DNA sequencing and characterization of a HSP-like 42 kDa protein gene of Mycoplasma hyopneumoniae

• Main Authors: Chou, Shih-Yu, 周時羽
• Other Authors: Shiuan, David
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/45180781884682927562

碩士 === 國立中山大學 === 生命科學研究所 === 84 === Mycoplasma hyopneumoniae is the etiologic agent of swine enzootic pneumonia (SEP), a chronic nonfatal but serious disease affecting pigs of all ages. Understanding about its pathogenicity as well as the control of the disease by vaccination are still preliminary. In addition, mycoplasma are the smallest and simplest self-replicating prokaryotes. Their rather small genome size (appox. 580-1300 kb) and low G+C% content result in very constrained genetic information avalible for cellular metabolism. These intriguing features prompt us to gain more insights into the structures and regulation patterns of its various genes. By immunoscreening the lEMBL3 genomic library of M. hyopneumoniae with purified antiserum, 5 positive clones which encode 18 kDa (P18), 36 kDa(P36), 38 kDa(P38), 42 kDa(P42) and 60 kDa(P60) protein genes were isolated. P42 clone was then chosen for furhter studies. The 4.4 kb subcloned HindIII-XmaI fragment containing the 42 kDa protein gene was sequenced after subcloning and Western blot analysis. According to mycoplasmal codon usage, three open reading frames(ORF) which may encode 16kDa (P16), 24 kDa (P24) and 65 kDa (P65) proteins were identified. Upstream of the putative initiation codon of these three ORFs show the consensus sequences TATA box and ribosomal binding site(RBS), but only the AT-rich segments were found in the -35 region of P16 and P24. The P65 can be viewed as two part, a 22 kDa invisible portion and 42 kDa positive signal portion in the western blot analysis due to a TGA codon inthe P65 ORF. However, using the purified anti-P42 monospecific antibody we found an approxmately 75 kDa band instead of the deduced 65kDa protein in M. hyopneumoniae total, suggesting posttranslational modification may present in the microorganisms. BLAST result of P65 showed relatively high similarity(appox. 60%) with Mycoplasma genitalium hsp70 gene, Erysipelothrix rhusiopathiae hsp70 gene and Lactococcus latis dnaK gene