Bring Improved HBV Pre-S1 Gene Expression Vector Construction And Correlation

Abstract:Objective:Liver disease is a common disease in the world, existing treatments are not ideal, gene therapy is the most effective treatment. However, the current slow progress in gene therapy of liver disease, of which the most important reason is lack of efficient, liver cell-specific carrier. The developed and efficient hepatotropic gene therapy vector is the key to the liver gene therapy made substantial progress. pre-S1 protein contains HBV and liver cell membrane binding sites, the sites identified in section 21～47 amino acids, the region is an important part of immune-mediated, liver cells, B cells and T cells all can be expressed for this peptide receptors, the peptide is rather conservative, mutation of the virus as long as in this whole section has infectious. In this research, we use recombinant DNA technology, making the surface protein of hepatotropic HBV components in the gene encoding for the modified non-immunogenic, building a portable modified HBVpre-S1 gene expression vector, providing experimental research base for the next hepato-tropic of recombinant adenoassociated virus micropreparation.Method:Using HBV strains adr of the DNA as a template, PCR ampli- fication extracted and encoded HBVpre-S1, and then used site-directed mutagenesis program which segment overlap extension method makes a more rigid flexible and strong large proline alanine substitutied.Completed HBV pre-Sl gene mutations. The mutated HBVpre-S1 gene fragment by restriction enzyme digestion, trans- formed into DH5a competent cells of prepared, in the phenol/chloroform extraction and purification, the recombinant plasmid was send to sequence, the use of transformed yeast cells AH 109 by lithium acetate method then transformed ceiling on SD/Trp screening. Pick (2～3) mm size of the colonies after overnight culture, the yeast protein extraction, analyzed by conventional methods and the expression product of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Westernblot analysis. Results:The HBV strains amplified adr's HBVpre-S1 gene fragment, PCR products were 1.5% agarose gel electrophoresis analysis revealed that ampli-fied fragments of about 357bp, consistent with the expected fragment, and nothing specific amplification phenomenon By PCR amplification HBV pre-S1 gene sequence analysis showed that sequence is correct. By overlap extension method, using two independent PCR amplification of DNA obtain-ed two overlapping fragments, the default build in the overlap region mutations found in two amplified fragments. Mixed overlapping fragments, respectively with the two combination of the two ends of the original fragment of a second primer PCR, successfully replaced by hard strong flexible proline alanine larger, and completely HBVpre-Sl gene mutations. The restriction endonucleases EcoRI and PstI were digested and connected to the same plasmid vector pGBKT7. By restriction enzyme analysis result is correct. That the recombinant plasmid pGBKT7-HBVpre-S1 was constructed successfully. Transformation of yeast by lithium acetate method after the SD/Trp medium for selecting and screening. Training a few days later, colonies were picked for PCR,the consequence showed that the transformation is success. Analy-sised expression products of the SDS-PAGE and Western blot, extractied plasmid and transformed not into the yeast plasmid proteins for SDS-PAGE and Western blot analysising, showed no expression in control but transform-ed pGBKT7-HBVpre-S1 of the Western blot analysis target band can be seen clearly and without hybrid zone. Conclusion:1.Successfully amplified gene fragments HBVpre-S1.2. In vitro site-directed mutagenesis by PCR,success-fully site-directed ccc mutagenesis to gcc,nearly 357bp displayed a single amplified bands after three reactions, successfully eliminatiated HBV pre-S 1 protein immunogenicity.3.We used restriction endonuclease EcoRI and PstI Double digestion,Plasmid pGBKT7-HBVpre-S 1 is constructed successfully. 4.Plasmid pGBKT7-HBVpre-Sl transformed yeast cells AH109 to which successfully shifted in the SD/Trp medium.