Bring Improved HBV pre-S1 Gene Expression Vector Construction and Correlation
|School||Luzhou Medical College|
|Keywords||HBVpre-S1 Gene mutation Eukaryotic expression vector Yeast cells|
Objective: liver disease is a common disease worldwide, existing treatments are not ideal, gene therapy may be the treatment of the most effective methods. However, the slow progress in gene therapy of liver disease, the most important reason is the lack of efficient, liver cell-specific carrier. Therefore, the development of efficient hepatotropic gene therapy vector, is the key to substantive progress in gene therapy of liver disease. containing HBV pre-S1 protein and liver cell membrane binding sites, this locus is located in the first 21-47 amino acids is an important site of immune-mediated liver cells, B cells and T cells can be expression of the peptides receptor, the mutant virus as long as this section intact have contagious. In this study, using recombinant DNA technology, the the hepatotropic ingredients HBV surface protein gene coding for non-immunogenic modify, build a portable improved HBVpre-S1 gene expression vector Next hepatotropic recombinant adeno-associated virus micro Preparation of the ball to provide the basis of the experimental study. : The DNA of the the HBV epidemic strain adr as a template, PCR extraction and amplification of the gene encoding HBV pre-S1 of the pre-S1 then extracted HBVpre-S1 gene using site-directed mutagenesis to extend by two independent PCR amplification reaction Construction of preset mutations in the overlap region, while there in the two amplified fragment, obtained by mixing the two overlapping DNA fragments, respectively in conjunction with the end of the two original fragments with two primers for the second PCR amplification, a strong rigid proline replaced with flexible larger alanine HBV pre-S1 gene mutation. Mutant HBV pre-S1 gene fragments by double digestion and transformed into DH5a competent cells prepared in the phenol / chloroform extraction and purification, the recombinant plasmid sequencing using the lithium acetate method into yeast cells AH109 decking SD / Trp filter. Pick (2-3) mm size of the colonies after overnight culture, extraction of yeast proteins, the expression product was analyzed by a conventional method, and the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot Analysis. Results: out the HBV epidemic strain adr HBVpre-S1 gene fragment was successfully amplified and amplified fragment of approximately 357 bp PCR product by 1.5% agarose gel electrophoresis analysis showed that the gene sequencing results show that the sequence is completely correct, in line with the expected fragment, and nothing more specifically amplified phenomenon. Fragment overlap extension method, successfully replaced by a strong rigid proline flexible alanine completed HBVpre-S1 gene mutations. Respectively with the enzymes EcoRI and PstI restriction endonuclease double digestion, connected to the same digested pGBKT7 plasmid vector by restriction enzyme digestion results are correct, successfully constructed recombinant plasmid pGBKT7-HBVpre-S1. SD / Trp medium transformed yeast using the lithium acetate method selection and screening. Training a few days later, picked colonies for PCR amplification HBVpre-S1 protein gene, the results showed a successful conversion. Extract the untransformed the plasmid transformed yeast plasmid proteins by SDS-PAGE and Western blot analysis, the results show that control expression and transformation the pGBKT7-HBVpre-S1 blot analysis showed obvious purpose strip and non-hybrid with. Conclusion: 1. Successfully amplified gene fragment HBVpre-S1. 2 by PCR in vitro site-directed mutagenesis, right locus CCC successfully directed mutagenesis to the GCC, a single amplified bands appeared at 357 bp near cubic PCR reaction, the successful elimination of the immunogenicity of the HBV pre-S1 protein. 3 double digested with restriction endonuclease enzymes EcoRI and PstI, successfully constructed recombinant plasmid pGBKT7-HBVpre-S1. 4 the recombinant plasmid pGBKT7-HBVpre-S1 transformed yeast cells AH109, ??transformation on SD / Trp medium.