DNA sequencing and Characterization of a GTP-binding like Protein 18 kDa Membrabe Protein of Mycoplasma Hyopneumoniae

• Main Author: 林鶴農
• Other Authors: 宣大衛
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/26197081938636056958

碩士 === 國立中山大學 === 生命科學研究所 === 85 === M. hyopneumoniae is the etiologic agent of swine enzootic pneumoniae (SEP), a chronic nonfatal but serious disease affecting pigs of all ages. M. hyopneumoniae is also one of the smallest self-replicating prokaryotes. The goal of the present research is to understand the gene structure and regulatory mechanism of the surface antigen gene and to compare them with other prokaryotes. Possible function and characterization of the antigens are also be studied as well. In our laboratory, we had screened and isolated five positive clones, P60 (60 kDa), P42 (42 kDa), P38(38 kDa), P36(36 kDa), and, P18(18 kDa) from M. hyopneumoniae λEMBL3 genomic library by immunoscreening. The P18 clone was chosen for further studies. The 1.5 kb SalI-HindⅢ fragment was found to express the 18 kDa protein after subcloning and Western blotting analysis. The entire 1.5 kb was sequenced bidirectionally and analyzed. According to the mycoplasmal codon usage pattern (UGA codes for tryptophan), three open reading frame (ORFs), ORf 1(9.2 kDa), ORF 2(10.6 kDa), and an interrupted ORF 3 (30kDa) were identified. Promoter like -10 region and -35 region are RBS were found in the upstream of the P30 start codon, and two TGA codon were found in the coding sequence. As TGA814 codon was mutated to TOG, the monospecific anti-P18 antibody can detect a 30 kDa protein instead of 18 kDa protein in E. coli. The BLAST (Basic Local Alignment Search Tool) comparsion results of P30 revealed a relatively high similarity with the GTP-binding domain of various LepA proteins. However, Far-Western blot analysis found no GTP- binding activity associated with the P30 protein probably because of protein folding. The 30 kDa protein was found to be a membrane protein by Triton X- 114 phase fractionation of M. hyopneumoniae total protein. In brief, P30 has been demonstrated to be a membrane protein with GTP-binding domain and may play an important role in the pathogenesis of M. hyopneumoniae