Abstract：Mycoplasma bovis (M.b) is one of the important pathogenic mycoplasmas. It can cause a variety of diseases if cow (Bos taurus) infected with the M.b. Pyruvate dehydrogenase complex E1 (PDHc E1) is a speed limit of enzymes that involve in biological sugar metabolic process, and widely exists in microbe, mammals and higher plants. It directly impact on pyruvate to acetyl coA. According to the gene sequences of M.b PG45 strain in GenBank, two pairs of primers were designed. PDHc E1 component subunit alpha (pdhα)(GenBank No.KU355295) and PDHc E1 component subunit beta (pdhβ) (GenBank No. KU3552956) genes of M.b Wuwei strain were amplified by PCR. After finished sequencing and point mutation, the genes were cloned into prokaryotic expression plasmid pET-28a(+), respectively. Then the recombinant plasmids pET-pdhα and pET-pdhβ were transformed into Escherichia coli BL21 (DE3), respectively. After induced expression by isopropyl β-D-1-thiogalactopyranoside (IPTG), the fusion protein recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were purified and anti-serum against rPDHA or rPDHB was prepared respectively through immunizing New Zealand rabbit (Oryctolagus cuniculus). Subsequently, subcellular location of E1α and E1β in M.b was performed using Western blot and enzyme linked immunosorbent assay (ELISA). The results showed that the recombinant proteins successfully expressed in Escherichia coli BL21 (DE3) and their molecular weights were approximately 40 and 37 kD, respectively. Western blot and ELISA analysis indicated that the E1α and E1β of M.b equally distributed in both membrane and the cytoplasm. This study provides foundational data for further investigation of biological functions of M.b.
[1] Koike M, Carroll W. R. Alpha-Keto acid dehydrogenation complexes. IV. Resolution and reconstitution of the Escherichia coli pyruvate dehydrogenation complex[J]. Journal of Biological Chemistry, 1963, 238: 30-39. [2] Yavlovich A, Rechnitzer H, Rottem S. Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen[J]. Infection and immunity, 2007, 75(12): 5716-5719. [3] 何随彬，谭磊，包世俊，等. 鸡毒支原体醛缩酶的原核表达及膜定位鉴定[J]. 中国兽医科学，2013，43（01）: 42-46.（He S B, Tan L Bao S J et al. Prokaryotic expression and membrane localization of aldolase of mycoplasma gallisepticum[J]. Chinese Veterinary Science, 2013, 43(01): 42-46.） [4] Thomas C, Jacobs E, Dumke R. Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumonia[J]. Microbiology, 2013, 159(Pt 2):352-365. [5] Martinez MA, Das K, Saikolappan S, et al. A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence[J]. BMC Microbiol, 2013, 13:44. [6] Corona L, Cillara G, Tola S. Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp. Capri[J]. Veterinary microbiology, 2013, 167(3-4): 434-439. [7] Hegde S, Rosengarten R, Chopra-Dewasthaly R. Disruption of the pdhB pyruvate dehyrogenase gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae[J]. PLOS ONE, 2015, 10(3): e0119706. [8] Rocha TS, Tramuta C, Catania S, et al. Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli[J]. Poultry science, 2015, 94(4): 621-627. [9] Churchward CP, Rosales RS, Gielbert A, et al. Immunoproteomic characterisation of Mycoplasma mycoides subspecies capri by mass spectrometry analysis of two-dimensional electrophoresis spots and western blot[J]. The Journal of pharmacy and pharmacology, 2015, 67(3): 364-371. [10] 吴移谋，叶元康. 支原体学[M]. 2008， 人民卫生出版社，北京. pp. 3-4.(Wu Y M, Ye Y K. Mycoplasmaology[M]. 2008, PMPH. Beijing. pp. 3-4) [11] Grundel A, Friedrich K, Pfeiffer M, et al. Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen[J]. PLOS ONE, 2015, 10(5):e0126600. [12] Dallo SF, Kannan TR, Blaylock MW, et al. Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumonia[J]. Molecular microbiology, 2002, 46(4): 1041-1051. [13] Burki S, Frey J, Pilo P. Virulence, persistence and dissemination of Mycoplasma bovis[J]. Veterinary microbiology, 2015, 179: 15-22. [14] Hale H et al. Bovine mastitis caused by a Mycoplasma species[J]. Cornell Veterinary, 1962, 52: 582-591. [15] 辛九庆，李媛，郭丹，等. 国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J]. 中国预防兽医学报，2008，30（9）: 662-664.(Xin J Q Li Y Guo D et al. First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(9): 662-664.) [16] Su H, Hutchison, C, Giddings, M. Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumonia[J]. 2007, BMC Microbiol, 7: 63. [17] Cerniglia G, Dey S, Gallagher-Colombo S, et al. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1alpha Phosphorylation[J]. Molecular cancer therapeutics, 2015, 44: 285-290. [18] Zhao P, He Y, Chu Y F, et al. Identification of novel immunogenic proteins in Mycoplasma capricolum subsp. Capripneumoniae strain M1601[J]. Journal of Veterinary Medical Science, 2012, 74(9): 1109-1115. [19] Axworthy D B, Hoffmann K J, Streeter A J, et al. Covalent binding of acetaminophen to mouse hemoglobin. Identification of major and minor adducts formed in vivo and implications for the nature of the arylating metabolites[J]. Chemico-Biological Interactions, 1988, 68(1-2): 99-116. [20] Lowe P N, Leeper F J, Perham R N. Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli[J]. Biochemistry, 1983. 22(1): 150-157.
