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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 Foot-and-mouth disease virus (FMDV) multi-epitope subunit vaccine of Cholera toxin B subunit (CTB) as molecular adjuvant in Pichia pastoris Expression, and evaluate its immunogenicity. Methods Pichia pastoris expression plasmid pPIC9K-P1 / 2A-CTB-TEpi was constructed and transformed into Pichia pastoris GS115. The recombinant plasmid was induced by methanol. The expressed product was purified by ammonium sulfate precipitation and analyzed by SDS-PAGE and Western blot. BALB / c mice were immunized with the purified P1 / 2A-CTB-TEpi protein and FMD inactivated vaccine, respectively. PBS was injected as a control and immunized at 0 and 21 days respectively. Blood was collected from the tail vein each week, and the serum was separated. Serum antibody levels were measured by ELISA. At 10 days after the second immunization, mice were sacrificed and splenic lymphocytes were isolated for lymphocyte proliferation and IFNγELPOT detection. Results The recombinant protein was expressed in Pichia pastoris and secreted into the culture supernatant. Two specific protein bands were observed at molecular weight of 82600 (P1 / 2A) and 40500 (CTB-TEpi) Accounting for 21% of the soluble protein in the supernatant. Purified target protein P1 / 2A and CTB-TEpi ratio of about 1: 2, and has good reactivity. The mice immunized with P1 / 2A-CTB-TEpi had strong humoral and cellular immune responses, and the specific serum antibody level was not significantly different from that of the inactivated vaccine (P> 0.05), but the level of lymphocyte proliferation And IFNγ secretion levels were significantly higher than inactivated vaccine group (P <0.01). Conclusion The construction of the Asia1 FMDV multi-epitope subunit vaccine with CTB as molecular adjuvant has good immunogenicity and lays the foundation for the study of FMD multi-epitope vaccines and subunit vaccines.