RESUMO
The interaction between recombinant Fab57P and the coat protein of tobacco mosaic virus was studied using quantitative structure-activity relationship (QSAR) method. The development of quantitative multivariate model has shown to be a promising approach for unraveling protein-protein interactions by designed mutations in peptide sequence. This approach makes it possible to stereo-chemically determine which residue properties contribute most to the interaction. A set of side-chain descriptors was proposed and applied in structural characterization of three positions (positions 142, 145 and 146) in the peptide antigen. Quantitative sequence-kinetics relationship (QSKR) models describing the dissociation rates (log k(d) ) were developed successfully using orthogonal signal correction-partial least squares method. The results showed that peptides will have high log k(d) values when the amino acids in position 142 and 145 have high net charge index, and when residue 145 has high hydrophobicity and residue 146 has low hydrophobicity.
Assuntos
Anticorpos Antivirais/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/genética , Interações Hidrofóbicas e Hidrofílicas , Cinética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
<p><b>OBJECTIVE</b>To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression.</p><p><b>METHODS</b>A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot.</p><p><b>RESULTS</b>HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1.</p><p><b>CONCLUSION</b>The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.</p>
Assuntos
Vacinas Anticâncer , Proteínas do Capsídeo , Genética , Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Papillomavirus Humano 16 , Genética , Proteínas Oncogênicas Virais , Genética , Proteínas Recombinantes de Fusão , GenéticaRESUMO
A set of 70 peptides with affinity for the class I MHC HLA-A*0201 molecule was subjected to quantitative analyses of structure-affinity relationship based on the SCORE function with good results (r(2)=0.6982, q(2)=0.6188, RMS=0.280). Then the 'leave-one-out' cross-validation (LOO-CV) and an outer test set including 18 outer samples were used to validate the QSAR model, and the results show that the QSAR model has good predictability for outside samples. Statistical analysis showed that the hydrophobic and hydrogen bond interaction played a significant role in peptide-MHC molecule binding. The study also provided useful information for structure modification of CTL epitope, and laid theoretical base for molecular design of therapeutic vaccine.