【摘要】 以猪传染性胸膜肺炎放线杆菌血清1型国内分离株721株基因组DNA为模板,用PCR方法扩增出ApxⅣA基因2.5kb特异片段,将其克隆于pMD18T中,经酶切鉴定筛选重组质粒后进行核苷酸序列测定,并与GenBank中登录的血清1型ApxⅣA(AX002405)基因进行比较,结果显示核苷酸同源性为99.5%。将该片段亚克隆到原核表达载体pGEX6P1的EcoRⅠ/SalⅠ位点,成功地构建了重组表达载体pGEXapxⅣA,并转化大肠埃希氏菌BL21,获得了表达。SDSPAGE检测结果显示,表达的融合蛋白分子质量约为117ku,与预期片段大小相符;Westernblotting分析证实,该融合蛋白具有免疫学活性。更多还原

【Abstract】 The DNA fragment of Actinobacillus pleuropneumoniae(APP) ApxⅣA was amplified from the genomic DNA of APP serotype 1 strain 72-1 by PCR and cloned into the pMD18-T vector. The sequence analysis showed that the cloned gene shared 99.5% nucleotide homology with that of APP serotype 1 reference strains in GenBank. After the gene was subcloned into the expression vector pGEX-6P-1, the recombinant plasmid pGEX-apxⅣA was obtained. By transformation of pGEX-apxⅣA into E.coli BL21 and subsequent induction with IPTG, an expected fusion protein was expressed properly. The fusion protein was immunogenic when detected by Western-blotting analysis.更多还原