铜绿假单胞菌疫苗候选株筛选及其类毒素对小鼠交叉保护性的初步评估

Screening of candidate strains for Pseudomonas aeruginosa vaccine and preliminary evaluation cross-protection of related toxoid in mice

目的筛选多株铜绿假单胞菌(Pseudomonas aeruginosa,PA)菌株,提供可作为疫苗研发使用的备选菌株,并对其进行小鼠交叉保护性的初步评估。方法通过抗生素耐药性、毒力试验筛选铜绿假单胞菌疫苗候选株,采用不同孔径膜包(相对分子质量分别为300000、500000及1000000)对类毒素(toxoid)进行纯化工艺研究,并对其免疫原性及同源交叉保护性进行研究,评估候选株是否适用于疫苗研发。结果铜绿假单胞菌疫苗候选株均具有多重耐药性,尤其对四环素和氯霉素耐药性较高,分别为96.30%和88.89%,但对亚胺培南、环丙沙星敏感,耐药性仅为14.81%和18.52%。其中,耐受5种以上的占83.33%;毒力较强的菌株分别为PA101和PA36。类毒素经相对分子质量分别为300000、500000及1000000的孔径膜包处理后,与对照组相比类毒素收率分别为89.95%、95.52%及97.99%;内毒素残留量分别为≤107.39 EU/μg蛋白、≤101.13 EU/μg蛋白和98.58~985.80 EU/μg蛋白;与对照组相比,500000组类毒素热原检测为阴性;免疫原性结果表明500000组、1000000组与对照组一致,均≥70%。与PA36比较,PA101类毒素对疫苗候选株免疫原性均≥70%。除PA15和PA46外,13株同源菌株交叉保护性在50%~100%之间,其中保护性≥70%的占92.31%。结论铜绿假单胞菌PA101类毒素抗原对候选菌株具有保护性,对同源其他菌株也具有一定的保护性作用,为后续铜绿假单胞菌医院获得性感染性的预防研究提供了数据支持。

Objective To screen several Pseudomonas aeruginosa strains in providing alternative candidate strains for vaccine development,and to evaluate their cross-protection in mice.Methods It was to obtain Pseudomonas aeruginosa vaccine candidate strains by means of antibiotic resistance and virulence test.The purification process of toxoid was performed by using cassettes with different sizes,in which the suitability was evaluated for candidate strains in vaccine development,by using of immunogenicity and the cross-immunological protection against homologous strains.Results Pseudomonas aeruginosa all had multiple drug resistance,especially to tetracycline and chloromycetin,which were 96.30%and 88.89%,respectively,but they were sensitive to penem and ciprofloxacin only 14.81%and 18.52%,among them,more than five accounted for 83.33%.The virulent strains were PA101 and PA36,respectively.Compared of the control group,the yields of toxoid were 89.95%,95.52%and 97.99%,respectively,after treated with 300000,500000 and 1000000 cassettes,respectively.The endotoxin residues were≤107.39 EU/μg protein,≤101.13 EU/μg protein and 98.58—985.80 EU/μg protein,the pyrogenic properties of 500000 toxoid was in negative.The immunogenicity showed that 500000,1000000 groups were the same as those of the control group(≥70%).Compared to PA36,the immunogenicity of PA101 was higher than 70%to the candidate vaccine strain.The cross protection of toxoid against 13 homologous strains were from 50%to 100%,except for PA15 and PA46,among which more than 70%was 92.31%.Conclusion The oxoid antigen of PA101 was in protective to both the candidate strain and the homologous strains,which provided a related data in supporting for the follow-up study on the prevention of nosocomial infection.