RESUMO
The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.
Assuntos
Humanos , Sequência de Aminoácidos , Fármacos Anti-HIV/síntese química , Linhagem Celular , Dicroísmo Circular , Cisteína/química , Dissulfetos/química , Proteína gp41 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , Concentração Inibidora 50 , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Objective To construct recombinant plasmid with p24 and gp41 gene, express fusion protein in E.coli. Methods To design primer with restriction endonuclease position,and amplify p24 and gp41 by RT-PCR, link both into pMD18-T vector.To choose correct clone with target gene.Then p24 fragment will be cleaved and linked into pMD18-T vector within gp41 gene. Both post-linked gene will be cleaved and linked into pET21a vector. The vector will be transformed into E.coli. And protein is highly effective expressed in E coli. Western blotting proved that the expressed product could react with 6?his antibody. Result Fusion protein p24-gp41 is highly effective expressed in E.coli. Conclusion Fusion protein p24-gp41 is highly effective expressed in E.coli in pET21a vector.