AbstractMycoplasma 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.
BAO Shi-Jun,DING Xiao-Qin,XING Xiao-Yong, et al. Prokaryotic Co-expression of pdhA and pdhB Gene of Mycoplasma synoviae WVU1853 Strain and Enzymatic Activity Analysis of Expressed Pruducts[J]. 农业生物技术学报, 2019, 27(9): 1673-1680.
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