| Streptococcus suis (SS) is an important zoonotic pathogens and a serious threat to the development of animal husbandry and public health, especially the safety of related employees. Therefore, it is essential to determine the virulence factors and pathogenic mechanisms of Streptococcus suis, which is the prerequisite for the prevention and control of the disease. Dihydrolipoamide dehydrogenase was found as a critical immunogen by the methods of immune-proteomics in previous work, which can induce protective immunity against challenging of virulent SS2 in zebrafish, but the biocharacteristics and biological function of dihydrolipoamide dehydrogenase in SS is still open.In order to reveal the distribution of dihydrolipoamide dehydrogenase gene in Streptococcus suis, according to the sequence of dihydrolipoamide dehydrogenase gene of Streptococcus suis type 2 (SS2) deposited in GenBank, two pairs of primers were designed and synthesized, then hm6 gene was assayed in 40 strains of Streptococcus suis, and 35 strains were demonstrated that hm6 gene was existed for the expected 461bp PCR product. The blast of hm6 gene sequence from 8 strains of S. suis (ZY05719, HA9801, 2-4, 2-N, 2-H, 2-1, 19-2 and T15) showed that these strains had 98% homology compared with that of in NCBI. It is proved that hm6 gene was widespread in S.suis, and highly homologous.For the expression and purification of dihydrolipoamide dehydrogenase, the whole hm6 gene of SS2 ZY05719 isolated from Sichuan was amplified by PCR and was cloned into prokaryotic expression vector pET-28a to conduct the expression vector pET28a-hm6. The recombinant protein HM6 of 70kD was successfully expressed in E.coliBL21 and purified by His·Bind? Purification Kit.To explore the biological properties and function of membrane protein HM6, based on the fundamental that protein HM6 can catalyzes the interconversion of dihylipoamide and lipoamide with concomitant interconversion of NADH and NAD+, the activity of the enzyme was measured. The result of activity assay indicated that the recombinant protein was able to catalyze the NAD+-dependent oxidation of lipoamide in the reverse reaction. In addition, the character of adhesion of SS to HEp-2 cells mediated by HM6 was studied. Both strains with hm6 gene (HM6+) and without hm6 gene (HM6-) could adhere to HEp-2 cells, but the number of adhered HM6+ strains was significantly higher than HM6- strains (P<0.05). Purified recombinant protein HM6 had no significant cell adhesion inhibitory effect on HM6-strains, but could significantly reduce the number of adhered HM6+ bacteria (P<0.05), although which could not block it completely. Anti-HM6 serum also could significantly reduce the number of adhered HM6+ bacteria (P<0.05). These results implied that the HM6 protein of SS2 seems to be involved in the bacterial adhesion to host cells.Gene engineered vaccine is important on the prevention of Streptococcus suis, vaccination of recombinant trivalent protein GAPDH-MRP-HM6 which was made up of protein HM6, the protective antigen GAPDH and MRP was evaluated in swine. The 4-week-old piglets were randomly divided into test and control groups and immunized at interval of two weeks. The results of antibody assay showed that the immunized piglets had good immune response to the recombinant trivalent protein including HM6. Two weeks after booster immunization, the immunized and control piglets were challenged with SS2 HA9801. And the control piglets struck seriously while the immunized piglets were normal as usual. The results demonstrated that the recombinant trivalent protein provided good protection against challenge with virulent SS2 strains in piglets and would be a candidate of SS2 subunit vaccine. |
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