绵羊肺炎支原体延伸因子EF-P基因的克隆、表达及其重组蛋白免疫原性研究

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29.5 ku;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.%In order to screen the related immune proteins of Mycoplasma ovipneumoniae (MO),EF-P gene of Mycoplasma ovipneumoniae (MO) isolated from sheep was amplified by PCR and it's molecular characteristics was analyzed after was sequenced in this study.It's epitope clustering region was screened,cloned and expressed in prokaryotic cells.and then gene EF-P was amplified by PCR.It was synthesized and subcloned to pET-32a (+) to generate pET-32a(+)-EF-P.Then the pET-32a(+)-EF-P was transformed into competent cells of E.coli BL21 (DE3) for expression after induced by IPTG.and it's reactogenicity and were analyzed.SDS-PAGE showed that the molecular mass of recombinant protein was 29.5 ku.Western blot analysis showed that the recombinant protein was specifically recognized by the MO positive serum,and it had good reactogenicity.The immunization tests in mice showed that the recombinant protein could induce anti-serum antibody with the IHA titer of 1 ∶ 64,which confirmed that the recombinant protein had a strong immunogenicity.