[Influence of JEV E-HSP70 (Mycobacterium tuberculosis) fusion protein on immune response in BALB/c mice].

JEV infection can cause severe central nerve system disease which result in high mortality or developing permanent neurological sequelae in more than half of the survivors. The envelope (E) protein of JEV is the major antigen peptide fused to it. A recombinant hsp70 protein expression vector pPICZalpha-E-HSP70 in methylotrophic yeast Pichia pastoris was developed that permits major antigenic segment of JEV E protein fused to the amino terminus of M. tuberculosis hsp70. This core vector avoided inclusion bodies formed in Escherichia coli and complex purification. Moreover,it ruled out contamination of LPS. Two other vectors pPICZalpha-E and pPICZalpha-HSP70 were also constructed. The two vectors were constructed by routine molecular technique. All vectors were transformed into yeast X-33 by electroporation. Expression of the fusion protein in yeast was induced by the addition of methanol every 24 hours and analysed by SDS-PAGE and western blot. Major antigenic segment of E protein was produced at a yield of 290 mg per litter of culture, hsp70 protein at a yield of 178 mg per litter of culture and E-HSP70 fusion protein at a yield of 33 mg per litter of culture in methylotrophic yeast Pichia pastoris. To examine cell and body immune response after BALB/c mice were immunized with E-hsp70 fusion protein expressed in Pichia pastoris, there were three groups with ten mice in each group. 5.7 microg (50pmol) of E-hsp70 fusion protein, 2.2 microg (50pmol) major antigenic segment of E protein and a mixture of hsp70 and major antigenic segment of E protein (1:1) including 3.5 microg (50pmol) Hsp70 and 2.2 microg (50pmol) major antigenic segment of JEV E protein were used per mouse i.p. on day 0 and day 21. The production of mIL-2 was quantitated by semi-quantitative RT-PCR. Besides, proliferation of lymphocytes was measured by MTT and titers of antibody was determined by ELISA. These data show that the fusion protein is a more powerful antigen than major antigenic segment of JEV E protein. So it also illustrates the effectiveness of hsp70 in eliciting a humoral and cellular response to an attached molecule in the absence of adjuvant and affirms the potential utility of hsp70 in vaccine development.