抗汉坦病毒单链抗体基因向拟南芥遗传转化的初步研究  

A study on the transformation of single chain Fv gene against hantavirus into Arabidopsis thaliana

作　　者：罗雯[1] 吴兴安[2] 李莺[1] 徐志凯[2] 

机构地区：[1]西安文理学院生命科学系,西安710065 [2]第四军医大学微生物学教研室,西安710032

出　　处：《中国人兽共患病学报》2007年第12期1202-1206,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No30471626);西安市科技局社会与发展计划(NoSF200345);西安文理学院专项科研基金(NoKY2004024)联合资助

摘　　要：目的构建抗汉坦病毒mAb1A8scFv的转基因拟南芥植株。方法从含有1A8scFv基因的重组质粒中酶切获得携带Nos终止子的抗体基因片段,将其与CaMV35S启动子相连克隆入载体pCAMBIA2301,构建1A8scFv基因的植物表达载体1A8scFv-pCAMBIA2301。通过农杆菌介导的花粉管法将其转入拟南芥,PCR及Dotblot检测是否获得转基因植株,ELISA和固相酶联斑点实验检测表达产物的生物学活性。结果酶切鉴定结果证明1A8scFv基因被成功克隆入植物表达载体pCAMBIA2301,构建获得1A8scFv-pCAMBIA2301重组质粒。PCR及Dotblot检测证明获得抗汉坦病毒mAb1A8scFv的转基因拟南芥植株。ELISA和固相酶联斑点实验结果证明在转基因拟南芥中成功表达了具有生物学活性的抗汉坦病毒mAb1A8scFv片段。结论成功地将外源基因转入拟南芥中并表达,为进一步研究利用植物表达医用抗体奠定了基础。This study aimed at constructing the transgenic Arabidopsis thaliana containing single chain Fv gene of monoclonal antibody 1AS against hantavirus. 1AS scFv gene fragment with Nos terminator was obtained from its recombinant vector by digestion with restriction enzyme and subsequently cloned into pCAMBIA2301 with CaMV 35S promoter to generate plant expression vector 1ASscFv-pCAMBIA2301which was proved by restriction enzyme analysis. The infection of Agrobacterium tumefaciens was used to transform Arabidopsis thaliana mean while PCR and Dot blot of transformed Arabidopsis thaliana DNA were used to select the transgenic ones. ELISA and Dot-ELISA were used to identify the biological activity of expressed products in transgenic plants. The results showed that the biologically active scFv fragment of mAb 1AS against hantavirus had been expressed in transformed Arabidopsis thaliana. It evident that the foreign gene was transformed into Arabidopsis thaliana successfully. This study has laid a foundation for further study on expression of certain antibody in plant.

关 键 词：汉坦病毒 单链抗体 植物表达载体 拟南芥 转基因植物 

分 类 号：Q78[生物学—分子生物学] R394.2[医药卫生—医学遗传学]