摘要目的 筛选并鉴定幽门螺杆菌外膜蛋白GroEL的优势T细胞和B细胞(T-B)联合抗原表位.方法 采用PCR扩增幽门螺杆菌NCTC11637株groEL基因并构建其原核表达系统,SDS-PAGE检查目的重组蛋白rGroEL表达情况.Ni-NTA亲和层析法提纯rGroEL.采用激光共聚焦显微镜法检测幽门螺杆菌NCTC 11637和SS1株中GroEL分布.Triton X-114法提取上述菌株外膜蛋白样本,采用Western blot法检测外膜蛋白样本中的GroEL.采用专业生物信息学软件预测GroEL的T-B联合抗原表位并构建其噬菌体展示系统.采用Western blot法和ELISA分别检测含T-B联合表位肽的重组噬菌体PⅢ蛋白和人工合成的T-B联合表位肽的免疫反应性.结果 幽门螺杆菌NCTC11637和SS1株groEL基因核苷酸和氨基酸序列相似性高达97.68％～99.63％.所构建原核表达系统能有效表达可溶性rGroEL.GroEL定位于幽门螺杆菌NCTC11637和SS1株外膜.GroEL168、GroEL258、GroEL288、GroEL365、GroEL396和GroEL438六个主要的预测T-B联合抗原表位中,GroEL168显示了很强的阳性杂交信号.ELISA结果显示,GroEL168免疫反应性最强(P＜0.01),其次为GroEL258和GroEL288 (P＜0.05).结论 GroEL是幽门螺杆菌外膜蛋白.GroEL168是GroEL的优势T-B联合抗原表位,可用于制备幽门螺杆菌多抗原肽疫苗.

abstractsObjective To screen and identify the predominant T-and B-cell (T-B) combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori (H.pylori).Methods The entire groEL gene of H.pylori strain NCTC11637 was amplified by PCR,and then a prokaryotic expression system for groEL gene was constructed.The expressed target recombinant protein rGroEL was analyzed by SDSPAGE and purified by Ni-NTA affinity chromatography.Laser confocal microscopy was applied to determine the distribution of GroEL in H.pyloristrains of NCTC11637 and SS1.The outer membrane protein (OMP) samples were extracted from the two strains using Triton X-114 method.GroEL in OMP samples was detected by Western blot assay.Special bioinformatic softwares were used to predict T-B combined antigenic epitopes on GroEL molecule,and phage display systems for every T-B combined antigenic epitope were established.Western blot assay and ELISA were used to detect the immunoreactivity of recombinant T-B combined antigenic epitope-containing phage PⅢ proteins and artificially synthesized T-B combined antigenic epitope peptides,respectively.Results The groEL genes from H.pylori strains of NCTC11637 and SS1 showed 97.68％ ～99.63％ identities in their nucleotide and amino acid sequences.The established prokaryotic expression system could efficiently express soluble rGroEL.GroEL located on the outer membrane of H.pylori strains of NCTC11637 and SS1.Among the six major predicted T-B combined epitopes (GroEL168,GroEL258,GroEL288,GroEL365,GroEL396 and GroEL438),GroEL168 showed the strongest positive immunoblotting signal.The results of ELISA showed that the immunoreactivity of GroEL168 was also the strongest (P＜0.01),followed by GroEL258 and GroEL288 (P＜0.05).Conclusion GroEL is an outer membrane protein of H.pylori.GroEL168 being the predominant T-B combined antigenic epitope of GroEL can be used to develop multiple antigenic peptide vaccines of H.pylori.