哈维氏弧菌外膜蛋白OmpK真核表达载体构建及在毕赤酵母中的表达

Construction of eukaryotic expression vector of OmpK of Vibrio harveyi and identification of protein expression in Pichia pastoris

采用已构建的原核重组质粒pET30a-OmpK为模板,通过PCR扩增OmpK成熟肽基因片段,引入酶切位点后克隆至分泌表达载体pPIC9K,经SalⅠ线性化后,氯化锂法转化至毕赤酵母GS115中,并经G418遗传霉素筛选高拷贝酵母转化子,最后进行甲醇诱导表达,表达上清经SDS-PAGE电泳检测及Western-blot鉴定,表明重组蛋白已经表达。

The existing research papers have demonstrated the strong immunogenicity of outer membrane proteins to aquacultured creatures.Using the previously constructed recombinant plasmid pET30a-OmpK as template,the peptide coding sequence of OmpK was amplified by PCR.After introducing restriction sites at both 5′ ends and 3′ends of the sequence,the gene was cloned into secreted expression vector pPIC9K,and formed the recombinant vector pPIC9K-OmpK.The vector was linearized by SalⅠ,and transformed into Pichia pastoris strain GS115 by the method of Lithium Chloride transformation.Transformants were selected by G418 and confirmed by PCR.The recombinant protein was expressed and secreted into the supernatant after inducing by methanol.SDS-PAGE analysis indicated the recombinant protein OmpK was hyperglycosylated with a molecular weight about 37 ku,larger than the expected size 29 ku.Western-blotting results showed that the recombinant protein OmpK could react with the antiserum against OmpK expressed by E.coli BL21.It indicated that the protein OmpK was glycosylated but remained its antigenicity.