表达LTB乳酸杆菌提高艰难梭菌类毒素疫苗黏膜免疫效应的研究

Improvement of mucosal immunity effect on Clostridium difficile toxin vaccine with expressing LTB Lactobacillus

目的通过构建大肠埃希菌热敏性肠毒素B亚单位(heat labile enterotoxin B subunit,LTB)乳酸杆菌表达载体,研究其对艰难梭菌类毒素疫苗的黏膜免疫保护效应。方法克隆LTB基因,制备pSIP411-LTB重组质粒,电转化到乳酸杆菌中,制备乳酸杆菌表达LTB载体;通过透析和层析等技术纯化艰难梭菌A毒素和B毒素,经甲醛处理获得的艰难梭菌A毒素和B毒素的类毒素作为疫苗候选抗原;在第0 d、第14 d和第28 d皮下接种小鼠艰难梭菌类毒素和口服乳酸杆菌表达LTB载体;在初次接种后第0 d、10 d、24 d和38 d检测抗A毒素和B毒素的血清IgG和粪便IgA;第34 d,小鼠经口感染艰难梭菌,观察疫苗保护效果。结果成功构建了LTB乳酸杆菌佐剂表达载体和制备了艰难梭菌A毒素和B毒素的内毒素;各疫苗组和疫苗+LTB佐剂组小鼠在感染艰难梭菌后,均无腹泻、体重减轻和死亡现象,而对照组老鼠腹泻发病率为100%,死亡率42.86%,5 d体重下降0.83 g;在初次接种后的10 d、24 d和38 d,与空白组比较,各疫苗组抗A毒素或B毒素的血清IgG及粪便IgA显著升高(F=284.93,1037.05,696.82,74,184.07,166.95,P<0.01);与疫苗组比较,接种疫苗+LTB佐剂组的血清IgG及粪便IgA显著升高(F=21.88,27.22,8.31,56.49,63.76,101.78,P<0.05)。结论艰难梭菌A毒素和B毒素的类毒素作为疫苗的候选抗原对艰难梭菌的感染具有很好的保护效应,LTB乳酸杆菌载体表达佐剂能够提高免疫动物的抗体效价,增强黏膜免疫效应。

To construct Lactobacillus heat labile enterotoxin B subunit(LTB)expressing vector,and study the mucosal immune protecting effect of LTB on Clostridium difficile(C.difficile)toxoid vaccine,LTB gene was cloned and pSIP411-LT recombinant plasmid was prepared.The Lactobacillus LTB expressing vector was prepared by recombinant plasmid electrotransforming to Lactobacillus.After A toxin(TcdA)and B toxin(TcdB)of C.difficile were purified by dialysis and chromatography,the toxoid of TcdA and TcdB were obtained by formaldehyde treatment for vaccine candidate antigen.On 0 day,14 days and 28 days,the mice were subcutaneously inoculated with Tcd toxoid and oral lactobacillus expressing LT adjuvant.On 0 day,10 days,24 days and 38 days of first inoculation,serum IgG and fecal IgA of anti-A-toxin and B-toxin were detected.On 38 days,the mice were infected by C.difficile by mouth,and the protective effect of the vaccine was observed.Adjuvant expressing vector of LTB Lactobacillus was constructed,and toxoid of TcdA and TcdB was prepared successfully.After infection with C.difficile,no diarrhea or weight loss occurred in all vaccine groups,while the diarrhea incidence was 100%,the mortality incidence was 42.86%,and the weight loss of 5 days was 0.83±0.11g in the control group.On the 10 days,24 days and 38 days after first inoculation,the serum IgG and fecal IgA levels of anti-TcdA or TcdB in each vaccine group were significantly higher than those in the blank group(P<0.01),and the serum IgG and fecal IgA levels in the vaccine+LTB adjuvant group were significantly higher than those in the vaccine group(P<0.05).In conclusion,as vaccine candidate antigens,toxoid of TcdA and TcdB has good protective effect on infection of C.difficile.The adjuvant LTB expressing of Lactobacillus can improve antibody titer and enhance mucous membrane immunity in immune animals.