Asia 1型FMDV复合多表位亚单位疫苗在毕赤酵母中的表达及其免疫原性

Expression of Multi-epitope Subunit Vaccine of Foot-and-mouth Disease Virus Type Asia 1 in Pichia pastoris and Immunogenicity of Expressed Product

目的构建以霍乱毒素B亚单位(Cholera toxin B subunit,CTB)为分子佐剂的Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)复合多表位亚单位疫苗,在毕赤酵母中进行表达,并评价其免疫原性。方法构建毕赤酵母表达质粒pPIC9K-P1/2A-CTB-TEpi,转化至毕赤酵母GS115中,甲醇诱导表达,表达产物经硫酸铵沉淀法纯化后,进行SDS-PAGE和Western blot分析。将纯化的P1/2A-CTB-TEpi蛋白和FMD灭活疫苗分别免疫BALB/c小鼠,以注射PBS作为对照,分别于0、21d各免疫1次。每周尾静脉采血,分离血清,ELISA法检测血清抗体水平;第2次免疫后10d,处死小鼠,分离脾脏淋巴细胞,进行淋巴细胞增殖试验和IFNγELISPOT检测。结果目的蛋白在毕赤酵母中经诱导表达后可分泌到培养上清中,在相对分子质量约为82600(P1/2A)和40500(CTB-TEpi)处可见2条特异性蛋白表达条带,占上清液中可溶蛋白的21%。纯化后的目的蛋白P1/2A和CTB-TEpi的比例约为1∶2,且具有良好的反应原性。P1/2A-CTB-TEpi蛋白免疫小鼠产生了较强的体液免疫和细胞免疫反应,其特异性血清抗体水平与灭活疫苗组差异无统计学意义(P>0.05),而淋巴细胞增殖水平和IFNγ分泌水平显著高于灭活疫苗组(P<0.01)。结论构建了以CTB为分子佐剂的Asia1型FMDV复合多表位亚单位疫苗,其免疫原性良好,为FMD多表位疫苗和亚单位疫苗的研究奠定了基础。

Objective To construct a multi-epitope subunit vaccine of foot-and-mouthdisease virus（FMDV）type Asia 1,using cholera toxin B subunit（CTB）as a molecular adjuvant,express in Pichia pastoris and evaluate its immunogenicity.Methods Recombinant plasmid pPIC9K-P1/2A-CTB-TEpi was constructed and transformed to P.pastoris GS115 for expression under induction of methanol.The expressed product was purified and analyzed by SDS-PAGE and Western blot.BALB/c mice were immunized with the purified P1/2A-CTB-TEpi protein and inactivated FMDV vaccine respectively,twice on days 0 and 21,using PBS as control.The venous blood of immunized mice was collected every week,from which sera were separated and determined for antibody level by ELISA.The mice were killed 10 d after the 2nd immunization,of which splenic lymphocytes were isolated for proliferation test and determined for IFNγ by ELISPOT.Results The target protein was expressed in P.pastoris and secreted into the culture supernatant,which showed two bands with relative molecular masses of about 82 600（P1/2A）and 40 500（CTB-TEpi）respectively on SDS-PAGE profile and contained 21% of soluble protein in supernatant.The ratio of P1/2A to CTB-TEpi was about 1：2 in the purified target protein which showed high reactogenicity.P1/2A-CTB-TEpi protein induced high humoral and cellular immune responses in mice.The serum antibody level of mice immunized with the protein showed no significant difference with（P 〉0.05）,while the lymphocyte proliferation and IFNγ secretion levels were significantly higher than those with inactivated FMDV vaccine（P〈 0.01）.Conclusion The multi-epitope subunit vaccine of FMDV type Asia 1,using cholera toxin B subunit（CTB）as a molecular adjuvant,was constructed and showed high immunogenicity,which laid a foundation of study on multi-epitope and subunit vaccines of FMDV.