Human defensin-β3 mutant gene and its expression in E. coli fusion.docVIP

 PAGE \* MERGEFORMAT 22 Human defensin-β3 mutant gene and its expression in E. coli fusion 【Abstract】 Objective To extract the normal human skin tissue β   3-defensin gene after site-directed mutagenesis in the fusion expression of E.coli. Method from normal human skin tissue extract total RNA, was amplified by RT  PCR encoding β   3-defensin mature peptide cDNA sequences, sequencing is correct in identifying a suitable mutations, design with mutation primers , using PCR overlap extension method, β   3-defensin mature peptide cDNA sequences of site-directed mutagenesis, the mutant product was cloned into pUC18 for sequencing and the fusion protein in E.coli. The results of sequencing results showed that the RT  PCR derived from β   3-defensin mature peptide sequence reported in GeneBank encoding human β   3-defensin mature peptide cDNA sequence identical. After mutation of β  mature peptide of defensin- 3 No. 29 by the CAG glutamine codon mutation of the arginine codon CGA, the remaining nucleotide sequences were not changed. The mutated β   3-defensin gene expression in E.coli was induced mutations seen after 3 ~ 5h β   3-defensin protein. Conclusion successfully constructed and expressed the β  defensin- 3 mutant gene, in order to further mutants eukaryotic expression, biological activity of the foundation. Keywords: β  human defensin- 3; site-directed mutagenesis; gene cloning; sequencing; gene expression Construction and expression of a site  directed mutant gene of human beta  defensin  3 ABSTRACT Objective To extract the gene encoding human beta  defensin  3 (hBD  3) from human foreskin tissue, make a site  directed mutagensis and expression with the mutant gene in E.coli. Methods The total RNA extracted from human foreskin tissue and the sequence of cDNA encoding hBD  3 was amplified by RT  PCR. After sequencing, a pair of mutant primers was designed and site  directed mutation was made by over