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| Prokaryotic Expression and Subcellular Localization of Methylenetetrahydrofolate Dehydrogenase (MTHFD) in Mycoplasma synoviae |
| ZHANG Hong-Yan, YUE Ya-Hui, XING Xiao-Yong, LONG Cui-Qin, WU Xiao-Chun, WEN Feng-Qin, BAO Shi-Jun* |
| College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China |
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Abstract Mycoplasma 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.
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Received: 02 March 2021
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Corresponding Authors:
*bsjdy@126.com
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