猪链球菌9型5'核苷酸酶克隆表达及特性分析

Previous studies showed that 5'-nucleotidase is a virulence-associated factor for Streptococcus suis serotype 9 (SS9), but its biological function is not clear. Since NT contains a 5'-nucleotidase domain, we tested whether it could hydrolyze AMP. The hydrolysis activity of the Δnt mutant was significantly reduced compared with the wild-type strain GZ0565 and NT complemented strain C-nt in the presence of 0.2 M AMP. Homology analysis revealed that NT is conserved in 19 different serotypes SS strains. NT fragment from C-terminal (1026bp) was amplified from SS9 strain GZ0565 genomic DNA and cloned into the PMD19-T vector, then inserted into expression vector pET32a. The recombinant plasmid was transformed into E.coli BL21(DE3). After induction by IPTG, a recombinant fusion protein about 57kDa was detected. A rabbit was immuned with recombinant NT protein (rtNT). After the third immunization, the titer of the serum is 1:12800. The bacterial cell wall proteins of the GZ0565 and the Δnt mutant were extracted and probed with rabbit anti-rtNT serum. An apparent band was detected in the cell wall protein of the GZ0565 strain, while no specific band was observed with the same serum when blotting Δnt cell wall proteins. In addition, NT was recognized by anti-SS9 convalescent sera from pigs. Taken together, we confirmed that NT is conserved in SS strains, a immunogenic protein expressed on the bacterial surface, and a promissing vaccine candidate. It also can hydrolyze AMP, which contributes to understanding its role in SS9 pathogenesis.