绵羊肺炎支原体膜蛋白p74基因的克

发布时间：2018-03-16 13:01

  本文选题：绵羊肺炎支原体　切入点：p基因　出处：《西南农业学报》2017年05期 　论文类型：期刊论文

【摘要】：【方法】利用PCR方法对绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)新疆分离株MO-XJ p74基因进行扩增、克隆及测序,分析其分子特征。将P74蛋白抗原集中区编码序列亚克隆至表达载体p ET-32a(+),构建重组表达载体p ET-32a(+)-p74C,转化至E.coli BL21(DE3)感受态细胞中,IPTG诱导表达。【结果】MO-XJ p74基因全长2016 bp,编码671个氨基酸;对编码的氨基酸序列分析发现,该蛋白不含信号肽序列,9~31位氨基酸序列含有1个跨膜区,有16个N-糖基化位点、7个N-酰基化位点、17个酪蛋白激酶Ⅱ磷酸化位点及5个蛋白激酶C磷酸化位点。同源性分析显示MO-XJ P74蛋白氨基酸序列与标准株MO-SC01氨基酸序列同源率为86.5%,其羧基端为P74蛋白抗原集中区。SDS-PAGE检测结果显示,表达的P74蛋白抗原表位集中区片段对分子质量约为35.5 k Da,与理论值相符;Western blot分析表明重组P74蛋白具有较强的反应原性。【结论】本研究为进一步筛选MO亚单位疫苗及血清学诊断的候选抗原奠定了前期基础。
[Abstract]:[methods] the MO-XJ p74 gene of Mycoplasma ovis pneumoniae MOA Xinjiang isolate was amplified, cloned and sequenced by PCR. By analyzing its molecular characteristics, the coding sequence of P74 antigen concentration region was subcloned into the expression vector pET-32a( pET-32a), and the recombinant expression vector pET-32a (pET-32a) was transformed into E. coli BL21DE3. [results] the full-length MO-XJ p74 gene was expressed in 166bp. Encoding 671 amino acids; The amino acid sequence analysis showed that the protein contained a transmembrane region without a signal peptide sequence. There were 16 N-glycosylation sites, 7 N-acylation sites, 17 casein kinase 鈪，

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