Vishwanath S, McDonald G A, Watkins N G
Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
Infect Immun. 1990 Mar;58(3):646-53. doi: 10.1128/iai.58.3.646-653.1990.
There are no vaccines against boutonneuse fever and Rocky Mountain spotted fever. Previous studies have identified a Rickettsia rickettsii surface protein as a vaccine candidate and shown that an antigenically related protein is present in R. conorii, which causes boutonneuse fever. The gene encoding the R. rickettsii protein has been cloned and expressed in Escherichia coli. We confirmed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rickettsial lysates followed by immunoblotting with a monoclonal antibody raised against the R. rickettsii protein that an analogous protein exists in R. conorii. Although these proteins were previously called 155-kilodalton (kDa) proteins, we found that their apparent molecular masses were 198 kDa for R. conorii Kenya tick typhus and 190 kDa for R. rickettsii R. Using the R. rickettsii gene probe, we cloned and expressed a 5.5-kilobase HindIII fragment from R. conorii Kenya tick typhus genomic DNA in E. coli JM107. The expressed recombinant product was recognized by a monospecific polyclonal rabbit antiserum prepared against the 198-kDa protein. Guinea pigs immunized with sonic lysates of the E. coli strain expressing the recombinant gene product developed antibodies recognizing R. conorii when tested by a microimmunofluorescence antibody assay. Upon immunoblotting of rickettsial lysates, those antisera specifically recognized the 198-kDa R. conorii protein and its 190-kDa analog in R. rickettsii. Guinea pigs immunized with sonic lysates of the recombinant E. coli expressing the 198-kDa protein were protected from experimental infections with the homologous R. conorii strain and partially protected from experimental infections with a strain of the heterologous species R. rickettsii. These findings show that the 198-kDa R. conorii protein is a candidate for a vaccine against boutonneuse fever.
目前尚无针对纽扣热和落基山斑疹热的疫苗。先前的研究已确定立氏立克次体表面蛋白为疫苗候选物,并表明在引起纽扣热的康氏立克次体中存在一种抗原相关蛋白。编码立氏立克次体蛋白的基因已被克隆并在大肠杆菌中表达。我们通过对立克次体裂解物进行7.5%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳,然后用针对立氏立克次体蛋白的单克隆抗体进行免疫印迹,证实康氏立克次体中存在类似蛋白。尽管这些蛋白以前被称为155千道尔顿(kDa)蛋白,但我们发现康氏立克次体肯尼亚蜱伤寒的这些蛋白表观分子量为198 kDa,立氏立克次体的为190 kDa。使用立氏立克次体基因探针,我们从康氏立克次体肯尼亚蜱伤寒基因组DNA中克隆并在大肠杆菌JM107中表达了一个5.5千碱基的HindIII片段。表达的重组产物被针对198 kDa蛋白制备的单特异性多克隆兔抗血清识别。用表达重组基因产物的大肠杆菌菌株的超声裂解物免疫的豚鼠,通过微量免疫荧光抗体试验检测时,产生了识别康氏立克次体的抗体。对立克次体裂解物进行免疫印迹时,这些抗血清特异性识别康氏立克次体的198 kDa蛋白及其在立氏立克次体中的190 kDa类似物。用表达198 kDa蛋白的重组大肠杆菌的超声裂解物免疫的豚鼠,对同源康氏立克次体菌株的实验感染具有抵抗力,对异源立氏立克次体菌株的实验感染有部分抵抗力。这些发现表明,198 kDa的康氏立克次体蛋白是预防纽扣热疫苗的候选物。