【作者】 巩雪俐；

【导师】 张建龙； 成军；

【作者基本信息】 新疆医科大学 ， 病理学与病理生理学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原