افزایش میزان بیان پروتئین متصل شونده به دافی در پلاسمودیوم ویواکس به عنوان پروتئین کاندید واکسن مرحله خونی مالاریا

Abstract Background: The cysteine rich region II of Plasmodium vivax Duffy Binding Protein (PvDBP-II) is essential during merozoite invasion into the human erythrocyte and because of this biological importance, PvDBP-II is a vaccine candidate and a target for protective immunity against vivax malaria. In the present study we improved the yield of this protein by using E. coli (M15/BL21) expression system, which is easier for handling than genetic engineering approaches. Methods: After cloning of PvDBP-II in pGEM T easy vector, sub-cloning was carried out in pET 28a, pQE30 and pET24d expression vectors. Then expression was performed on the 1mM IPTG-induced M15 (pREP) and BL21(DE3) strains of E. coli containing expression constructs in different expression conditions such as different culture medias and temperatures. Purification was performed by metal affinity chromatography using Ni-NTA agarose under denaturing conditions. Elutes were separated on SDS-PAGE and western blotting was carried out to detect rPvDBP-II. Yields of purified rPvDBP-II were estimated by measuring optical density at 280 nm. Results: Expression of rPvDBP-II was successfully performed using M15-pQE30 expression system at OD600 = 0/6, 1mM IPTG, in LB2 and 37oC. Size of the recombinant protein on SDS-PAGE was 47 KD (a.a 198-588) and the highest amount of expression was 4h after induction. Western blotting with anti-His antibodies and human P. vivax infected serum, confirmed expression of PvDBP-II. Expression of PvDBP using M15-pQE30 yielded about 1200 μg/ml. Conclusion: In the present investigation, production of expressed PvDBP-II in E. coli (M15/BL21) expression system, in comparison with other two expression system, was in sufficient amounts for further evaluation without using complicated and expensive methods, with least process of transformation. Such a recombinant protein is highly needed for development of a recombinant vaccine and sero-epidemiological study of P. vivax malaria. Keywords: Type Plasmodium vivax, Duffy Binding Protein, Expression, pQE30-E.coli Expression system.