Vemulapalli R, Biswas B, Dutta S K
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA.
Microb Pathog. 1998 Jun;24(6):361-72. doi: 10.1006/mpat.1998.0208.
Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens of E. risticii in strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed in E. coli. Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a ;10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues of E. coli GroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains of E. risticii. The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a ;2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed in E. coli using a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S. A.). The purified recombinant proteins cross-reacted with antisera to E. canis and E. sennetsu.
马波托马克热的病原体里氏埃立克体是一种专性细胞内立克次氏体微生物。为了解里氏埃立克体55千道尔顿和51千道尔顿免疫显性抗原在菌株变异中的作用，从25 - D和90 - 12菌株中克隆了它们的基因，进行测序，并在大肠杆菌中表达。序列分析表明，55千道尔顿抗原的基因与一个约10千道尔顿蛋白质的基因一起存在于一个热休克操纵子中。同源性搜索表明，55千道尔顿抗原和10千道尔顿蛋白质分别是大肠杆菌GroEL和GroES蛋白的同源物。来自两种里氏埃立克体菌株的55千道尔顿抗原基因之间以及整个操纵子之间均无核苷酸序列差异。基于序列对90 - 12和25 - D菌株推定成熟51千道尔顿抗原大小的估计分别为52.7千道尔顿和52.9千道尔顿。90 - 12和25 - D菌株的51千道尔顿抗原在推导的氨基酸序列上有98%的同一性。某些氨基酸的差异可能是它们在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上迁移率不同的原因，其中25 - D菌株的51千道尔顿抗原向约低2千道尔顿的分子量区域迁移。在蛋白质免疫印迹中，鉴定出一种155千道尔顿的蛋白质，它似乎是51千道尔顿抗原的三聚体产物。使用商业表达系统pRSET A、B、C（美国加利福尼亚州圣地亚哥的英杰公司）在大肠杆菌中过表达55和51千道尔顿抗原。纯化的重组蛋白与犬埃立克体和赛氏埃立克体的抗血清发生交叉反应。
