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目的:构建能够表达胞壁结合型贝氏柯克斯体 27 000外膜蛋白的重组卡介苗。方法:用PCR技术从卡介苗基因组扩增分枝杆菌 19 000抗原的细胞壁结合区基因和从贝氏柯克斯体基因组扩增 27 000外膜蛋白基因,将它们分别插入大肠杆菌-分枝杆菌穿梭质粒 (pSMT3)的XbaⅠ /BamHⅠ和BamHⅠ /HindⅢ位点。将重组的穿梭质粒转化大肠杆菌并从大肠杆菌提取重组穿梭质粒转化卡介苗。结果:从含潮霉素的培养基筛选到具有该抗生素抗性的重组卡介苗菌落,用免疫印迹分析重组质粒转化的卡介苗,发现一约 27 000的蛋白带与贝氏柯克斯体 27 000重组蛋白免疫兔血清发生特异性反应; 27 000重组蛋白免疫兔血清做间接免疫荧光检测重组卡介苗,结果为阳性。结论:重组卡介苗能表达贝氏柯克斯体 27 000外膜蛋白,表达的 27 000外膜蛋白存在于卡介苗菌体表面,卡介苗菌体表面的 27 000抗原可能诱导抗Q热免疫应答。
OBJECTIVE: To construct a recombinant BCG that expresses 27 000 outer membrane proteins of cell wall-bound C. cercis. Methods: PCR was used to amplify the cell-wall binding region gene of mycobacterium 19 000 antigen from BCG genome and amplify 27 000 outer membrane protein genes from C. bassiana genome and insert them into E. coli-Mycobacterium shuttle The XbaI / BamHI and BamHI / HindIII sites of the plasmid (pSMT3). The recombinant shuttle plasmid was transformed into Escherichia coli and the recombinant shuttle plasmid was extracted from Escherichia coli to transform BCG. Results: The recombinant BCG cell lines with this antibiotic resistance were screened from hygromycin-containing medium and the recombinant plasmid-transformed BCG was analyzed by immunoblotting. About 27,000 protein bands were found to be 27,000 recombinants Protein-immunized rabbit serum specific reaction; 27 000 recombinant protein immunized rabbit serum indirect immunofluorescence detection of recombinant BCG vaccine, the result was positive. CONCLUSION: Recombinant BCG could express 27 000 outer membrane proteins of C. cynostella and 27 000 outer membrane proteins expressed on the surface of BCG cells. 27 000 antigens on BCG surface may induce anti-Q thermal immune response.