迟钝爱德华菌鞭毛基因克隆表达及其免疫学活性分析

从鳗源迟钝爱德华菌Edwardsiella tarda菌株ETY的基因组克隆到其鞭毛基因(flagella gene,ETF)。该基因开放阅读框为1 257bp,编码419个氨基酸,推导的蛋白分子量为43.951kD。ELISA和Western-blotting试验证实表达的蛋白与迟钝爱德华菌菌株ETY表达的鞭毛蛋白具有相同的抗原性和免疫原性。免疫攻毒保护试验证明:经表达产物(ETF-rxA融合蛋白)与ISA佐剂结合免疫的日本鳗鲡对爱德华菌ETY的免疫保护率可达到100%。本试验首次成功实现了ETF基因的高效表达,并初步证实了迟钝爱德华菌鞭毛可诱导产生免疫保护,为进一步研制合适的高效的迟钝爱德华菌鞭毛亚单位疫苗奠定了基础。

Prokaryotic Expression and Immunological Analysis of the Flagella Gene of Edwardsiella tarda

Edwardsiella tarda strain ETY was isolated from Japanese eel.With two pairs of specific primers,the flagella gene(ETF) was amplified from the strain ETY via nest-PCR.After sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software.The analysis results showed that ETF had a longest open reading frame(ORF) of 1 257 bp,which was predicted to encode a 419-aa protein with the molecular weight of 43.951 kD.Considering ELISA(enzyme-linked immunosorbent assay) analysis and Western-blotting analysis,it was proved that the expressed gene products shared similar antigenicity and immunocompetence with the natural flagella.The protective rate in the Japanese eel immunized with the ETF-TrxA fusion protein and ISA adjuvant is 100%.It is the first time that the ETF gene was efficient expressed in E.coli.,and it also primarily studied the ETF function of Edwardsiella tarda in invasion and inducing immunoreaction.The results enable the development and formulation of an appropriate and effective subunit vaccine(s) against Japanese eel edwardsiellosis.