PVX介导的槟榔坏死环斑病毒CP蛋白在烟草中的表达和纯化

Expression and Purification of PVX-mediated Areca Palm Necrotic Ringspot Virus Coat Protein in N.benthamiana

槟榔坏死环斑病毒(areca palm necrotic ringspot virus,ANRSV)是新发现的一种槟榔黄化病致病病毒。本研究利用马铃薯X病毒(potato virus X,PVX)表达载体pgR107,在烟草中表达ANRSV的外壳蛋白(coat protein,CP),旨在分离纯化该蛋白并开展对病毒蛋白的功能研究和利用该蛋白制备多克隆抗体。为方便纯化,将8肽Strep蛋白标签序列融合到ANRSV-CP编码基因的3’端,利用In-Fusion无缝克隆策略将其克隆到PVX载体,构建重组表达质粒PVX-ANRSV-CP-Strep。采用电转化法将质粒PVX-ANRSV-CP-Strep转入根瘤农杆菌GV3101中,通过农杆菌介导注射侵染接种本氏烟,14 d后对收集的16 g表达ANRSV-CP-Strep蛋白、PVX侵染的本生烟草样品进行了蛋白提取,采用SDS-PAGE和Western blot检测ANRSV-CP-Strep蛋白的表达情况。测序结果显示植物病毒表达载体PVX-ANRSV-CP-Strep构建成功。经Strep-Tactin■XT High Capacity亲和层析柱纯化,SDS-PAGE和Western blot检测结果显示ANRSV-CP-Strep重组蛋白可通过PVX载体在烟草中有效表达,其表达量为22μg/g(鲜叶质量)。本研究为后续利用该蛋白制备多克隆抗体并建立ELISA方法进行病毒检测提供了理论依据。

Areca palm necrotic ringspot virus(ANRSV)is a novel virus related to betel nut yellowing disease.In this study,the potato virus X(PVX)expression vector pgR107 was used to express the ANRSV coat protein(CP)in tobacco,aiming to isolate and purify the protein and carry out functional research.In order to facilitate purification,the 8-peptide Strep protein tag sequence was fused to the 3’end of the ANRSV-CP encoding gene,and it was cloned into the PVX vector using the In-Fusion seamless cloning strategy to construct the recombinant expression plasmid PVX-ANRSV-CP-Strep.Then,the plasmid PVX-ANRSV-CP-Strep was transformed into Agrobacterium tumefaciens GV3101 by electroporation,and the Agrobacterium-mediated injection was used to inoculate Nicotiana benthamiana.After 14 days,the protein was extracted from 16 g tobacco samples infected with PVX and expressed ANRSV-CP-Strep protein.The expression of ANRSV-CP-Strep protein was detected by SDS-PAGE and Western blot.Sequencing results showed that the plant virus expression vector PVX-ANRSV-CP-Strep was successfully constructed.The final results showed that the recombinant protein ANRSV-CP-Strep could be effectively expressed in tobacco through PVX vector,and the expression level was 22μg/g(Fresh leaf quality).This study provides a theoretical basis to prepare polyclonal antibodies and establish an ELISA method for virus detection.