Research For The Flagllin Of Edwardsiella Tarda

A single bacteria strain was isolated from one Japonica eel suffering from ascites. Using biochemical testing it was confirmed as Edwardsiella tarda (ET). The ETY was injected into the abdomen of more Japonica eels to observe symptoms. This was used to prove that Edwardsiella tarda ETY is a bacterial pathogen of Japonica eel.By the nest-PCR and the biochemical method, the suspended Edwardsiella tarda strains preserved in our lab were checked as well. The results of the nest-PCR were the same as the results from the biochemical method. The results confirmed that the strains ETY,ETV,ETLi and ET030906 were Edwardsiella tarda, the strains ET1 and ET2 were not. This shows the nest-PCR technique can detect Edwardsiella tarda quickly and accuratly.With the anti-ETY serum and the anti-ETV serum, the assured strains were distinguished into two serum types: ETY,ETLi, and ET030906 in one type, and ETV in the other.Using several methods the flagellin of Edwardsiella tarda (ETFP) were distilled from Edwardsiella tarda. Finally, the most effective method was chosen, while the mouse anti- ETFP serum and anti- ETY serum was being prepared. The antigenicity and immunogenicity of the ETFP were analyzed using ELISA and Western-blotting. The results showed that the method of Acidification and high-speed centrifuging could get the high-purity ETFP weighted to about 44kD. The protein could be recognized by the mouse anti- ETFP serum and anti-ETY serum, while the mouse anti- ETFP serum could recognize the 44kD protein band of the whole protein of ETY This proved the ETFP had antigenicity and immunogenicity, and thus it could be considered a candidate for the subunit vaccine against Japonica eel edwardsiellosis.To get a considerable amount of ETFP, a flagellin gene (ETF) was amplified by means of nest-PCR from the genomic DNA of Edwardsiella tarda strain ETY. The sequencing analysis showed that ETF had a full-length open reading frame (ORF) of 1257nt, which was predicted to encode a 43.951kDa protein made up of 418 amino acids. The ETF was inserted into the expressional plasmid pET32b(+), and the recombined plasmid was transfered into E.coli BL21 (DE3). The yield of the expressed ETFP-TrxA fusion protein was as much as 50.1% total bacterial protein. The results of the ELISA and Western-blotting demonstrated that the expressed ETFP-TrxA fusion protein shared similar antigenicity and immunogenicity with the native flagellin protein. The immunotrial proved that the Japonica eel immunized with the ISA763A adjuvant emulsified ETFP-TrxA fusion protein gave 100% protection when they were challenged at day 21 post-injection. In these experiments, the gene ETF of Edwardsiella tarda ETY was successfully cloned and the high efficiency expression of the gene product was obtained by transforming the E.coli BL21 (DE3) with the recombined plasmid. It was also proven that the flagellin protein of Edwardsiella tarda could induce the immunized eel to generate protective immunity against Edwardsiella tarda. The results provide a valuable basis for the development of an effective subunit vaccine against Japonica eel edwardsiellosis.The established nest-PCR can detect Edwardsiella tarda quickly. In China, our first procedure was to distill the flagellin of Edwardsiella tarda, and then choose the best method for distilling the protein. The gene ETF of Edwardsiella tarda ETY was successfully cloned and the high efficiency expression of the gene product was obtained. This proves that the flagellin protein of Edwardsiella tarda can induce the immunized eel to generate protective immunity. The results provide a valuable basis for the clinical quick test of edwardsiellosis and the development of an effective subunit vaccine with the flagellin of Edwardsiella tarda.