LTB-2xSTN12S融合蛋白联合cGAMP和dmLT双佐剂对小鼠的免疫保护效果

Immune protection of LTB-2xST fusion protein combined with cyclic guanosine monophosphate-adenosine monophosphate and double mutant heat-labile enterotoxin adjuvant in mice

目的在原核系统中表达肠产毒性大肠埃希菌(enterotoxigenic Escherichia coli,ETEC)不耐热肠毒素B亚基(heat labile enterotoxin B subunit,LTB)和耐热肠毒素(heat stable enterotoxin)突变体(STN12S)的融合蛋白,联合环鸟-腺二核苷酸(cyclic guanosine monophosphate-adenosine monophosphate,cGAMP)和dmLT(double mutant heat-labile enterotoxin)佐剂初步评价其保护效果并进行免疫反应分析。方法构建表达质粒pET28α-LTB-2xSTN12S,转化至E.coli BL21(DE3),IPTG诱导表达,镍柱纯化,将纯化的重组LTB-2xSTN12S蛋白联合dmLT和cGAMP佐剂(为抗原+dmLT+cGAMP组;同时设空白对照组、感染对照组、抗原组),均经皮下途径免疫BALB/c小鼠,免疫2针,间隔2周,末次免疫2周后滴鼻攻击ETEC-HN临床分离株(LT+/ST+),2周后称重,取眼眶静脉血及脾淋巴细胞进行体液和细胞免疫分析。结果重组质粒pET28-LTB-2xSTN12S基因测序正确,纯化的重组LTB-2xSTN12S蛋白相对分子质量约20000,纯度为93%,与霍乱毒素(cholera toxin,CT)和ST多抗均能发生特异反应。抗原+dmLT+cGAMP组小鼠诱导的免疫保护均高于感染对照组和抗原组(P均<0.05),诱导的针对LTB-2xSTN12S血清IgG抗体水平及针对LTB和ST抗原特异性IgG抗体水平均高于其他组(P均<0.05);脾淋巴细胞培养上清中抗LTB-2xSTN12SIgG抗体水平及针对LTB-2xSTN12SLTB抗原特异性IgG抗体水平均显著高于其他组(P均<0.01),诱导的脾淋巴细胞抗原特异性IL-4、IL-6、IL-17和IFNγ细胞因子分泌细胞数量均高于其他组(P均<0.05)。结论经原核表达系统成功表达并纯化了LTB-2xSTN12S融合蛋白,LTB和ST抗原性良好,联合cGAMP和dmLT佐剂对同时表达LT和ST临床分离株的攻击具有保护效果,并检测到显著的特异性体液和细胞免疫反应。

Objective To express the fusion gene of heat-labile enterotoxin B subunit(LTB)and heat-stable enterotoxin mutant(STN12 S)of enterotoxigenic coli(ETEC)in prokaryotic system and preliminarily evaluate the protective effect and analyze the immune response of the protein combined with cyclic guanosine monophosphate-adenosine monophosphate(cGAMP)and double mutant heat-labile enterotoxin(dmLT)adjuvant in mice.Methods Expression vector pET28α-LTB-2 xSTN12 Swas constructed,transformed to E.coli BL21(DE3)and induced with IPTG.The expressed fusion protein was purified by nickel ion affinity chromatography.BALB/c mice were randomly divided into four groups.The mice in blank control group were untreated,while those in antigen group were immunized s.c.with purified LTB-2 xSTN12 Sprotein,and those in antigen+adjuvant group with the purified LTB-2 xSTN12 Sprotein in combination with dmLT and cGAMP,for 2 times at an interval of 2 weeks,and challenged i.n.with clinically isolated ETEC-HN strain(LT+/ST+)2 weeks after the last immunization.However,the mice in infection control group received no immunization before challenge.The mice in various groups were weighed 2 weeks after challenge,of which the venous blood and splenic lymphocytes were collected to analyze the humoral and cellular immune responses.Results Gene sequencing proved that recombinant plasmid p ET28-LTB-2 xSTN12 Swas constructed correctly.The purified recombinant LTB-2 xSTN12 Sprotein,with a relative molecular mass of about 20000,reached a purity of 93%and showed specific reactions with polyclonal antibodies against cholera toxin(CT)and ST.The immune protection levels induced by purified LTB-2 xSTN12 Sprotein in combination with dmLT and cGAMP was higher than that in infection control group and that by purified LTB-2 xSTN12 Sprotein alone(each P<0.05),while the serum IgG antibody levels against LTB-2 xSTN12 Sas well as LTB and ST antigens were higher than those in other groups(each P<0.05).The IgG antibody levels against LTB-2 xSTN12 Sand LTB in culture supernatant of splenic lymphocytes were significantly higher(each P<0.01),while the number of cells secreting antigen-specific cytokines including IL-4,IL-6,IL-17 and IFNγwas larger(each P<0.05),than those in other groups.Conclusion LTB-2 xSTN12 Sfusion protein was successfully expressed by procaryotic expression system and purified,which showed good LTB and ST immunogenicity,provided protection against the challenge with ETEC(LT+/ST+)clinical isolate in combination with cGAMP and dmLT as adjuvant and induced significantly humoral and cellular immune responses.