AbstractMycoplasma synoviae (MS) is an important pathogenic mycoplasma in poultry, which can cause exudative synovitis, arthritis, tarsometatarsal swelling and respiratory tract inflammation in chickens (Gallus domesticus) and turkeys (Meleagris gallopavo), causing great economic losses in chicken industry. Methylenetetrahydrofolate dehydrogenase (MTHFD) is a key enzyme in folic acid metabolism, catalyzing 5,10-methylenetrahydrofolate to 5,10-methoxytetrahydrofolate and participating in the synthesis of purine nucleotides. To study the immunogenicity of MTHFD protein of MS and its distribution in MS, primers were designed according to the folD (bifunctional 5, 10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase) gene of MS WVU1853 strain in GenBank. On the basis of sequence analysis and gene optimization, the full-length folD gene was successfully obtained through PCR, which the length of CDS of folD is 837 bp and the sequence similarity was as high as 99.6% with other MS strains. The prokaryotic expression vector pET-folD was constructed and transformed into E.coli BL21 (DE3). After induction, the recombinant protein (r) MS MTHFD was expressed through SDS-PAGE and molecular weight of rMS MTHFD was approximately 36 kD. The antiserum was prepared by immunizing New Zealand rabbits (Oryctolagus cuniculus) , Western blot, ELISA and immunofluorescence test were used to analyze the distribution of MTHFD in MS. The results showed that MTHFD protein was distributed in MS cell membrane and cytoplasm, but more in cytoplasm. The study build up a basis for in-deepth research of biological characteristics of MS MTHFD.
[1] 包世俊. 2014. 滑液支原体膜表面丙酮酸脱羧酶的生物学特性研究[D]. 博士学位论文, 中国农业科学院, 导师: 丁铲, pp. 1-58. (Bao S J.2014. Study on the biological characteristics of pyruvate decarboxylase on the membrane surface of Mycoplasma synoviae[D]. Thesis for Ph.D., Chinese Academy of Agricultural Sciences, Supervisor: Ding C, pp. 1-58.) [2] 包世俊, 朱彩宏, 邢小勇, 等. 2020. 牛支原体NOX2的原核表达及黏附特性[J]. 畜牧兽医学报, 51(11): 2895-2902. (Bao S J, Zhu C H, Xing X Y, et al.2020. Prokaryotic expression and adhesion properties of Mycoplasma bovis NOX2[J]. Chinese Journal of Animal and Veterinary Sciences, 51(11): 2895-2902. [3] 柴东奇, 王卫星. 2021. 一碳代谢与肿瘤治疗的研究进展[J].肿瘤防治研究, 48(03): 288-292. (Chai D Q, Wang W X.2021. Progress in one-carbon metabolism and tumor therapy[J]. Cancer Research on Prevention and Treatment, 48(03): 288-292) [4] 陈鸿军, 沈欣悦, 陈丹清, 等. 2012. 鸡毒支原体烯醇化酶单克隆抗体研制及黏附阻断[J]. 中国兽医学报, 32(06): 862-865, 870. (Chen H J, Shen X Y, Chen D Q, et al.2012. Preparation of monoclonal antibody against Mycoplasma gallisepticum enolase and its adhesion blocking[J]. Chinese Journal of Veterinary Science, 32 (06): 862-865 ; 870) [5] 董亚旗, 朱习芳, 陈颖钰, 等. 2018. 牛支原体PepO原核表达及功能验证[J].中国奶牛, (08):1-6. (Dong Y Q, Zhu X F, Chen Y Y, et al. 2018. Prokaryotic expression and functional verification of Mycoplasma bovis PepO[J]. Chinese Dairy Cattle, (08): 1-6.) [6] 郭升伟. 2020. 一例白羽肉鸡共感染H9亚型禽流感、滑液支原体病及细菌性疫病的诊治[J]. 