猪源A组轮状病毒重组质粒pW425et-Vp7在乳酸菌中的表达及免疫原性分析

Expression and Immunogenicity Analysis of Recombinant Plasmid pW425et-Vp7 of Porcine Rotavirus A in Lactobacillus

摘要:

摘要：【目的】在乳酸菌中表达猪源A组轮状病毒重组质粒pW425et-Vp7，并检测其抗原性，为进一步制备猪轮状病毒乳酸菌疫苗奠定基础。【方法】摸索电转化条件，将已构建的重组质粒pW425et-Vp7转化至thyA基因缺陷型的嗜酸乳杆菌中。筛选阳性克隆，采用SDS-PAGE和Western blot检测其表达情况。并将BALB/c小鼠口服免疫该重组菌，间接ELISA检测其免疫原性。另外，给免疫后小鼠攻以纯化的猪轮状病毒(蛋白含量15 mg/mL),初步检测该重组菌的免疫保护作用。 【结果】Vp7基因在乳酸菌中获得表达，大小约40.77 kDa，且该蛋白可与猪轮状病毒抗体反应，具有反应原性。ELISA检测小鼠肠黏膜样品结果显示免疫后小鼠能产生明显的抗轮状病毒SIgA抗体，表明该重组菌具有较好的免疫原性。且攻毒后，免疫组仅23%的小鼠发生腹泻，初步证明该重组菌对猪轮状病毒的攻击具有较好的保护作用。【结论】在乳酸菌中成功地表达了猪轮状病毒Vp7基因，制备了猪轮状病毒Vp7重组乳酸菌，该重组菌具有较好的抗原性，初步证明其作为疫苗的可行性。

Abstract:

Abstract: [Objective] We tested the antigenicity of porcine rotavirus Vp7 gene expressed in Lactobacillus. [Methods] We studied the condition of electroporation and transformed the plasmid pW425et-Vp7 into thyA gene-mutant L. acidlophilus. Then the positive clone was selected and its expression was detected by SDS-PAGE and Western blot. In our test, the BALB/c mice were drenched by the above-mentioned recombinant strain and SIgA was detected by ELISA. In addition, we also tested the protection of the recombinant strain through attacking porcine rotavirus to the mice immunized (content of virus was 15 mg/mL). [Result] Vp7 gene expressed in Lactobacillus with approximate 40.77 kDa fusion protein was observed by SDS-PAGE. The protein had the reactionogenicity with the antibody of porcine rotavirus through Western blot. We also detected the specific SIgA from the mice immunized by ELISA. Moreover, we observed that only 23% mice had diarrhea after attacking porcine rotavirus, which was immunized by recombinant strain. [Conclusion] Vp7 gene was expressed in Lactobacillus successfully. It had good immunogenicity and protection against porcine rotavirus. Moreover, the test further proved that the recombinant strain could be applied in the study on porcine rotavirus vaccine in the future.