CAJ | 학술논문

目的:构建尘螨变应原Der f1植物表达载体并侵染烟草叶片表达.方法:从保存的含pET28a(+)-Der f1的甘油菌株中扩增Der f1基因,并克隆到质粒载体中,提取质粒,进行测序;以ClaⅠ、SalⅠ双酶切,将Der f1基因克隆到马铃薯X病毒(PVX)载体中,构建植物病毒表达载体;将PVX-Der f1转化脓杆菌,挑取Kan、Tet抗性阳性的菌株侵染烟草叶片进行蛋白表达,采用SDS-PAGE和Western blot对表达蛋白进行鉴定和分析.结果:经SDS-PAGE分析,有4株烟草叶片蛋白提取物在34Mr处有特异性蛋白条带;经Western blot进一步验证其变应原,结果显示,烟草叶片中获得的重组蛋白在34M_r处与阳性血清发生特异性结合,而与阴性血清并不发生结合.结论:成功构建了植物病毒表达载体PVX-Der f1并获得表达,为尘螨变应原Der f1的研究提供新思路.
목적:구건진만변응원Der f1식물표체재체병침염연초협편표체.방법:종보존적함pET28a(+)-Der f1적감유균주중확증Der f1기인,병극륭도질립재체중,제취질립,진행측서;이ClaⅠ、SalⅠ쌍매절,장Der f1기인극륭도마령서X병독(PVX)재체중,구건식물병독표체재체;장PVX-Der f1전화농간균,도취Kan、Tet항성양성적균주침염연초협편진행단백표체,채용SDS-PAGE화Western blot대표체단백진행감정화분석.결과:경SDS-PAGE분석,유4주연초협편단백제취물재34Mr처유특이성단백조대;경Western blot진일보험증기변응원,결과현시,연초협편중획득적중조단백재34M_r처여양성혈청발생특이성결합,이여음성혈청병불발생결합.결론:성공구건료식물병독표체재체PVX-Der f1병획득표체,위진만변응원Der f1적연구제공신사로.
Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.