空肠弯曲菌PEB1-LTB DNA疫苗构建及其对小鼠免疫保护性的研究

目的:研究粘膜佐剂大肠埃希菌不耐热肠毒素B亚单位(LTB)辅助的空肠弯曲菌(C.jejuni)外膜蛋白PEB1基因重组DNA疫苗诱导小鼠免疫应答水平。方法:构建pcDNA3.1(-)-PEB1-LTB真核重组表达载体,转染Hela细胞,Westernblot鉴定蛋白表达。滴鼻免疫BALB/c小鼠,末次免疫后2周,测定小鼠血清中IgG、IgA及气管、小肠粘膜冲洗液sIgA抗体;脾细胞培养上清中IFN-γ、IL-4水平。末次免疫后4周,采用空肠弯曲菌重复攻击的方式进行灌胃攻击,攻击后,根据动物疾病指数评价疫苗临床保护率。结果:构建的重组表达质粒能在Hela细胞内表达,重组蛋白。疫苗免疫小鼠后,不仅诱导了高水平血清IgG、IgA抗体,而且诱导了高水平粘膜sIgA抗体。其诱导的特异性免疫应答能有效保护免疫后小鼠免遭空肠弯曲菌的感染攻击。结论:构建的DNA疫苗经粘膜免疫能诱导小鼠产生较高水平的特异性免疫应答,能有效预防空肠弯曲菌感染。

Construction of Campylobacter jejuni PEB1-LTB DNA vaccine and study on its immune responses in mouse

Objective:To investigate the specific immune response induced by DNA vaccine with Campylobacter jejuni gene PEB1 fused with B subunit of Escherichia coli heat-labile enterotoxin B(LTB) in mice.Methods:Target genes of PEB1,LTB and PEB1-LTB were amplified by polymerase chain reaction(PCR) and cloned into eukaryotic vector pcDNA3.1(-),respectively.The vectors were transfected into Hela cells,and expressed proteins were checked by SDS-PAGE and Western blot.The BALB/c mice were intranasally immunized with DNA vaccines.Two weeks after the final immunization,the antibodies and cytokines were detected,including the specific IgG and IgA antibodies in serum,sIgA in mucosa douche,IFN-γ and IL-4 in splenocyte culture supernatant.Mice were attacked repeatedly through intragastric administration of C.jejuni at the 4th week after the immunization and the protective efficacy were evaluated by detecting the Animal Disease Index.Results:The PCR fragements of PEB1,LTB and PEB-LTB were about 780,370 bp and 1 180 bp,which had high homology with those related sequences in GenBank.The recombinant plasmids pcDNA3.1(-)-PEB1-LTB were constructed.The recombinants were expressed in Hela cells,which had a molecular weight(Mr) 43×103.Western blot showed the protein had a specific reaction with C.jejuni antiserum.The specific antibodies of IgG(0.636±0.101),IgA(1.092±0.145)in serum,and sIgA(0.889±0.165)in mucosa douche,cytokines IFN-γ [(2021±256) pg/ml] and IL-4[(224±35) pg/ml] in splenocyte culture supermatant in group D were all higher than those in other groups(P<0.05).The DNA vaccines induced strongest level of protection in BALB/c mice against challenge with C.jejuni.Conclusion:The results of this study indicate that mucosal immunization of the vaccines could induce high level specific immune response.The constructed DNA vaccines could induce significant protective immunity against C.jejuni challenge in the mice model.