鲍曼不动杆菌外膜蛋白OmpW保护性表位的预测与验证  

Prediction and validation of protective epitopes on Acinetobacter baumannii outer membrane protein OmpW

作　　者：杨宁 朱成华[1] 邵明玥 陈思锡 周润露 何宇聪 杜兴冉 冯旰珠[1] Yang Ning;Zhu Chenghua;Shao Mingyue;Chen Sixi;Zhou Runlu;He Yucong;Du Xingran;Feng Ganzhu(Department of Respiratory and Critical Care Medicine,the Second Affiliated Hospital of Nanjing Medical University,Nanjing 210011,China;Department of Respiratory and Critical Care Medicine,the Affiliated Jiangning Hospital of Nanjing Medical University,Nanjing 211100,China;Department of Infectious Disease,the Second Affiliated Hospital of Nanjing Medical University,Nanjing 210011,China)

机构地区：[1]南京医科大学第二附属医院呼吸与危重症医学科,南京210011 [2]南京医科大学附属江宁医院呼吸与危重症医学科,南京211100 [3]南京医科大学第二附属医院感染性疾病科,南京210011

出　　处：《中华微生物学和免疫学杂志》2024年第4期330-335,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(82100014,82070017);江苏省自然科学基金(BK20210981);江苏省高等学校基础科学(自然科学)面上项目(21KJB320002)。

摘　　要：目的构建和筛选鲍曼不动杆菌外膜蛋白OmpW的B细胞和T细胞表位。方法运用生物信息学软件预测OmpW的小鼠B细胞及T细胞表位并合成相应肽段;构建重组质粒pET28a-ompW并原核表达、纯化获得OmpW蛋白;以OmpW蛋白皮下免疫BALB/c小鼠,2周免疫1次,共3次。第3次免疫后1周,收集小鼠血清、分离小鼠脾细胞培养。分别用OmpW蛋白及不同的T细胞表位肽刺激脾细胞,72 h后收集脾细胞上清。间接ELISA法检测小鼠血清中的特异性IgG抗体水平,双抗夹心ELISA法检测脾细胞上清液中IFN-γ浓度。结果筛选并构建了5个B细胞表位及4个T细胞表位,B细胞表位分别是OmpW B1、OmpW B2、OmpW B3、OmpW B4、OmpW B5,T细胞表位分别是OmpW T1、OmpW T2、OmpW T3、OmpW T4;间接ELISA法检测结果显示,OmpW B1和OmpW B5能够与OmpW全长蛋白质免疫小鼠的血清发生抗原抗体反应,其A450值与对照组相比显著升高(P<0.001);双抗夹心ELISA法结果显示OmpW T4表位刺激的脾细胞上清中的IFN-γ浓度最高,与对照组相比差异有统计学意义(P<0.001)。结论成功构建并筛选出OmpW蛋白的两个B细胞表位OmpW B1及OmpW B5和一个T细胞表位OmpW T4。Objective To construct and screen B-cell and T-cell epitopes on Acinetobacter baumannii outer membrane protein OmpW.Methods Mouse B-cell and T-cell epitopes on OmpW were predicted using bioinformatics software and synthesized.A recombinant plasmid pET28a-ompW was constructed to express OmpW in a prokaryotic expression system.BALB/c mice were subcutaneously immunized three times with purified OmpW with an immunization interval of two weeks.Mouse serum samples were collected,and spleen cells were isolated and cultured one week after the third immunization.Spleen cells were stimulated with OmpW or different T cell epitopes,and spleen cell culture supernatants were collected 72 h after stimulation.The levels of specific IgG antibodies in serum were detected by indirect ELISA.The levels of IFN-γin the cell culture supernatants were detected by double antibody sandwich ELISA.Results Five B-cell epitopes(OmpW B1,OmpW B2,OmpW B3,OmpW B4 and OmpW B5)and four T-cell epitopes(OmpW T1,OmpW T2,OmpW T3 and OmpW T4)were selected and constructed.Indirect ELISA results showed that OmpW B1 and OmpW B5 could react with the serum of OmpW-immunized mice and the A450 values were significantly higher than that of the control group(P<0.001).The level of IFN-γin the culture supernatant of spleen cells stimulated by OmpW T4 was the highest and significantly higher than that of the control group(P<0.001).Conclusions Two B-cell epitopes OmpW B1 and OmpW B5 and one T-cell epitope OmpW T4 are successfully screened and constructed.

关 键 词：鲍曼不动杆菌 OmpW 表位 生物信息学 

分 类 号：R392[医药卫生—免疫学]