淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性

Construction, expression and immunocompetence of Neisseria gonorrhoeae porB and Escherichia coli ltB fusion gene

【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 prokaryotic fusion gene expression vector pET-30a /ltB-porB,express the recombinant fusion protein LTB-PorB and analyze the immunocompetence of the recombinant fusion protein in female BALB /c mice through intranasally immunization.MethodsB subunit of Escherichia coli heat-labile enterotoxin（LTB） and Neisseria gonorrhoeae Porin B（PorB） fusion gene,LTB gene and PorB gene were cloned into prokaryotic vector pET-30a.The recombinants were identified by Polymerase Chain Reaction（PCR）,enzyme digestion and DNA sequencing,and then expressed efficiently in Escherichia coli BL21 in the form of inclusion bodies.The renatured recombinant proteins had antigenicity,which was confirmed by Western blot.Female BALB /c mice were inoculated with renatured recombinant proteins without endotoxin through intranasally immunization at the days 0,14,28.Next,humoral immunoresponse and cellullar immunologic response were detected in female BALB /c mice by enzyme linked immunosorbent assay（ELISA） and methyl thiazolyl tetrazolium（MTT） colorimetric assay.ResultsThe level of PorB specific sIgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in LTB-PorB group,A450of sIgA in LTB-PorB group was 0.66 at the day 42,which was significantly higher than controls（P 0.01）,and the titer was up to 1 ∶ 1280.A450 of serum IgG in LTB-PorB group was 0.60 at the day 28,which was significantly higher than the LTB and the Solution Buffer controls（P 0.01）,and the titer was up to 1∶ 2560.However,the IgG between LTB-PorB group and PorB control（A450 ∶ 0.57）had no significant difference（P 0.05）.Stimulation index of the splenic lymphocyte in LTB-PorB group was significantly higher than the LTB and the Solution Buffer controls（P 0.05）.But the level of IFN-γ induced by splenic lymphocyte between LTB-PorB group and controls had no significant difference（P 0.05 ）.Conclusion The recombinant fusion protein LTB-PorB could induce high level of humoral immunoresponse and slightly cellullar immunologic response in female BALB /c mice through intranasally immunization.For the first time to our knowledge,the mucosal adjuvant LTB could assist PorB to induce high level of mucosal immune response in the genital tract mucosa of mice.