牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

Study on Cloning,Prokaryotic Expression and Immunogenicity of P48 Gene of Mycoplasma bovis Xinjiang Isolate

试验旨在确定牛支原体P48基因的免疫原性,为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象,运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因,构建原核表达载体pET-32a(+)-P48,转化大肠杆菌BL21(DE3)感受态细胞,在诱导剂ITPG的诱导下获得重组蛋白P48,纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体,运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明,试验成功构建原核表达载体pET-32a(+)-P48,重组蛋白P48大小约为66ku,纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应,血清抗体滴度达到较高水平(D450nm值为1.126)。Western blotting结果显示,抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应,表明P48重组蛋白具有良好的免疫原性与反应原性,可作为牛支原体新型疫苗的候选基因,且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高,亲缘关系较近。

In order to determine the immunogenicity of Mycoplasma bovis P48 gene and lay the foundation for further screening of the immunoprotective gene of Mycoplasma bovis.In this study,Mycoplasma bovis Xinjiang isolate as the research object,Overlap PCR method was used to amplify P48 gene point mutation of Mycoplasma bovis Xinjiang isolate and construct the prokaryotic expression vector pET-32 a（＋）-P48,which was transformed into Escherichia coli BL21（DE3）,the recombinant protein P48 was obtained under the induction of ITPG.BALB/c mice was immunized by purified recombinant P48 protein to produce polyclonal antibody,the reactivity and immunogenicity of the recombinant protein were tested using Western blotting and ELISA methods.The results showed that the prokaryotic expression vector pET-32 a（＋）-P48 was successfully constructed,and the recombinant protein P48 was 66 ku.The purified recombinant P48 protein of Mycoplasma bovis immunized in mice could produce good immune response,and serum antibody titers reached a higher level（D450 nm=1.126）.Western blotting results showed that there was obvious antibody response of anti-MycoplasmabovisP48 recombinant protein of mouse serum,My-coplasma bovis P48 recombinant protein and Mycoplasma bovis total protein antigen,P48 recombinant protein had good immunogenicity and reactogenicity,which could be used as a candidate gene of Mycoplasma bovis vaccine.The homology of P48 gene was high between Mycoplasma bovis Xinjiang isolate and 5 domestic Mycoplasma bovis,and the genetic relationship was closer.