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试验构建猪肺炎支原体重组表达质粒pET30a-LppT,通过快速定点突变试剂盒将LppT基因中513、945、1 860、2 547 bp处的A突变为G,使改造后的LppT基因能在大肠杆菌系统中正确表达,IPTG诱导重组蛋白的表达并进行了蛋白纯化,纯化的目的蛋白与佐剂混合、乳化制备免疫原,免疫新西兰大白兔制备LppT蛋白多克隆抗体。SDS-PAGE分析结果显示,IPTG诱导表达后获得1条特异性蛋白条带,分子质量约为110 ku,与预期蛋白大小一致。可溶性分析结果表明目的蛋白以可溶形式表达于上清中,纯化的蛋白经SDS-PAGE分离后,用制备的LppT多克隆抗体进行Western Blot检测,结果表明制备的LppT多克隆抗体具有较强的特异性。
To construct the recombinant plasmid pET30a-LppT of Mycoplasma hyopneumoniae, the A site of 513,945,1 860,2 547 bp in LppT gene was mutated to G by rapid site-directed mutagenesis kit so that the transformed LppT gene could be expressed in Escherichia coli system IPTG was used to induce the expression of the recombinant protein and the protein was purified. The purified target protein was mixed with the adjuvant, emulsified to prepare the immunogen, and immunized New Zealand white rabbits to prepare LppT polyclonal antibody. SDS-PAGE analysis showed that one specific protein band was obtained after induced by IPTG. The molecular weight was about 110 ku, which was consistent with the expected protein size. Soluble analysis showed that the target protein was expressed in soluble form in the supernatant. The purified protein was separated by SDS-PAGE and detected by Western Blot with the prepared LppT polyclonal antibody. The results showed that the prepared LppT polyclonal antibody had stronger Specificity.