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透明质酸酶(HylB)是无乳链球菌重要的毒力因子。根据本实验室分离并公布在GenBank上的罗非鱼源无乳链球菌GD201008-001全基因组序列,PCR扩增获得长为2 346 bp,覆盖hylB保守功能区的基因片段。将该片段采用限制性内切酶BamHⅠ和XhoⅠ消化后亚克隆至表达载体pET28a(+),经双酶切、测序鉴定后转化至大肠杆菌BL21(DE3)中,进行体外原核表达,获得88.5 ku的目的蛋白。将纯化后的目的蛋白免疫家兔制备多克隆抗体,经ELISA和Western blot鉴定该蛋白具有良好的免疫原性。
Hyaluronidase (HylB) is an important virulence factor of Streptococcus agalactiae. According to the whole genome sequence of Streptococcus agalactiae GD201008-001 isolated in our laboratory and published in GenBank, a 2 346 bp fragment covering the hylB conserved functional region was obtained by PCR. The fragment was subcloned into the expression vector pET28a (+) after digestion with restriction endonucleases BamH I and Xho I, and then double-digested and sequenced. The fragment was transformed into E. coli BL21 (DE3) for prokaryotic expression in vitro to obtain 88.5 ku Of the target protein. The purified target protein was immunized rabbit to prepare polyclonal antibody, and the protein was identified as good immunogenicity by ELISA and Western blot.