马腺疫链球菌FNE基因的克隆及原核表达

Cloning and Prokaryotic Expression of Truncated FNE Gene from Streptococcus equinus

采用PCR方法从马腺疫链球菌新疆分离株中扩增FNE基因片段,克隆至pMD19-T载体。利用分子生物学软件分析测序结果,将FNE截短基因亚克隆至原核表达载体pET-30a中,转化到大肠埃希菌(E.coli)BL21(DE3)感受态细胞,以IPTG诱导FNE重组蛋白的表达,用SDS-PAGE和Western blot法分析蛋白表达及其反应原性。序列分析显示,其与GenBank收录的马腺疫链球菌4047(登录号YP002747216)核苷酸序列和氨基酸序列同源性均为99%。采用PCR法扩增到675bp的FNE截短基因片段构建重组表达载体获得重组蛋白,经SDS-PAGE分析在46ku处出现明显条带,Western blot分析显示具有反应原性。成功克隆和表达了马腺疫链球菌的FNE截短基因,为重组FNE蛋白亚单位疫苗的研制奠定了基础。

A fragment of FNE gene was amplified from Streptococcus equinus isolated from Xinjiang by PCR and cloned into pMD19-T vector.After sequence analysis by molecular biology softwares,the trun-cated FNE gene was subcloned into the prokaryotic expression vector pET-30a and resulting plasmid was transformed into host BL21 （DE3）cells,in which a recombinant protein was expressed by inducing with IPTG.Then the expression and reaction of the recombinant proteins were detected by SDS-PAGE and Western blot.Results：Sequence analysis indicated that a 675 bp fragment of FNE gene was obtained by amplification by PCR,whose relative similarities in nucleotide and amino acids compared to the published sequences of Streptococcus equinus 4047（GenBank accession number YP002747216）all were 99%,respec-tively.After transformation of recombinant expression plasmid into host BL21 （DE3）cells,the SDS-PAGE analysis revealed that the expressed recombinant protein was 46 ku.Western blot showed its reac-tivity.It is concluded that the expression of truncated FNE protein lays foundation for the development of recombinant subunit vaccines against equine strangles.