Molecular characterization and recombinant expression of carboxypeptidase B2 of Anopheles stephensi as a transmission blocking vaccine candidate molecule

Malaria is one of the important infectious diseases in the world. According to the malEra guidelines; developing Vaccines that Interrupt Malaria Transmission (VIMTs) is one of the main objectives to reach the global malaria eradication. In EMR/WHO region, An. stephensi is the main malaria vector. Lavazec et al. reported that the expression pattern of another member of cpb gene family which was nominated as cpb2 is increased after infection with P. falciparum. In this study, the active segment of CPB2 from An. stephensi was expressed in E. Coli origami to provide the basic required information for designing an efficient VIMT in parallel with CPB-1 molecule.
 First, two specific primers with two cutting sites for EcoRI and HindIII restriction enzymes were designed. Then, the active segment of the cpbAs2 was amplified, ligated into the pTG-19 cloning vector, sub-cloned into the pET23a expression vector and transformed into the Ecoli origami bacteria. Four colonies were selected for expression evaluation by SDS-PAGE method and the best clone was selected for further analysis. The expressed protein was confirmed using the western blotting analysis and purified by Ni-NTA purification.
 The active segment of the CPBAs2 was expressed in E. coli origami with 36 kD molecular weight and confirmed by western blotting method.
 This study provides the fundamental information about the molecular and structural features of CPBAs2 as an effective VIMTs candidate molecule alone and in parallel with CPBAs1 molecule to evaluate their synergy in blocking activity.