Lahdenne P, Porcella S F, Hagman K E, Akins D R, Popova T G, Cox D L, Katona L I, Radolf J D, Norgard M V
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Infect Immun. 1997 Feb;65(2):412-21. doi: 10.1128/iai.65.2.412-421.1997.
Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide. Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa). The DNA sequences of the corresponding ORFs in B. burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide. Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600. The lp6.6 gene, which was localized to the 49-kb linear plasmid of B. burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B. burgdorferi lipoprotein reported previously (L. I. Katona, G. Beck, and G. S. Habicht, Infect. Immun. 60:4995-5003, 1992). Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B. burgdorferi. Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B. burgdorferi failed to develop antibodies against this antigen. We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection.
利用伯氏疏螺旋体297的分离外膜获取一种低分子量、外膜相关多肽的部分氨基酸序列信息。基于该信息设计的简并寡核苷酸引物用于扩增一个100 bp的探针，以在伯氏疏螺旋体全基因组文库中检测相应的全长基因。相关的开放阅读框（ORF）编码一种多肽，该多肽由一个17个氨基酸的推定信号肽组成，其末端为LFVAC，这是脂蛋白修饰的一个可能的共有序列，以及一个51个氨基酸的成熟蛋白（预测分子量为5.8 kDa）。伯氏疏螺旋体297和B31中相应ORF的DNA序列相同；N40菌株中的相应ORF仅相差一个核苷酸。假设进行常规加工和酰化，这种脂蛋白（命名为lp6.6）的分子量约为6600。lp6.6基因定位于伯氏疏螺旋体的49 kb线性质粒上，随后被克隆并在大肠杆菌中作为与谷胱甘肽S-转移酶的融合蛋白进行表达。用单克隆抗体240.7进行的免疫印迹分析表明，lp6.6与先前报道的一种低分子量、高度保守的伯氏疏螺旋体脂蛋白相同（L. I. Katona、G. Beck和G. S. Habicht，《感染与免疫》60:4995 - 5003，1992）。间接免疫荧光试验、生长抑制试验、被动免疫和主动免疫的结果表明，这种外膜相关抗原在伯氏疏螺旋体中并非表面暴露。特别有趣的是，长期感染伯氏疏螺旋体的小鼠和恒河猴未能产生针对该抗原的抗体。我们提出，lp6.6的高水平表达与螺旋体生命周期的节肢动物阶段相关，并且该基因的表达在哺乳动物感染期间下调。
