不可分型流感嗜血杆菌OMP6优势T-B联合抗原表位及其免疫原性研究

摘要

The aim of this study is to investigate the distribution and sequence conservation of P6 outer membrane protein (OMP6)-encoding gene of nontypeable Haemophilus influenzae isolates as well as screen and identify the predominant T-and B-cell (T-B)-combined antigenic epitopes in OMP6 sequences and their immunogenicity.The entire omp6 genes of NTHi isolates were amplified by PCR and the amplification products were sequenced after T-A cloning.By using bioinformatic softwares,the sequence conservation and membrane location of OMP6 were analyzed as well as the T-B-combined antigenic epitopes in OMP6 were predicted.The immunogenicity and immunoreactivity of T-B-combined antigenic epitope peptides displayed by recombinant phage PⅢ proteins (rPⅢ) were determined by Western Blot assay and ELISA.The PCR showed that all the 35 NTHi isolates tested were detectable for omp6 gene.The identities of nucleotide and amino acid sequences of omp6 genes from 28 strains in the NTHi isolates were 98.3％-100％ and 99.3 ％-100％,respectively.OMP6 of NTHi was predicted as an outer membrane superficial protein that contains OMP6-2-25,OMP6-61-86 and OMP6-98-126 predicted T-B-combined antigenic epitopes.The immunoblotting assay and ELISA confirmed that OMP6-2-25 presented stronger hybridization band with NTHi antisera while 96.9％ (59/62),69.4％ (43/62) and 74.2％ (46/62) of serum samples from NTHi-infected children were positive for OMP6-2-25,OMP6-61-86 and OMP6-98-126 T-B-combined antigenic epitope peptides,respectively.All the results lead to a conclusion thatomp6 is an extensive distribution and sequence conserved gene of NTHi,and OMP6-2-25 is the predominant T-B-combined antigenic epitopes which can be used as the candidates for developing multiple antigenic peptide vaccine against NTHi.%目的 了解无荚膜不可分型流感嗜血杆菌(NTHi)临床菌株P6外膜蛋白(OMP6)编码基因(omp6)分布及其序列保守性,筛选并鉴定OMP6序列中优势T和B细胞(T-B)联合抗原表位及其免疫原性.方法 采用PCR扩增NTHi临床菌株全长omp6基因,T-A克隆后测序.采用生物信息学软件分析OMP6序列保守性与膜定位并预测其T-B联合抗原表位.采用噬菌体展示联合免疫印迹法和ELISA分别检测重组噬菌体PⅢ蛋白(rPⅢ)展示的OMP6中T-B联合抗原表位肽免疫原性和免疫反应性.结果 35株NTHi临床菌株均能检出omp6基因,其核苷酸和氨基酸序列相似性分别为98.3％～100％和99.3％～100％.OMP6为外膜表面蛋白,具有OMP6-2-25、OMP6-61-86和OMP6-98-126三个预测T-B联合抗原表位.Western Blot和ELISA结果显示,OMP6-2-25能与NTHi抗血清产生较强的杂交条带,96.9％(59/62)、69.4％(43/62)和74.2％ (46/62) NTHi感染患儿血清标本OMP6-2-25、OMP6-61-86和OMP6-98-126抗原表位肽抗体阳性.结论 OMP6是分布广泛且序列保守的NTHi跨膜蛋白,OMP6-2-25为OMP6优势T-B联合抗原表位,可作为NTHi多抗原肽疫苗的候选表位.