嗜水气单胞菌鞭毛蛋白基因flaB的克隆、表达及其免疫原性分析

Cloning,expression and antigenicity analysis of recombinant Aeromonas hydrophila flagellar FlaB protein

目的克隆、表达及纯化嗜水气单胞菌鞭毛FlaB蛋白,比较了它与天然鞭毛蛋白的抗原性,为以后鱼类弧菌病的防治及其疫苗的制备奠定了基础。方法利用PCR扩增出嗜水气单胞菌鞭毛蛋白基因flaB,经确定后将该基因克隆到原核表达载体pET-30a中,在大肠杆菌BL21(DE3)中获得了表达,并用电洗脱法对表达的蛋白进行纯化,用纯化的重组蛋白免疫家兔制备抗血清,并进行Western-blot分析。结果嗜水气单胞菌鞭毛蛋白flaB基因全长912bp,编码303个氨基酸,预测分子量为32kDa,Western-blot结果表明兔抗FlaB血清不仅能与重组鞭毛蛋白发生反应,而且能与天然鞭毛蛋白发生反应。结论鞭毛蛋白FlaB可能是嗜水气单胞菌的重要保护抗原之一,为下一步疫苗的制备奠定了基础。

We expressed flagellar FlaB protein of Aeromonas hydrophila in E .coli and analyzed its immunogenicity .In this study ,the flagellar gene f laB of Aeromonas hydrophila was amplified by PCR from the Aeromonas hydrophila genome ,and cloned into the prokaryotic expression vector pET30a .After being sequenced ,the recombinant plasmid pET30a‐flaB was transformed into strain BL21 （DE3） ,and FlaB protein was expressed by the recombinant strain after IPTG induction .By SDS‐PAGE and electrophoresis elution purification ,the purificated recombinant protein was inoculated to New Zealand rabbits .The full‐length of Aeromonas hydrophila FlaB flagellin genes was 912 bp ,which encodes 303 amino acids .The molecular weight of the induced protein was about 32 kDa as expected .Our result showed that the rabbit antibodies raised against the recombinant could recognize the natural flagellin and the recombinant protein ,suggesting flagellin FlaB may be an important protective anti‐gen of Aeromonas hydrophila and has potential for a novel target for vaccine development .