Abstract：Mycoplasma synoviae (MS) is an important avian pathogenic Mycoplasmas that can lead to respiratory tract diseases, arthritis and synovitis in chickens (Gallus gallus) and turkeys (Meleagris gallopavo), which resulted in reduction in egg production and hatchability, poor egg quality, growth retardation, low feed conversion rate and carcass condemnation, thus caused serious economic losses to the poultry industry. Therefore, the study on detection method and good immune preparation for MS would provide good support for the rapid diagnosis and effective prevention of MS infection. Base on this, the primers were designed according to the sequence of the pdhA and pdhB gene of MS WVU1853 strain (GenBank No. CP011096.1) in GenBank, and the gene pdhA, pdhB and junction fragments AB of both in a single nucleotide overlap were amplified. After completing the point mutations of pdhA using over-lap PCR, the optimized pdhA and pdhB were respectively cloned into the pETDuet-1, the fragments AB was cloned into pET-28a(+), and the prokaryotic co-expression vectors pETDuet-AB and pET-AB were constructed. Then the pETDuet-AB and pET-AB were transformed into Escherichia coli BL21 (DE3), and the recombinant proteins were expressed with induction by isopropyl β-D-thiogalactoside (IPTG). Subsequently, the expression products were purified and its enzyme activity was analyzed. The results of SDS-PAGE have showed that the pdhA and pdhB of MS WVU1853 strain were co-expressed in E. coli BL21 (DE3), and both recombinant proteins were highly expressed in BL21 (DE3) transformed by pET-AB. However, the expression level of recombinant proteins pyruvate dehydrogenase E1 alpha subunit (PDHA) was significantly lower than that of pyruvate dehydrogenase E1 beta subunit (PDHB) in E.coli transformed by pETDuet-AB. The results of the enzyme activity analysis had showed that the purified expression products from BL21 (DE3) transformed by the pETDuet-AB or pET-AB had enzyme activity. This study provides a prospective material for further study of the biological functions of pyruvate dehydrogenase E1 alpha subunit and beta subunit from MS, also provides possible markers for the establishment of diagnostic methods and possible vaccine candidates for MS infection, as well as provides a new idea for the co-expression of the 2 genes.
1 包世俊, 丁小琴, 邢小勇, 等. 2017. 滑液支原体WVU1853株免疫相关膜蛋白的初步分析[J].畜牧兽医学报 48(2):316-323. (BAO S J, Xing X Y, Ding X Q, et al.2017. Preliminarily analysis of immune-related membrane proteins from Mycoplasma synovia WVU1853 strain[J]. Acta Veterinaria et Zootechnica Sinica, 48(2):316-323.) 2 耿风廷, 赵晓瑜, 高珊. 2007. 多基因在大肠杆菌中的共表达策略[J]. 生物技术通讯,18(2):339-341. ( Geng F T, Zhao X Y, Gao S.2007. The strategy of coexpression in Escherichia coli[J].Letters in Biotechnology, 18(2):339-341.) 3 马蓉,徐昊,丁锐,等.2012.大肠杆菌多基因共表达策略[J].中国生物工程杂志,32(4):117-121. (Ma R, Xu H, Ding R, et al.2012. The strategy of gene coexpression in Escherichia coli[J].China Biotechnology, 32(4):117-121.) 4 Bolha L, Bencina D, Cizelj I, et al.2013. Effect of Mycoplasma synoviae and lentogenic Newcastle disease virus coinfection on cytokine and chemokine gene expression in chicken embryos[J]. Poultry Science, 92(12): 3134-3143. 5 Cavan G, MacDonald D.1995. Cloning and sequence of a gene encoding the pyruvate dehydrogenase E1 beta subunit of Schizosaccharomyces pombe[J]. Gene,152(1):117-120. 6 Dallo S F, Kannan T R, Blaylock M W, et al.2002. Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae[J]. Molecular Microbiology,46(4):1041-1051. 7 Hinz K H, Luders H.1969. Occurrence of infectious synovitis (Mycoplasma synoviae infection) complicated by Staphylococcus aureus in chickens in northern Germany[J]. Dtsch Tierarztl Wochenschr,76(5): 120-123. 8 Marois C, Savoye C, Kobisch M, et al.2002. A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples[J]. Veterinary Microbiology, 89(1): 17-28. 9 Nemeria N S,Trevukhina R V.1993. Activity of the pyruvate dehydrogenase complex in Walker-256 carcinosarcoma[J]. Ukrainskii Biokhimicheskii Zhurnal (Kiev), 65(4): 28-33. 10 Pinto P M, Chemale G, de Castro L A, et al.2007. Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins[J]. Veterinary Microbiology 121 (1-2): 83-93. 11 Springer W T, Luskus C, Pourciau S S.1974. Infectious bronchitis and mixed infections of Mycoplasma synoviae and Escherichia coli in gnotobiotic chickens. Ⅰ. Synergistic role in the airsacculitis syndrome[J]. Infection and Immunity, 10(3): 578-589. 12 Springer W T, Luskus C, Pourciau S S.1980. Lymphoid organ and serologic responses of gnotobiotic and conventional chickens to infectious bronchitis virus and mixed infections of Mycoplasma synoviae and Escherichia coli[J]. Comparative Immunology, Microbiology and Infectious Diseases, 3(3): 363-371. 13 Thomas C, Jacobs E, Dumke R.2013. Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae[J]. Microbiology, 159(Pt2): 352-365. 14 Yagihashi T, Nunoya T, Otaki Y.1983. Effects of dual infection of chickens with Mycoplasma synoviae and Mycoplasma gallinaceum or infectious bursal disease virus on infectious synovitis[J]. Nihon Juigaku Zasshi. The Japanese Journal of Veterinary Science, 45(4): 529-532. 15 Yep A, Kenyon G L, McLeish M J.2006. Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase[J]. Bioorganic & Medicinal Chemistry Letters, 34(6): 325-336.
