Uchiyama T, Zhao L, Uchida T
Department of Virology, University of Tokushima, Japan.
Microbiol Immunol. 1996;40(2):133-9. doi: 10.1111/j.1348-0421.1996.tb03328.x.
Genomic libraries of Rickettsia japonica were cloned into an expression vector lambda gt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as lambda Rj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with beta-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and lambda gt11 lacZ gene amplified the lambda Rj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.
日本立克次氏体的基因组文库被克隆到表达载体λgt11中。筛选出一个表达与抗120千道尔顿（kDa）蛋白抗血清发生反应的蛋白的克隆，该蛋白是热可修饰和热稳定多肽的混合物，命名为λRj120-1。表达的蛋白分子量为180 kDa。蛋白质免疫印迹法表明，表达的蛋白是与β-半乳糖苷酶的融合蛋白。抗120-kDa蛋白的抗血清被诱导的溶原菌吸收，导致对热稳定的120-kDa多肽的反应性消失。抗表达蛋白的抗血清除了与日本立克次氏体反应外，还与斑点热群（SFG）立克次氏体的热稳定120至130 kDa多肽反应。这些发现表明，从日本立克次氏体克隆基因表达的蛋白具有SFG立克次氏体共有的抗原性基团。根据编码康氏立克次氏体热稳定120-kDa蛋白的基因（Schuenke，K.W.，和Walker，D.H.，《感染与免疫》62：904 - 909，1994）和λgt11 lacZ基因设计的引物通过聚合酶链反应（PCR）扩增了λRj120-1 DNA。对PCR扩增产物的限制性片段长度多态性（RFLP）分析表明，克隆的DNA对应于编码康氏立克次氏体热稳定120-kDa蛋白的基因的一部分，从开放阅读框的第190个核苷酸开始，有2519个核苷酸。RFLP表明克隆的基因与康氏立克次氏体的相应基因高度同源。
