Abstract
Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder.
Highlights
Streptococcus iniae is a Gram-positive pathogen, which infects a wide range of marine and freshwater fish [1], including flounder (Paralichthys olivaceus) [2], resulting in serious economic losses around the world [3, 4]
The results showed that recombinant proteins of PDHA1 (rPDHA1), rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher relative percent survival (RPS) (62.5% and 75%, respectively) again
To screen the immunogenic proteins, the whole-bacterial proteins of S. iniae was analyzed by western blotting using flounder anti-S. iniae antibodies, and the results showed that two obvious protein bands with molecular weight (MW) of approximate 37 kDa and 40 kDa were recognized by flounder anti-S. iniae antibodies, and no band was observed in negative control (Fig 1A)
Summary
Streptococcus iniae is a Gram-positive pathogen, which infects a wide range of marine and freshwater fish [1], including flounder (Paralichthys olivaceus) [2], resulting in serious economic losses around the world [3, 4]. The high protective capacity of ΔsimA and ΔPGM mutant as a live attenuated vaccine candidate against S. iniae are demonstrated in aquaculture [11, 12]. These two kinds of vaccines have been developed as commercial vaccines for prevention of Streptococcus disease [13]. DNA vaccine is well-known for its advantages of inducing humoral and cellular immune responses, requiring no adjuvants and providing longer protection [14, 15], and a few proteins have been constructed as DNA vaccine against S. iniae, such as Sia and enolase, which could provide high protective efficacy to fish [16, 17]. It is important to identify immunogenic proteins to develop highly protective vaccines against S. iniae
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