副猪嗜血杆菌单克隆抗体的制备及其B细胞抗原表位的鉴定

Preparation of monoclonal antibody against Haemophilus parasuis and the epitope identification

为筛选具有临床诊断价值的副猪嗜血杆菌（H.parasuis）单克隆抗体（MAb）,本研究采用H.parasuis 5型强毒株（HPS-5）免疫BALB/c小鼠,利用常规细胞融合技术制备了一株稳定分泌MAb的杂交瘤细胞株.Western blot结果表明该MAb能够特异性识别所有H.parasuis标准菌株;免疫共沉淀技术与蛋白质谱分析显示该MAb所识别的蛋白为ATP结合盒转运蛋白（ABCT）家族的寡肽透过酶（OppA）;利用MAb对噬菌体12肽库进行淘选,8个阳性噬菌体克隆展示有“KTPAE-R”保守序列,对应于H.parasuis SH29755株和SH0165株的469位～475位的氨基酸残基.对OppA氨基酸序列比对结果表明H.parasuis与其它10种猪常见细菌病病原序列一致性为9.1％～74.5％,本研究结果为进一步研究H.parasuis感染的病原学诊断建立了良好的基础.

To prepare monoclonal antibody （MAb） and identify epitope of related antigen in Haemophilus parasuis, a hybridam secreting MAb against H.parasuis was generated by fusion of the SP2/0 cells with the splenic cells from H.parasuis immunized BALB/c mice. Western blot analysis showed that the MAb was group specifically antibody which reacted with 15 serotype standard strains of H.parasuis. In addition, immunoprecipitation and protein mass spectrum displayed that the MAb recognized the oligopeptidepermease A （OppA） which belongs to the ATP binding cassette transporter family. Fifteen randomly positive phage clones were identified by ELISA which obtained after three panning of the phage 12-mer peptide library with the MAb, and the sequence analysis indicated that eight phage clones displayed a conserved sequence of KTPSE-R corresponding to 469KTPAE-R475 in OppA of H.parasuis. Aligned amino acid sequences of OppA showed that H.parasuis shared 9.1% to 74.5% homologous with other related pathogenic bacteria in swine. These results provided a basis for further study on etiological diagnosis of H.parasuis infection.