Optimization Of PCR Conditions For Detection Of Hepatitis Virus And Research On The Interaction Of Phycobiliprotein

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.