【作者】 张莉华；

【导师】 陈思礼；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 病毒性肝炎是对人类健康危害严重的传染病之一。由于HBV和HCV混合感染不仅是原发性肝癌发生的原因,也是导致肝组织纤维变,加快向肝硬化发展的重要因素。所以研究HBV、HCV的病理机制为临床早期抗病毒干预治疗,控制肝组织纤维化进程,提供了理论依据,对肝癌防治具有重要意义。一般对HBV和HCV检测有免疫学方法和核酸检测法,免疫学方法主要包括放射性标记免疫分析技术、酶免技术分析（ELISA）、时间分辨荧光免疫分析（TRFIA）和化学发光免疫分析（CLIA）等;核酸检测方法主要有斑点杂交技术和PCR技术,其中PCR技术检测存在假阳性和假阴性的情况,本实验通过优化PCR的检测条件达到快速灵敏检测,降低假阳性和假阴性的目的。本实验用已经构建好的pGEM-T-HBV（C区/C基因）和pGEM-T-HCV（核心蛋白）作为PCR检测的质粒模板,通过改变Mg²⁺浓度、退火温度以及采用不同的酶进行PCR检测条件的优化。其中检测HBV病毒时共设了1.5μl-4.0μl共6个Mg²⁺浓度梯度,设了58.0～62.0℃共12个温度梯度,质粒模板稀释浓度从10^-1-10^-9共9个浓度梯度。检测HCV病毒时,共设了1.5～2.0μl共6个Mg²⁺浓度梯度,设了56.0～60.0℃共12个温度梯度,质粒模板稀释浓度从10^-1～10^-9共9个浓度梯度。实验结果证明检测HBV病毒时,4.0μl为最佳Mg²⁺浓度,59℃为最佳退火温度,天源公司的Taq DNA聚合酶灵敏性最高,对质粒浓度的要求达到10^-9。当检测HCV病毒时,1.5μl为最佳Mg²⁺浓度,57℃为最佳退火温度,Bio-Rad公司的iTaq聚合酶灵敏性最高,对质粒浓度的要求达到10^-6。藻胆体（phycobilisome）是存在于蓝藻和红藻中的一类捕光蛋白复合物,它由藻胆蛋白（phycobiliprotein）和连接蛋白组成。藻胆蛋白由藻胆色素（phycobilin）与相应的脱辅基蛋白中保守性半胱氨酸的巯基以硫醚键共价结合而成。藻胆蛋白是一类结构相似的色素蛋白。目前已发现了一种能催化β-CPC84、β-PEC84、α-APC82和β-APC82位半胱氨酸残基与PCB连接的裂合酶基因cpeS,其编号为alr0617。为了研究PCB与层理鞭枝藻藻红蓝蛋白连接的机制β亚基（β-PEC）第84位Cys连接机制,现通过同源性分析,筛选出四个同源性较高的基因片段,即alr0617,all5339, all5292,alr0647。现根据国际标准,将编号为alr0617,all5339,all5292,alr0647基因编码的蛋白命名为CpeS1,CpeT1,CpeS2,CpeT2, CpeT1能分别与CpeT2、CpeS1、PecE形成比较明显的复合物,但是只有CpeT1与CpeT2之间形成大约1:1的复合物。本研究成功构建质粒pBT- CpeS1、pBT- CpeS2、pBT- CpeT2、pBT- 3357、pTRG- CpeS1、pTRG- CpeT₁、、pTRG- 3357基因。将pBT- CpeS₁、pBT- CpeS₂、、pBT- CpeT₂、pBT- 3357和pTRG- CpeT₁;pBT- CpeS₁、pBT- CpeS₂、pBT- CpeT₂和pTRG- 3357;pBT- CpeS₂、、pBT- CpeT₂和pTRG- CpeS₁共转化大肠杆菌,利用细菌双杂交系统,证明pBT- CpeT2、pBT-CpeS2、pBT-CpeS1和pBT-3357的重组子与pTRG-CpeT1的重组子有相互作用,pBT-CpeS2和pBT-CpeS1与pTRG-3357的重组子的蛋白质间有相互作用,而pBT-CpeS2和pTRG-CpeS1间没有相互作用。更多还原

【Abstract】 Hepatitis is one of infectious diseases which could harm human health seriously. The mixed infection of HBV and HCV is not only the causes of primary liver cancer, but also the main factor of making liver tissue fibosis and accelerating the development of liver cirrhosis. Research on the pathological mechanisms of HBV, HCV which provides the theoretical basis of doing early anti-virus treatment and controlingf liver fibrosis process, has great significance for prevention and treatment of liver cancer.General detection methods of HBV and HCV are immunological methods and nucleic acid testing. Immunological methods includes radioactive immune analysis technology, ELISA, TRFIA and CLIA; Nucleic acid testing methods includes speckle hybrid technology and PCR technology,and the PCR technology exists of false positive and false negative situation. This work can approach rapider detection, greater sensitivity, lower false positive and lower false negative through optimizing of PCR test conditions.This work uses the constructed plasmid （pGEM-T-HBV （C District/C gene） and pGEM-T- HCV （core）） as the template, optimizes the PCR test conditions by changing Mg²⁺ concentration, annealing temperature and types of