福建畜牧兽医, 42(02): 64-66. (Guo S W.2020. Diagnosis and treatment of a white-feathered broiler co-infected with H9 subtype avian influenza, Mycoplasma synoviae and bacterial blight[J]. Fujian Journal of Animal and Veterinary, 42(02): 64-66.) [7] 雷元元, 郭亚男, 郭磊, 等. 2019. 一起禽滑液囊支原体病的诊断分析[J]. 中国动物传染病学报, 27(06): 103-107. (Lei Y Y, Guo Y N, Guo L, et al.2019. Diagnosis and analysis of a Mycoplasma synovialis infection[J]. Chinese Journal of Animal Infectious Diseases, 27(06): 103-107.) [8] 李安平, 李福伟, 朱连勤, 等. 2020. 一例蛋鸡滑液囊支原体病的诊治[J]. 家禽科学, (04): 27-29. (Li A P, Li F W, Zhu L Q, et al. 2020. Diagnosis and treatment of a case of Mycoplasma synovialis in laying hens[J]. Poultry Science, (04): 27-29.) [9] 李浩然, 祁晶晶, 王宇, 等. 2020. 滑液支原体NADH氧化酶的酶学活性及亚细胞定位研究[J]. 微生物学通报, 47(03): 801-812. (Li H R, Qi J J, Wang Y, et al.2020. Enzymatic activity and subcellular localization of NADH oxidase from Mycoplasma synoviae[J]. Microbiology China, 47(03): 801-812.) [10] 刘佳, 张生英, 邢小勇, 等. 2020. 滑液支原体DnaK的原核表达及免疫原性分析和亚细胞定位[J].中国兽医科学, 50(08):1029-1036. (Liu J, Zhang S Y, Xing X Y, et al.2020. Prokaryotic expression, immunogenicity analysis and subcellular localization of DnaK of Mycoplasma synoviae[J]. Chinese Veterinary Science, 50(08): 1029-1036.) [11] 刘婷. 2016. 鸡滑液囊支原体JS1株的分离鉴定及禽支原体、大肠杆菌、沙门菌多重PCR检测方法的建立[D]. 硕士学位论文,山东农业大学, 导师: 马卫明, pp. 5. (Liu T.2016. Isolation and identification of chicken Mycoplasm synoviae JS1 strain and establishment of multiplex PCR method for detection of avian mycoplasma, Escherichia coli and Salmonella[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Ma W M, pp. 5.) [12] 尚原冰, 祁晶晶, 王宇, 等. 2021. 鸡滑液支原体磷酸甘油酸激酶的克隆表达及酶学活性测定[J].中国动物传染病学报, 29(03):71-79. (Shang Y B, Qi J J, Wang Y, et al.2021. Cloning, expression and enzymatic activity determination of Mycoplasma synoviae phosphoglycerate kinase[J]. Chinese Journal of Animal Infectious Diseases, 29(03): 71-79.) [13] 汪企再, 王鸿超, 陈海琴, 等. 2018. 过表达亚甲基四氢叶酸脱氢酶对高山被孢霉脂质合成的影响[J]. 中国生物工程杂志, 38(09): 12-18. (Wang Q Z, Wang H C, Chen H Q, et al.Effects of overexpression of methylene tetrahydrofolate dehydrogenase on lipid synthesis of Morphella alpine[J]. China Biotechnology, 38(09): 12-18.) [14] 吴旭锦, 朱小甫, 邢蕾, 等. 2020. 安卡拉、传染性喉气管炎、鸡毒支原体和滑液囊支原体四重混合感染病例的诊治分析[J]. 陕西农业科学, 66(02): 86-87, 97. (Wu X J, Zhu X F, Xing L, et al.2020. Diagnosis and treatment of mixed infection of Ankara, infectious laryngotracheitis, Mycoplasma gallisepticum and Mycoplasma synoviae[J]. Shaanxi Journal of Agricultural Sciences, 66(02): 86-87, 97.) [15] 王宇, 祁晶晶, 刘婷, 等. 2019. 滑液支原体醛缩酶的亚细胞定位及免疫原性[J].微生物学通报, 46(04): 780-789. (Wang Y, Qi J J, Liu T, et al.2019. Subcellular localization and immunogenicity of aldolase from Mycoplasma synoviae[J]. Microbiology China, 46(04): 780-789) [16] 赵刘碧琦, 徐子力, 刘宗谕, 等. 2019. 叶酸代谢通路相关酶基因多态性与先天性心脏病的关系研究[J]. 中国妇幼保健, 34(14): 3371-3374. (Zhao L B Q, Xu Z L, Liu Z Y, et al.2019. Study on the relationship between folate metabolism pathway related enzyme polymorphism and congenital heart disease[J]. Maternal and Child Health Care of China, 34(14): 3371-3374.) [17] 张日欣. 2018. 一例庄河大骨鸡滑液囊支原体与非典型新城疫混合感染的诊治[J]. 现代畜牧兽医, (10): 37-39. (Zhang R X. 2018. Diagnosis and treatment of Mycoplasma synoviae fluid mixed with atypical Newcastle disease in Zhuanghe chicken[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, (10): 37-39.) [18] Bao S J, Guo X Q, Yu S Q, et al.2014. Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J]. BMC Veterinary Research, (10): 223-226. [19] Bekő K, Kreizinger Z, Yvon C, et al.2020. Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates[J]. PLOS ONE, 15(10): e0241647. [20] Cisneros-Tamayo M, Kempf I, Coton J, et al.2020. Investigation on eggshell apex abnormality (EAA) syndrome in France: Isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum[J]. BMC Veterinary Research, 16(1): 747-754. [21] D'Ari L, Rabinowitz J C.1991. Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli[J]. The Journal of Biological Chemistry, 266(35): 23953 [22] Emam M, Hashem Y M, El-Hariri M, et al.2020. Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt[J]. Veterinary World, 13(7): 1410-1416. [23] Gao X, Bao S J, Xing X Y, et al.2018. Fructose-1,6-bisphosphate aldolase of Mycoplasma bovis is a plasminogen-binding adhesin[J]. Microbial Pathogenesis, 124(11): 230-237. [24] Huang J, Zhu H M, Wang J Y, et al.2019. Fructose-1,6-bisphosphate aldolase is involved in Mycoplasma bovis colonization as a fibronectin-binding adhesin[J]. Research in Veterinary Science, 124: 70-78. [25] Hum D W, Bell A W, Rozen R, et al.1988. Primary structure of a human trifunctional enzyme. Isolation of a Cdna encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase[J]. Journal of Biological Chemistry, 263(31): 15946-15950. [26] Li P, Zhang Y, Li X, et al.2019. Mycoplasma hyopneumoniae Mhp597 is a cytotoxicity, inflammation and immunosuppression associated nuclease[J]. Veterinary Microbiology, 235(8): 53-62. [27] Liu R, Xu B, Yu S Q, et al.2020. Integrated transcriptomic and proteomic analyses of the interaction between chicken synovial fibroblasts and Mycoplasma synoviae[J]. Frontiers in microbiology, 11(4): 576-590. [28] Oven I, Resman R K, Dušanić D, et al.2013. Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses[J]. Veterinary Research, 44(1): 1-11. [29] Petrone-Garcia V M, Tellez-Isaias G, Alba-Hurtado F, et al.2020. Isolation and antimicrobial sensitivity of Mycoplasma synoviae and Mycoplasma gallisepticum from vaccinated hens in Mexico[J]. Pathogens, 9(11): 924-936. [30] Sah S, Varshney U.2015. Impact of mutating the key residues of a bifunctional 5,10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase from Escherichia coli on its activities[J]. Biochemistry, 54(22): 3504-3513. [31] Song Z Q, Li Y, Liu Y, et al.2012. α-Enolase, an adhesion-related factor of Mycoplasma bovis[J]. PLOS ONE, 7(6): e38836. [32] Sun S K, Lin X, Chen F, et al.2017. Epidemiological investigation of Mycoplasma synoviae in native chicken breeds in China[J]. BMC Veterinary Research, 13(1): 1-9. [33] Xu B, Liu R, Ding M, et al.2020. Interaction of Mycoplasma synoviae with chicken synovial sheath cells contributes to macrophage recruitment and inflammation[J]. Poultry Science, 99(11): 5366-5377. [34] Yavlovich A, Rechnitzer H, Rottem S.2007. Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen[J]. Infection and Immunity, 75(12): 5716-5719.
