Analysis Of The Protective Mechanism Of GAPCH And Identification Of Its Key Region

Edwardsiella tarda is a Gram-negative pathogen which causes systemic infection in fish, which has caused great damage to aquaculture. Unfortunately, until now there is no an effective vaccine that could prevent E. tarda’s infection.In our previous study, GAPDH had been proved to be an potential protective antigen, and has a good protective effect for pathogens from five mariculture fish. For good understanding the protective mechanisms of GAPDH, the gene of GAPDH from E. tarda was cloned, expressed and purified in Escherichia coli. The RPS of GAPDH-vaccinated turbot was above60%when challenged with EIB202. The level of specific IgM was higher in the serum of vaccinated-fish and enhanced bactericidal activities of sera were observed, these results showed that humoral immunity was obvious induced by GAPDH-vaccination. Moreover, obvious cross-reactions were found between IgM antibodies induced by recombinant GAPDH of EIB202and other five different pathogens in aquaculture.We also analyzed the transcription levels of four immune-related genes in liver, spleen and kidney of GAPDH-vaccinated turbot by qRT-PCR. At the end, the expression of four genes(MHC-Ⅰ, MHC-Ⅱ, IL-1βand IgM) were up-regulated after vaccination, among which, therelative expression of MHC-Ⅰand MHC-Ⅱ wereboth raised up to the peak (20-fold and70-fold, respectively) in liver in1dpv, while the relative expression of IgM was up-regulated to14-fold in spleen after3dpv. Also therelative expression of MHC-II was raised up to the peak (10-fold) in kidney in14dpv. These results showed that both humoral and cellular immune responses were soundly aroused in vaccinated fish.Finally, we analyzed the key region of GAPDH. We successfully constructed eight different recombinant plasmids which have different regions of GAPDH, and the proteins were successfully purificated. Fish vaccinated with a deletion of GAPDH exhibited different RPS when challenged with E. tarda EIB202, among which, GapA(3), GapA(4) and GapA(5) row in the back of the RPS. The relevant deletions are Ⅲ (333-489bp), Ⅳ (490～660bp) and V (661～840bp), respectively. These results showed that the key region of GAPDH may exist in333-840bp or111～280AA.In a word, these results suggested that GAPDH could be a broad spectrumprotective antigen and would be a potential candidate for a subunit vaccine in the furture.