摘要
在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,研究酵母表达的该乙脑病毒(Japaneseencephali-tisvirus)E蛋白主要抗原片段与结核杆菌热休克蛋白70(Hsp70)的融合蛋白以及该抗原肽与Hsp70上的一个功能域-肽连接区(Peptidebindingdomain,以下简称BD)融合形成的蛋白,用这三种蛋白分别免疫BALB/c小鼠,以酵母单独表达的E蛋白主要抗原片段免疫作为对照,比较它们对小鼠抗E蛋白主要抗原片段特异性的细胞免疫和体液免疫的影响。采用腹腔内注射蛋白的方法免疫小鼠,主要从IL-2的mRNA水平,淋巴细胞的增殖和抗体滴度这三个方面进行比较,最后我们得出E-BD融合蛋白在免疫效果方面比E-Hsp70略好一些,所以在本试验中肽连接区是完全可以代替Hsp70独立行使其佐剂功能。
Further investigation of this interesting adjuvant-free carrier effect is necessary to ascertain whether peptide-binding portion alone can replace the whole M. tuberculosis Hsp70 acting as carrier. In our study, we selected a portion of E protein with higher index of antigenic determinants dependent on analysis of computer software and con-structed two chimeric vectors of pPICZα-E-Hsp70 and pPICZα-E-BD. Vectors were transformed into yeast X-33 by electroporation. Expression of fusion protein in yeast was induced by the addition of methanol every 24 hours and analysed by SDS-PAGE and Western blotting. We produced E-HspT0 fusion protein at a yield of 33 mg per litter of culture and E-Bindingdomain fusion protein at a yield of 97 mg per litter of culture in methylotrophic yeast Pichia pastoris, with specific antigenicitys. To compare the immune response induced by E-HspT0 fusion protein with that induced by E-bindingdomain, mice were immunized i.p. on day 0 and day 21. Mice immunized with E-bindingdomain had higher amount of mIL-2 and titers of antibody than that of mice immunized with E-HspT0 fusion protein. But their proliferation of lymphocytes was almost the same. We concluded that peptide-binding portion alone can replace the whole M. tuberculosis Hsp70 acting as carrier. Besides, this vector in Pichia pastoris avoided inclusion bodies formed in Escherichia coli and complex purification. Moreover, it ruled out contamination of LPS.
出处
《中国病毒学》
CSCD
2005年第4期357-361,共5页
Virologica Sinica
