绵羊肺炎支原体延伸因子EF-P基因的克

发布时间：2018-07-02 22:17

本文选题：绵羊肺炎支原体 + EF-P　；参考：《西北农业学报》2017年11期

【摘要】：为筛选绵羊肺炎支原体(MO)免疫相关蛋白,以MO新疆流行株基因组DNA为模板,通过PCR扩增其EF-P基因,测序后对其编码蛋白进行分子特征分析;用生物信息学软件预测其抗原表位集中区编码片段;进一步将该基因片段克隆入pET-32a(+)中,构建原核表达载体pET-32a(+)-EF-P,将pET-32a(+)-EF-P质粒转化至感受态细胞Escherichia coli BL21(DE3)中,IPTG诱导目的蛋白表达后,检测重组蛋白反应原性和免疫原性。SDS-PAGE结果显示,EF-P重组蛋白分子质量为29.5ku;Western blot结果显示,该重组蛋白可被MO阳性血清特异性识别,证实其具有良好的反应原性;小鼠免疫试验结果显示,该重组蛋白可诱导机体产生特异性抗体,采用正向间接血凝方法检测效价可达1∶64,提示其具有较强的免疫原性。
[Abstract]:In order to screen mycoplasma pneumoniae (MO) immune-related protein, the EF-P gene was amplified by PCR using genomic DNA of MO Xinjiang epidemic strain as template, and its encoding protein was analyzed by molecular characterization after sequencing. Using bioinformatics software to predict the encoding fragment of epitope concentration region, the gene fragment was further cloned into pET-32a (), and the prokaryotic expression vector pET-32a () -EF-P was constructed. The plasmid pET-32a () -EF-P was transformed into Escherichia coli BL21 (DE3), and the target protein was induced by IPTG. The reactivity and immunogenicity of the recombinant protein. SDS-PAGE showed that the molecular weight of EF-P recombinant protein was 29.5ku.Western blot showed that the recombinant protein could be specifically recognized by MO-positive serum, which confirmed that the recombinant protein had good reactivity. The results of mouse immunological test showed that the recombinant protein could induce the production of specific antibody, and the titer of positive and indirect hemagglutination could reach 1: 64, which indicated that the recombinant protein had strong immunogenicity.
【作者单位】：石河子大学动物科技学院;中国农业科学院兰州兽医研究所;
【基金】：国家国际科技合作专项(No.2014DFR31310) 兵团中青年科技创新领军人才计划(2016BC001)~~
【分类号】：S852.62

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