胸膜莫肺炎放线杆菌APXIV部分基因片段的表达及纯化

参照GenBank中已发表的ApxⅣ基因序列,以自行分离的AppDNA为模板,利用PCR方法扩增出ApxⅣ3’端,大小为552bp的保守基因序列。将PCR产物克隆到pMD18-T Simple Vector中,获得重组质粒pMD-ApxⅣ,对其重组质粒pMD-ApxⅣ进行BamHI、HindIII双酶切,并将酶切产物克隆到原核表达载体pET-32a(+)中,构建了重组表达质粒pET-ApxⅣ。将表达质粒转化至大肠杆菌BL21中,用IPTG诱导表达,通过SDS-PAGE和Western blot分析,结果表明pET-ApxⅣ在BL21中成功表达,并能被App阳性血清所识别,具有良好的免疫原性。表达蛋白的分子质量约为39.5KDa。利用HiTrapFFcrude columns将表达的蛋白进行了纯化。

Expression and Purification of Part ApxIV Genetic Fragment of Actinobacillus Pleuropneumoniae

According to the ApxⅣ sequence in GenBank and using DNA from the local isolate as template.Conservative gene fragment landed 3’ tip of ApxⅣ was amplified by PCR.The resulting product was cloned into pMD18-T Simple Vector and the recombinant plasmid pMD-Apx Ⅳ was obtained.The recombinant plasmid pMD-ApxⅣ was digested with BamHI and HindIII,and then the fragment was sub-cloned into the prokaryotic expressing vector pET-32a(+),obtaining recombinant plasmid pET-ApxⅣ.Then the pET-ApxⅣ was transformed into the BL21(DE3)competent cell of E.coli and was induced for protein expression by IPTG.SDS-PAGE and Western blot analysis indicated that the ApxⅣ had been expressed in BL21(DE3),and the expressed production had good immunogenicity.Molecular mass of the expression product was39.5KDa.Large quantities of expressed protein was purified by HiTrap FF crude columns.