辣椒脉斑驳病毒外壳蛋白多克隆抗体的制备和应用

Preparation and Application of Polyclonal Antibody of Chilli Veinal Mottle Virus Capsid

【目的】辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)是茄科植物生产上危害最严重的病毒之一,也是口岸检疫的对象。本研究旨在制备特异性强的ChiVMV外壳蛋白的多克隆抗体并用于田间病株的检测。【方法】通过RT-PCR方法扩增ChiVMV贵州烟草分离物外壳蛋白基因的全长(861 bp)和部分片段(396 bp),分别重组至原核表达载体pET28a中,转化Escherichia coli BL21后进行诱导表达,表达产物层析分离和透析纯化后免疫大耳兔制备多克隆抗体,最后使用酶联免疫吸附法(ELISA)、Western blot等方法检测抗体效价和特异性。【结果】成功制备了ChiVMV外壳蛋白的两种多克隆抗体antiCP^(1-287aa)、antiCP^(1-132aa),抗体效价分别为1∶6400、1∶12800;两种抗体antiCP^(1-287aa)、antiCP^(1-132aa)均能通过Western blot方法检测到抗原;田间病株的间接ELISA检测显示,antiCP^(1-132aa)能特异性检测ChiVMV病株,未能检测到马铃薯Y病毒(Potato virus Y,PVY)病株,而antiCP^(1-287aa)无法区分这两种病株。【结论】多克隆抗体antiCP^(1-132aa)的特异性较强,可用于ChiVMV田间病株的检测,也为ChiVMV外壳蛋白的功能研究奠定基础。

【Objective】A highly specific polyclonal antibody of the capsid of chilli veinal mottle virus(ChiVMV)was prepared for field detection and quarantine inspection on infected Solanaceae plants.【Methods】The full-length(861 bp)and partial fragment(396 bp)of the gene from a ChiVMV GZ-Tabacco isolate were amplified using RT-PCR,recombined into the prokaryotic expression vector pET28a,and transformed into E.coil BL21 for induced expression.After chromatographic isolation and dialysis purification,the products were used to immunize Dahl rabbits for the antibody preparation.Potency and specificity of the candidate antibodies were evaluated by ELISA and western blot.【Results】Two polyclonal antibodies,namely antiCP^(1-287aa)and antiCP^(1-132aa)with the titers of 1∶6400 and 1∶12800,respectively,were obtained.Both positively detected the antigen as shown by western blot.But the indirect ELISA on the field strains revealed that antiCP^(1-132aa)specifically detected ChiVMV not potato virus Y(PVY),whereas antiCP^(1-287aa)failed to differentiate the two.【Conclusion】The identified polyclonal antibody,antiCP^(1-132aa)was highly specific in detecting ChiVMV in field tests.It could become a useful tool for further studies on the infectious virus of chilli peppers.