the enzyme,The detection of HBV virus has established a Mg²⁺ concentration gradient from 1.5 to 4.0μl, a annealing temperature gradient from 58.0℃to 62.0℃, and a template plasmid diluted concentrations gradient from 10^-1 to 10^-9. The detection of HCV virus has established a Mg²⁺ concentration gradient from 1.5 to 2.0μl , a temperature gradient from 56.0℃to 60.0℃, and a template plasmid diluted concentrations gradient from 10^-1 to 10^-9. The result of experiments shows that the optimal conditions of HBV virus detection is 4.0μl as the best Mg²⁺ concentration, 59℃as the best annealing temperature. The Taq DNA polymerase （Tiangene company） has the highest sensitivity, which requires optimal plasmid concentration of 10^-9.The result of experiments shows that the optimal conditions of HCV virus detection is 1.5μl as the best Mg²⁺ concentration, 57℃as the best annealing temperature. The iTaq DNA polymerase （Bio-Rad company） has the highest sensitivity, which requires optimal plasmid concentration of 10^-6. Phycobilisomes serve as the light-harvesting antenna in cyanobacteria and red algae. These protein complexes, are primarily composed of phycobiliproteins, a brilliantly colored family of proteins bearing covalently attached, open-chain tetrapyrroles known as phycobilins. In additional, phycobilisomes also contain smaller amounts of linker peptides,which are required for proper assembly and functional organization of phycobilisomes. Phycobilin chromophores are generally bound to apoprotein at conserved positions by cysteinyl thioether linkages to generate phycobiliproteins. Has now found a catalyst toβ-CPC84,β-PEC84,β-APC82 cysteine residues Connected with the PCB crack synthaseβ- APC82 and gene cpeS , its No. alr0617. In order to study the PCB and bedding whip sticks was phycoerythrocyanin PEC No. Cys84 connecting mechanism, subunit connecting mechanism,Is through homology analysis, selected four high homology of the gene fragments, That is, alr0617, all5339, all5292, alr0647. In accordance with international standards, will be No. alr0617, all5339, all5292, alr0647 genes encoding proteins called CpeS1, CpeT1, CpeS2, CpeT2. CpeT1 respectively and CpeT2, CpeS1, PecE a more obvious complex, but only CpeT1 and CpeT2 about 1:1 between the formation of the complex.This study successfully constructed plasmid pBT-CpeS1,pBT-CpeS2, pBT-CpeT2, pBT-3357,pTRG-CpeS1,pTRG-CpeT1,pTRG-3357.pBT-CpeS1,pBT-CpeS2,pBT-CpeT2.pBT-3357 and pTRG-CpeT1; pBT-CpeS1, pBT-CpeS2, pBT-CpeT2 and pTRG-3357; pBT-CpeS2, pBT-CpeT2 and pTRG-CpeS1 Were transformed into E. coli, bacteria use two-hybrid system that pBT-CpeT2, pBT-CpeS2, pBT-CpeS1 and pBT-3357 and the restructuring of pTRG-CpeT1 interactions with the recombinant, pBT-CpeS2,pBT-CpeS1 and pTRG-3357 Restructuring of the interaction between proteins and pBT-CpeS2 and pTRG-CpeS1 no interaction between.更多还原