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【目的】构建融合基因原核表达载体pET-30a/ltB-porB,并表达重组融合蛋白LTB-PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据。【方法】构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平。【结果】在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性sIgA水平随免疫时间呈上升趋势,第42天A450值达0.66,明显高于对照组(P<0.01),效价高达1∶1280;血清中产生的PorB特异性IgG第28天达最高,A450值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P<0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A450:0.57)无明显差异(P>0.05)。LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和SolutionBuffer对照组(P<0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P>0.05)。【结论】重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫。首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫。
【Objective】 To construct recombinant prokaryotic expression vector pET-30a / ltB-porB and express the recombinant fusion protein LTB-PorB. The immunization of female BALB / c mice by nasal feeding was carried out to analyze the immunological activity of the recombinant fusion protein. Provide experimental basis. 【Methods】 The prokaryotic expression vector pET-30a of LTB and PorB fusion gene of LTB and PorB gene was constructed and expressed in E. coli BL21 Protein; Female BALB / c mice were immunized by nasal route to detect humoral and cellular immunity. 【Results】 The recombinant protein with high expression was obtained in E. coli BL21. PorB-specific sIgA levels in reproductive tract mucosa of LTB-PorB group increased with the time of immunization after immunization with mice. On the 42nd day, A450 (P <0.01), the titer was as high as 1:1280. PorB-specific IgG produced in serum reached the highest on the 28th day and the A450 value was 0.60, which was significantly higher than that of LTB and protein solution (P <0.01). The titer was as high as 1: 2560, but there was no significant difference with the PorB control group (A450: 0.57) (P> 0.05). The spleen lymphocyte stimulating index of LTB-PorB group was significantly higher than that of LTB and SolutionBuffer control group (P <0.05), but the level of IFN-γ induced by splenic lymphocytes was not significantly different from that of control group (P> 0.05). 【Conclusion】 Recombinant fusion protein LTB-PorB can induce high level of humoral immunity and a certain level of cellular immunity after immunization of female BALB / c mice via nasal route. For the first time confirmed mucosal adjuvant LTB can help PorB induced mice to produce high levels of genital mucosal immunity.