中文摘要：

本研究以牛分枝杆菌valleeⅢ菌株的基因组DNA为模板,PCR扩增hbha和hsp65基因,采用重叠延伸剪接技术（SOE）获得融合基因hbha-hsp65,构建重组表达质粒p ET-hsp65、p ET-hbha、p ET-hsp65-hbha,转化至大肠杆菌BL21（DE3）中进行诱导表达。表达蛋白纯化后与等体积的弗氏不完全佐剂混合,制备成浓度为150 g/m L的HBHA-HSP65亚单位疫苗。用其免疫BALB/c小鼠,评价其免疫保护效力。结果显示,表达蛋白的分子质量分别为65、28、95 ku,主要以可溶状态存在;ELISA测定的HBHA-HSP65的血清抗体效价达1∶16 000,显著高于HBHA、HSP 65和BCG组（P〈0.05）;经MTT法测定,平均刺激指数为2.45,HBHA-HSP65组显著高于HSP65（P〈0.05）、HBHA和BCG免疫组（P〈0.01）和PBS（P〈0.01）;HBHA-HSP65的脾、肺荷菌量检测结果显示,该亚单位疫苗可明显降低牛分枝杆菌在小鼠脾的荷菌量（P〈0.05）,几乎完全切断对肺的感染（P〈0.01）。结果表明,HBHA-HSP65亚单位疫苗能够在小鼠体内激发细胞免疫和体液免疫应答,对牛分枝杆菌感染小鼠有一定的预防保护效果,具有良好的免疫原性。

英文摘要：

To study the expression of fusion adhesin protein HBHA-HSP65 of Mycobacterium bovis, the genes of hbha and hsp65 were amplified from genome of valle III by PCR and the fusion gene hbha-hsp65 was obtained using splicing overlap extension（SOE）.hbha,hsp65,and hbha-hsp65 were cloned into the prokaryotic expression vector p ET28a（＋）,and then the recombined plasmids were transformed into Escherichia coli BL21（DE3）to induce its expression. After purified, the expressed proteins were mixed with Freund＇s incomplete adjuvant in equal value and then were used to prepared subunit vaccine ‘HBHA- HSP65＇ with a concentration of 150 g/m L. The antibody titer of vaccine was determined by ELISA and its immune protective efficacy was determined by the immunized BALB/c mice. SDS-PAGE analysis showed that the fusion proteins, hbha,hsp65, and hbha-hsp65, expressed as a soluble proteins with molecular weight of 65 ku, 28 ku, and 95 ku,respectively. The serum antibody titer of HBHA-HSP65 group was 1 ∶ 16 000 and was significantly higher than the groups of HBHA, HSP65, and BCG（Bacille Calmette- Guerin）（P〈0.05）.The average stimulation index of HBHA-HSP65 group（2.45） was significantly higher than the groups of HBHA, BCG, PBS（P〈0.01）, as well as HSP65（P〈0.05）. Spleen and lungs bacteria detection showed that this subunit vaccine could significantly reduce the amount of M.bovis in the spleens of mice and thus cut off the lung infections almost completely.These results showed that the subunit vaccine ‘HBHA-HSP65＇ could stimulate cellular and humoral immune responses in mice, which provided a good immunogenicity for the infections of mycobacterium.