Zhang Nian-Zhang, Chu Yue-Feng, Gao Peng-Cheng, Zhao Ping, He Ying, Lu Zhong-Xin
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
J Vet Med Sci. 2012 Aug;74(8):983-7. doi: 10.1292/jvms.11-0260. Epub 2012 Mar 26.
Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESP-like protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 10(9) bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection.
已知副猪嗜血杆菌可产生一组与毒力相关的自转运蛋白（AT），即VtaAs；然而，尚未对其他自转运蛋白进行表征。基于已报道的副猪嗜血杆菌细胞外丝氨酸蛋白酶（ESP）样蛋白的假定espP2基因序列，成功从副猪嗜血杆菌血清型5的田间菌株HPS0819中扩增出该假定的自转运蛋白基因，并进行克隆和测序。确认的开放阅读框（ORF）序列与已报道的假定espP2基因显示出100%的同一性。利用pET表达系统从大肠杆菌中纯化出的重组ESP样蛋白用于免疫特性分析。用纯化的ESP样蛋白抗血清在培养的副猪嗜血杆菌中检测到一种约85 kDa的抗原，并且在患有实验性副猪嗜血杆菌感染的猪的选定血清中检测到了针对重组ESP样蛋白的抗体。结果表明，副猪嗜血杆菌可在体外和体内产生ESP样蛋白。在一项使用豚鼠的免疫保护研究中，用重组ESP样蛋白免疫的10只动物中有6只在受到5×10⁹个HPS0819菌株细菌攻击后存活，而用福尔马林灭活的H0819菌苗免疫的10只动物中有7只在受到攻击后存活。结果表明，ESP样蛋白可能是副猪嗜血杆菌感染的疫苗抗原候选物之一。
