毒感染与IL-6、肿瘤坏死因子水平的相关性.世界感染杂志2005,5(02).
[31] Cosentino F,Assenza GE. Diabetes and inflammation. Herz 2004;29:749-759.
[32] Savage DB,Petersen KF,Shulman GI. Mechanisms of insulin resistance in humans and possible links with inflammation. Hypertension 2005;45:828-833.
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