摘要
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34Mr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。
Objective：To construct the plant expression vector of Der fl allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina. Methods：The Der fl gene was amplified from the glycerin bacterium which contained pE3ESa（ ＋ ）-Der fl plasmid, cloned into the pMD 19-T plasmid, and then sequenced. The Der fl gene was digested by Cla Ⅰ and Sal Ⅰ , and cloned into potato virus X （PVX） to construct plant expression vector PVX-Der fl, and then was transformed agrobacterium tumefaciens. The positive one was selected to infect tobacco lamina for expressing tan＇get 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 34Mr and it could specific binding with positive serum. Conclusion： The plant expression vector of Der fl is successfully constructed and the recombinant protein is also produced.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第3期250-253,共4页
Chinese Journal of Immunology
基金
国家自然科学基金(30860261)
海南省自然科学基金(807070)
