【摘要】 为增强单个蛋白的抗原性,利用(Gly4Ser)3柔性连接肽将牛结核杆菌MPB70和ESAT-6融合。采用重叠延伸PCR技术将牛结核杆菌mpb70与esat-6基因连接,获得融合基因mpb70-esat-6,连接至T-Vector pMD19中,获得克隆质粒pMD-70-esat-6。经Bam HⅠ、EcoRⅠ酶切、纯化,并与p ET28a(+)载体连接,构建了pET-70-esat-6重组表达质粒。SDS-PAGE发现,在27.2 ku处表达了融合蛋白MPB70-ESAT-6,Western blotting证实,MPB70-ESAT-6与牛结核阳性血清反应性良好。MPB70-ESAT-6融合蛋白的研究为牛结核病诊断抗原及相关疫苗研究奠定了基础。更多还原

【Abstract】 To enhance the antigenicity of individual protein,Mycobacterium bovis( M. bovis) strain MPB70 was fused with ESAT-6 via a flexible linker,( Gly4 Ser)3. The fusion gene mpb70-esat-6 was obtained using overlap extension PCR and linked to pMD19 T-Vector. After Bam H Ⅰ and EcoR Ⅰ restriction enzyme digestion,the cloned plasmid pMD-70-esat-6 was connected with pET28 a( +) vector to construct a recombinat plasmid p ET-70-esat-6. SDS-PAGE showed that fusion protein MPB70-ESAT-6 was expressed at 27.2 ku. MPB70-ESAT-6 exhibited good reactivity with M. bovis positive serum confirmed by Western blotting analysis. The current study provides experimental basis for research of fusion protein MPB70-ESAT-6 as bovine tuberculosis diagnostic antigen and vaccine.更多还原