乙脑E蛋白与结核杆菌HSP70的融合蛋白对BALB/c小鼠特异性免疫的影响

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

研究酵母表达的乙脑病毒(JapaneseEncephalitisvirus)E蛋白主要抗原片段与结核杆菌热休克蛋白70 (hsp70 )形成的融合蛋白对小鼠细胞免疫和体液免疫的影响。采用腹膜内注射蛋白的方法免疫小鼠,以直接免疫E蛋白主要抗原片段和以E蛋白主要抗原片段和单独表达的hsp70两者均以5 0pmol的量进行混合后的蛋白免疫的小鼠作为对照,用半定量RT PCR检测细胞免疫因子IL 2mRNA的水平,IL 2是细胞介导的免疫反应和巨噬细胞激活中的关键分子,MTT法检测淋巴细胞的增殖情况以及通过ELISA检测抗体水平,从这3个方面来评价融合与未融合蛋白以及等量混合后的免疫效果。结果E蛋白主要抗原片段与结核杆菌hsp70重组后,mIL 2 ,淋巴细胞的增殖以及抗体水平均较重组前明显升高,因此,以乙脑E

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 to elicit neutralizing antibodies and protection in hosts. Hsp70 can potentiate specific immune response to some antigenic peptide fused to it. A recombinant hsp70 protein expression vector pPICZα-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 pPICZα-E and pPICZα-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 inPichia pastoris, there were three groups with ten mice in each group. 5.7μg (50pmol) of E-hsp70 fusion protein, 2.2μg (50pmol) major antigenic segment of E protein and a mixture of hsp70 and major antigenic segment of E protein(1∶1) including 3.5μg (50pmol) Hsp70 and 2.2μg (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 quantitiated 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.