转禽冠状病毒免疫原基因马铃薯及其对小鼠的免疫原性  

Transgenic Potato Containing Immunogenic Gene of Avian Coronavirus and Its Immunogenicity in Mice

作　　者：吴建祥[1] 周继勇[1] 周雪平[2] 

机构地区：[1]浙江大学动物预防医学研究所,杭州310029 [2]浙江大学生物技术研究所,杭州310029

出　　处：《生物化学与生物物理学报》2003年第11期1011-1015,共5页

基　　金：国家自然科学基金 (No .3 0 0 70 5 70 );教育部重点项目 (No .0 3 0 86)资助~~

摘　　要：为了探索禽冠状病毒免疫原基因在植物中表达的可行性 ,将传染性支气管炎病毒 (IBV) ZJ971毒株S1基因定向克隆到pBI1 2 1质粒的 35S启动子下游。用三亲交配法将重组质粒导入EHA1 0 5根癌农杆菌 ,建立了一种由农杆菌介导的马铃薯转化体系 ,其愈伤形成率达 1 0 0 % ,芽的分化率达 95 %。经PCR和Southern印迹检测证明 ,IBVS1基因已整合到植物基因组内 ,获 4 7株转基因植株。Northern印迹和ELISA分析表明 ,转基因植株中IBVS1基因能正常转录和翻译。转基因马铃薯的BALB/C小鼠免疫试验结果表明 ,0 .5g和 1 g剂量转基因马铃薯免疫小鼠 ,2 1天后的抗体ELISA效价分别达 1∶2 0～ 1∶4 0和 1∶80 1∶1 6 0 ,中和抗体效价分别达 1∶1 6 5～ 1∶5 6 2和 1∶330～ 1∶2 0 4 8。To check the feasibility of expression of the immunogenic gene of avian coronavirus infectious bronchitis virus (IBV) in plants, the transformation of S1 gene of IBV into potato and the immunogenicity of its expression product was studied. The S1 gene of IBV ZJ971 strain was inserted into plasmid pBI121 under the control of 35 S promoter. Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri parental mating method. So, an efficient potato transformation system mediated by Agrobacterium fumefaciens was established. The rates of calli and shoots differentiation were 100%, and more than 95% respectively, for transgenic potato with S1 gene of IBV. PCR and Southern blot analyses showed that IBV S1 gene was integrated into genomic DNA of the potato plant and most transgenic plants had two copies of S1 gene of IBV. In our experiments, 47 transgenic plantlets have been obtained. Northern blot and ELISA analyses indicated that most transgenic plants could normally transcribe and translate S1 gene of IBV, though the levels of transcription and translation were different in various transgenic plants. Immunity assay with BALB/C mice showed that expression products of transgenic potato with S1 gene of IBV were immunogenic, and ELISA antibody titer reached 1∶20 to 1∶40 and 1∶80 to 1∶160 with doses of 0.5 g and 1 g, respectively. Virus neutralization (VN) antibodies were detected by tracheal organ cultures, and the results showed that VN titers reached respectively 1∶160 to 1∶320 and 1∶320 to 1∶2048 with doses of 0.5 g and 1 g.

关 键 词：禽 冠状病毒 免疫原 基因 转基因马铃薯 抗体 传染性支气管炎 

分 类 号：S852.5[农业科学—基础兽医学]