Construction Of A Recombinant Vector PBV 220/PfCMR-IL-2, Its Expression And Characterization Of The Expressed Products

Malaria remains a leading cause of human morbidity and mortality due to the inability of insecticides and chemotherapy/chemoprohylaxis to eliminate the Anopheline mosquito vectors or disease caused by protozoan parasite belonging to genus Plasmodium. In an effort to develop new methods of control, vaccines targeted to the various stages of the complex life cycle of Plasmodium have been developed. While an effective malaria vaccine is not yet available, considerable insight has been gained in the complex, multiple immune mechanisms induced by the different developmental stages of these parasites, their complex antigenic makeup and their variability. Recent experimental approaches such as multiple antigen peptides (SPf66), recombinant live vectors, and new more potent adjuvants are considered for the development of more effective malaria vaccine formulations.In order to obtain an efficacious polyvalent malaria vaccine with multiprotective functions and high efficient immunological activities, the present study was carried out in our laboratory in which the hybrid gene PfCMR containing CSP, MSA₁ MSA₂ and RESA epitopes of Plasmodium falciparum was recombinated with human interleukin-2 (IL-2) gene and the expression vector pBV 220/PfCMR-IL-2 was constructed. The expressed products were isolated and identified by SDS-PAGE, Western blot, Dot ELISA, T lymphocyte transformation test and CTLL -MTT methods.The plasmid pWR 450-1/PfCMR was digested using restriction endonucleases EcoR1 and Sal1. The hybrid gene fragment PfCMR was recovered by 5% PAGE. PfCMR was cut off the stop codon, 255bp long and inserted into pBV220/IL-2 plasmid at the upstream of human IL-2 gene EcoRl site. The recombinant clones containing the hybrid gene PfCMR-IL-2 were screened by ampicillin and PCR technique.The positive clones were further identified with restriction endonuclease treatment, so that the PfCMR be confirmed to be inserted at the right side in the recombinant vector. The recombinant vector pBV220/PfCMR-IL-2 was used for the expression of protective antigens of P. falciparum and human IL-2.E. coli DH_5a was transferred with pBV220/PfCMR-IL-2, the genetically engineered bacteria were shaken at 30 degree centigrade and expressed at 42 degree centigrade. The exogenous gene products accounted for 37% of the total bacteria proteins. Biologically active IL₂ of the protein (molecular weight 25kDa) was upto 5×10⁵ u/l. Moreover, analysis by Western blot and Dot ELISA demonstrated that the expressed protein could strongly and specifically react with poly-clonal antibody against P.falciparum. T lymphocyte transformation test also confirmed the ability to induce the proliferative response of the Balb/c mouse spleen cells when stimulated by the expressed protein in vitro. Whereas the proliferative response of the immunized spleen cells from Balb/c mice was remarkably higher than that of the normal ones, the transformation percentages were 27.45%±2.69 and 3.20%±1.11 respectively.