副猪嗜血杆菌外膜蛋白P5的B细胞抗原表位鉴定

副猪嗜血杆菌外膜蛋白P5能诱导机体产生免疫保护反应，同时可以用于特异性的血清学诊断，本试验选取P5蛋白进行抗原表位鉴定。首先通过PCR扩增P5F1（1～204aa），P5F2（170～296aa）及P5F3（280～371aa）3个片段，PCR产物分别定向克隆到表达载体pET32a（＋）中表达纯化。根据ELISA和Western blotting结果确定P5F3片段（280—371aa）是OmpP5的免疫优势决定区。为了进-步对该免疫优势决定区进行抗原表住鉴定，设计了-套11个部分重叠的短肽，这些短肽覆盖全部280～371aa片段。每-个短肽合成1对寡核苷酸链，退火后插入表达载体pGEX-6p-1，与GST进行融合表达。用HPS阳性血清进行ELISA和Western blotting扫描，鉴定出其表位位于”。TGNTCDAVKGRKALIT351。通过序列分析证实该抗原表位在不同的HPS菌株中高度保守。本试验确定了位于HPSOmpP5上的-个抗原表位，为建立-种方便、快捷、适用于现地大规模样品检测的鉴别诊断方法奠定了基础，同时也为HPS新型亚单位疫苗的研制，以及研究病原茵感染和机体免疫过程中P5蛋白与宿主体内相应分子之间的相互作用提供了有用信息。

Identification of a conserved B-cell epitope on outer membrane protein P5 of Haemophilus parasuis

Outer membrane protein P5 of Haemophilus parasuis （HPS） is an important protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool,but the epitopes on P5 of HPS have not been identified. In this study, three truncated fragments,P5F1 （1-204aa） ,PSF2 （170-294 aa） and PSF3 （280-371 aa） of the HPS were expressed by fusion with 6-His in a pET32a （＋）vector respectively. ELISA and Western blotting results both demonstrated that the P5F3 fragment of the P5 protein was the antigenic dominant region. To map the antigenic epitope of this antigenic dominant region, a set of 11 partially overlapping fragments spanning the 280-371aa fragment were synthesized and fused with GST and expressed respectively. Through ELISA,linear epitope-eontaining fragment P5F3-8 with the motif of 3a6TGNTCDAVKGRKALIT3Sl was located. Western blotting showed that this epitope could be recognized by HPS-positive serum from pigs. Furthermore,it was found that the epitope is highly conserved among HPS strains through sequence analysis. This is the first time to identify a specific epitope on Omp P5 of HPS.