O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB、STⅠ肠毒素基因融合表达产物的免疫应答

The Immune Responses of the Fusion Protein Consisted of Two Copies of T-cell and B-cell Epitopes of Food and-mouth Disease Virus VP1 Type O and LTB and STⅠ Enterotoxins of Escherichia coli

合成O型口蹄疫病毒VP1蛋白中与细胞免疫（21～40表位肽）及体液免疫（141～160表位肽）相关的基因序列2020VP1，运用基因工程技术构建了含有肠毒素大肠杆菌LTB、STⅠ基因及双拷贝2020VP1的融合表达载体r2020-B-2020-STⅠ，转化宿主菌BL21（DE3）RIL后的表达产物经SDS—PAGE分析，结果显示重组融合蛋白的分子量约为45kDa，表达量较高。ELISA实验结果显示，融合蛋白能与霍乱毒素（cholera toxin）CTB抗体特异结合。动物实验表明，融合蛋白能够诱发兔体产生较强的FMDV中和抗体，免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应，说明融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应；同时，融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击，免疫兔体能够产生STⅠ中和抗体，且融合蛋白不具STⅠ毒性，证明融合蛋白具有良好的LTB、STⅠ免疫原性。实验结果表明，此融合蛋白具有开发成为口蹄疫及肠毒素腹泻联合疫苗的应用价值。

Abstract ：A nucleotides fragment 2020 VP1 related to T-cell epitope （aa21-40） and B-cell epitope （aa141-160） of VP1 of Food-and-mouth disease virus（FMDV）type O was synthesized, and the recombined expression vector r2020-B-2020- ST [ was correctly constructed, which was used to express the fusion protein consisted of two copies of aa21-40, aa141-160 of FMDV VP1 and LTB and ST [ Enterotoxins of Escherichia co//. The fusion protein with molecular weight of about 45 kDa was successfully expressed in E. coli BL21 （DE3） RIL at high level and confirmed by SDS-PAGE. The purified fusion protein could be specifically recognized by CTB antibody. The purified protein was then used to vacci- nate guinea pigs, rabbits and Balb/c mice at 6-8 weeks age, and immune responses were finally observed. The fusion protein could induce proliferation of spleen T cells in vaccinated guinea pigs and elicit a high level of neutralizing anti- body in rabbits. It could be concluded that the fusion protein could activate FMDV-specific cellular immune-response and humoral immune-response simultaneously. At the same time, the vaccinated mice could survive challenge of Enterotoxigenic Escherichia coli C83902, and the sera of vaccinated rabbits could neutralize the ST Ⅰ toxin. Moreover, the fusion protein had no ST Ⅰ toxicity, indicating that the fusion protein had LTB and ST Ⅰ immunogenicities. Therefore, it can be concluded that the fusion protein could be explored as a potentially efficient FMDV and ETEC vaccine.