Tan H Y, Nagoor N H, Sekaran S D
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Trop Biomed. 2010 Dec;27(3):430-41.
The major outer membrane protein (OmpH) of 4 local Malaysian strains of Pasteurella multocida serotype B:2 were characterized in comparison to ATCC strains. Three major peptide bands of MW 26, 32 and 37 kDa were characterized using SDSPAGE. Two of these fragments, the 32 kDa and 37 kDa were observed to be more reactive with a mouse polyclonal antiserum in all of the local isolates as well as the ATCC strains in a Western blot. However, the 32 kDa fragment was found to cross react with other Gram negative bacteria. Therefore, the 37 kDa OmpH was selected as vaccine candidate. The 37 kDa ompH gene of the isolated strain 1710 was cloned into an Escherichia coli expression vector to produce large amounts of recombinant OmpH (rOmpH). The 37 kDa ompH gene of strain 1710 was sequenced. In comparison to a reference strain X-73 of the ompH of P. multocida, 39bp was found deleted in the 37 kDa ompH gene. However, the deletion did not shift the reading frame or change the amino acid sequence. The rOmpH was used in a mice protection study. Mice immunized and challenged intraperitoneally resulted 100% protection against P. multocida whilst mice immunized subcutaneously and challenged intraperitoneally only resulted 80% protection. The rOmpH is therefore a suitable candidate for vaccination field studies. The same rOmpH was also used to develop a potential diagnostic kit in an ELISA format.
对4株马来西亚本地多杀性巴氏杆菌B:2血清型菌株的主要外膜蛋白（OmpH）进行了表征，并与美国典型培养物保藏中心（ATCC）菌株进行了比较。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）对分子量分别为26、32和37 kDa的三条主要肽带进行了表征。在蛋白质免疫印迹法中，观察到其中两个片段，即32 kDa和37 kDa的片段，在所有本地分离株以及ATCC菌株中与小鼠多克隆抗血清的反应性更强。然而，发现32 kDa的片段与其他革兰氏阴性菌发生交叉反应。因此，选择37 kDa的OmpH作为疫苗候选物。将分离菌株1710的37 kDa ompH基因克隆到大肠杆菌表达载体中，以大量生产重组OmpH（rOmpH）。对菌株1710的37 kDa ompH基因进行了测序。与多杀性巴氏杆菌ompH的参考菌株X-73相比，在37 kDa ompH基因中发现缺失了39个碱基对。然而，这种缺失并未改变阅读框或氨基酸序列。rOmpH用于小鼠保护研究。经腹腔免疫和攻击的小鼠对多杀性巴氏杆菌有100%的保护作用，而经皮下免疫和腹腔攻击的小鼠只有80%的保护作用。因此，rOmpH是疫苗现场研究的合适候选物。同样的rOmpH也被用于开发一种酶联免疫吸附测定（ELISA）形式的潜在诊断试剂盒。
