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利用PCR技术克隆人乳头瘤病毒HPV 16L1蛋白编码基因 ,将其重组于pUCmT和pBI12 1中 ,构建含HPV 16L1基因的植物双元表达载体pBI L1,L1基因由CaMV 35S启动子控制表达。采用叶盘共培育法经根瘤农杆菌介导转化烟草 (NicotianatobacumL .) ,获得HPV 16L1转基因烟草植株。经PCR及Southern杂交分析 ,HPV 16L1基因整合到烟草基因组中 ;Westernblot和ELISA分析检测显示转基因烟草叶片蛋白可与HPV 16L1单克隆抗体特异性反应 ,且定位于 5 5kD处 ,其最高表达量占烟草叶片总可溶蛋白的 0 0 76 %。小鼠红细胞凝集试验 (HA)及小鼠红细胞凝集抑制试验 (HAI)显示转基因烟草叶片蛋白可引起小鼠红细胞凝集。结果表明已成功地构建了HPV 16L1的植物双元表达载体 ,并证实了利用转基因烟草植物能够表达出HPV 16L1蛋白 ,所表达的L1蛋白具有良好的抗原性并具有介导小鼠红细胞凝集的生物活性
The HPV16L1 protein coding gene was cloned by PCR and recombined into pUCmT and pBI12 1 to construct the plant binary expression vector pBI L1 containing the HPV 16L1 gene. The L1 gene was controlled by the CaMV 35S promoter. Tobacco (Nicotianatobacum L.) mediated by Agrobacterium tumefaciens was transformed by leaf disc co-culture method to obtain HPV 16L1 transgenic tobacco plants. The HPV16L1 gene was integrated into the genome of tobacco by PCR and Southern blot analysis. The results of Western blot and ELISA showed that the tobacco leaf protein of transgenic tobacco could specifically react with HPV16L1 monoclonal antibody and localized at 55kD, Leaf total soluble protein 0 0 76%. Mouse Hemagglutination Test (HA) and Mouse Hemagglutination Inhibition Assay (HAI) showed that transgenic tobacco leaf protein can cause mouse erythrocyte agglutination. The results showed that the plant binary expression vector of HPV 16L1 has been successfully constructed and confirmed that the use of transgenic tobacco plants can express the HPV 16L1 protein, the expressed L1 protein has good antigenicity and has the biological activity of mouse erythrocyte aggregation active