RESUMO
OBJECTIVE: To clone and express a novel protein analogous to TIP (T cell immunomodulatory protein) of Plasmodium berghei ANKA and prepare its polyclonal antibody. METHODS: The PbTIP encoding nucleotide sequence was searched from the Plasmodium berghei genomic database and amplified by PCR. The gene was sub-cloned into prokaryotic expression vector pGEX4T-1 and expressed in E.coli BL21 (DE3). After induction by IPTG, the expression of PbTIP-GST fusion protein was characterized by SDS-PAGE and Western blotting. The inclusion bodies of GST-PbTIP fusion protein were injected into BALB/c mouse. Anti-sera were identified by indirect fluorescent antibody test and western blotting. RESULTS: The PbTIP-GST fusion protein was successfully expressed in the form of inclusion bodies, by controlling the temperature and concentration of IPTG. Anti-PbTIP-GST sera were acquired with high titer. The sera specifically recognized the PbTIP with a band of 60 000 in P.berghei infected erythrocyte protein. CONCLUSION: PbTIP/GST fusion protein and polyclonal antibody have been obtained.
Assuntos
Anticorpos Antiprotozoários/imunologia , Plasmodium berghei/genética , Proteínas de Protozoários/metabolismo , Animais , Clonagem Molecular , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
AIM: To screen the possible HLA-A*0201 restricted low-affinity CTL epitopes derived from HIV-1 pol antigen and to predict and identify the possible change of the affinity between epitope and the HLA-A*0201 molecule when the epitope is modified. METHODS: HLA-A*0201 restricted low-affinity CTL epitopes were predicted by CTL epitope prediction software based on super motif, proteasome cleavage probability, HLA affinity and so on. The candidates were modified acid substitution and analyzed by computer. T2 cells were used to determine the peptide by amino binding affinity and HLA-A*0201-peptide complex stability. RESULTS: Among the three predicted peptides by softer ware, YVSLSFPQI (pol52-60Y1), YVSQIIEQL pol(673-681Y1), YIQKETWEA(pol548-556Y1)could bind to HLA-A*0201 with high affinity, and the dissociation time of 50% HLA-A*0201 peptide complex was over to 8 h. CONCLUSION: Our results suggest that the three predicted peptides, as modification, might be HLA-A*0201 restricted CTL epitopes.