奇异变形杆菌疫苗候选抗原的鉴定及其免疫保护效果评价

Identification of candidate antigens of Proteus mirabilis vaccine and evaluation of immune protection effect

目的 鉴定奇异变形杆菌疫苗候选抗原,并评价其在小鼠体内的免疫保护效果。方法 采用细菌膜蛋白提取试剂盒提取奇异变形杆菌膜蛋白,进行Western blot检测及质谱分析。化学法合成外膜蛋白F(outer membrane protein F,OmpF)及丝氨酸水解酶(serine hydrolase,SH)两种膜蛋白的编码基因,克隆至载体pET-28a,于E.coli BL21(DE3)诱导表达重组蛋白,进行Western blot检测。重组蛋白通过Ni Sepharose High Performance纯化后,经腿部肌内注射15只雌性昆明小鼠,100μg/(0.2 mL·只),共免疫3次,间隔2周。末次免疫后15 d,经眶前静脉窦采血,分离血清,间接ELISA法检测特异性抗体水平;经小鼠腹腔接种致死剂量的奇异变形杆菌,接种1个月,观察小鼠存活情况,并计算抗原的免疫保护率。结果 提取的膜蛋白可与奇异变形杆菌免疫小鼠抗血清发生特异性结合,经质谱鉴定与奇异变形杆菌APB87305(OmpF)、WP_004247605(SH)的同源性为100%。原核表达的重组蛋白OmpF及SH可与奇异变形杆菌免疫小鼠抗血清发生特异性结合,免疫小鼠后均可诱导较高水平的特异性抗体,对致死性奇异变形杆菌感染的保护率分别为100%和86.67%。结论 OmpF和SH是宿主免疫保护性抗原,有望成为制备奇异变形杆菌疫苗的新靶点。

Objective To identify the candidate antigens of Proteus mirabilis vaccine and evaluate their immune protection effect in mice.Methods The membrane proteins of P.mirabilis were extracted by bacterial membrane protein extraction kit,and were detected by Western blot and analyzed by mass spectrometry.The genes encoding outer membrane protein F(OmpF)and serine hydrolase(SH)were synthesized by chemical method,cloned into vector pET⁃28a,and induced to express in E.coli BL21(DE3).The recombinant proteins were detected by Western blot,purified by Ni Sepharose High Performance and then injected into the leg muscle of 15 female Kunming mice at a dose of 100µg/(0.2 mL per mouse)for three times with an interval of two weeks.The blood samples were collected through the anterior orbital vein 15 d after the last immunization,the serum was separated,and the specific antibody level was detected by indirect ELISA.The mice were intraperitoneally inoculated with a lethal dose of P.mirabilis.One month after inoculation,the survival of mice was observed,and the im⁃mune protection rates of antigens were calculated.Results The extracted membrane proteins showed specific binding with the antiserum of mice immunized with P.mirabilis,and the homology with APB87305(OmpF)and WP_004247605(SH)was 100%as identified by mass spectrometry.The recombinant proteins OmpF and SH expressed in prokaryotic cells displayed specific binding with the antiserum of mice immunized with P.mirabilis.The immunized mice induced high levels of specific antibodies,and the protection rates against fatal P.mirabilis infection were 100%and 86.67%respectively.Conclusion OmpF and SH are host immune protective antigens and promising new targets for P.mirabilis vaccines.