Preparation and Application of Polyclonal Antibody of Chilli Veinal Mottle Virus Capsid
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摘要:
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antiCP1-287aa、antiCP1-132aa，抗体效价分别为1∶6400、1∶12800；两种抗体antiCP1-287aa、antiCP1-132aa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，antiCP1-132aa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antiCP1-287aa无法区分这两种病株。 结论 多克隆抗体antiCP1-132aa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。 Abstract: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 antiCP1-287aa and antiCP1-132aa 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 antiCP1-132aa specifically detected ChiVMV not potato virus Y (PVY), whereas antiCP1-287aa failed to differentiate the two. Conclusion The identified polyclonal antibody, antiCP1-132aa 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. -
图 2 两种抗原相应基因片段的扩增及其表达载体构建
M1、M2，核酸分子标准DL2000、DL5000 (宝生物)；A：1、2，ChiVMV CP1-287aa和CP1-132aa序列扩增；B：3、4，ChiVMV CP1-287aa和CP1-132aa重组质粒双酶切
Figure 2. Amplification and expression vector construction of corresponding gene fragments of two antigens
M1, M2: DL2000 DNA marker DL2000, DL5000 (Takara); A: 1, 2, amplification of ChiVMV CP1-287aa and CP1-132aa fragment; B: 3, 4, double enzyme digestion of recombinant plasmid of ChiVMV CP1-287aa and CP1-132aa fragment
图 3 抗原蛋白的纯化
M，预染蛋白分子量标准26616（赛默飞）；A为CP1-132aa蛋白纯化；B为CP1-287aa蛋白纯化；1、11，未层析样品；2、12，过柱的未层析样品；3～10、13～21，不同浓度（依次为30、40、50、60、70、80、90、100、110 mmol·L−1）的咪唑洗脱液。
Figure 3. Purification of antigenic proteins
M: Prestained protein Ladder 26616 (Thermo Scientific); A: for CP1-132aa protein purification; B: for CP1-287aa protein purification; 1 and 11: unstratified samples; 2 and 12: unstratified samples over the column; 3-10 and 13-21: imidazole eluates of different concentrations (i.e., 30, 40, 50, 60, 70, 80, 90, 100, and 110 mmol·L−1 in that order).
图 4 两种多克隆抗体（antiCP1-132aa，antiCP1-287aa）的效价测定
A，antiCP1-132aa的效价测定； B，antiCP1-287aa的效价测定。
Figure 4. Antibody titers of antiCP1-132aa and antiCP1-287aa
A: Antibody titer of antiCP1-132aa; B: antibody titer of antiCP1-287aa.
图 5 Western blot检测多克隆抗体的灵敏度
M，预染蛋白分子量标准26616（赛默飞）；A为使用antiCP检测感染ChiVMV叶片； B为antiCP灵敏度检测。
Figure 5. Sensitivity of polyclonal antibodies detected by western blot
M: Prestained protein Ladder 26616 (Thermo Scientific); A: detection of infected ChiVMV leaves using antiCP; B: determination of antiCP sensitivity.
表 1 RT-PCR扩增引物
Table 1. Primers for PCR amplification
引物名称
Primer names序列 （5′-3′）
Primer sequences (5′-3′)扩增位置及大小
Region and length of
PCR amplificationChiVMV-CP-F CGGAATTCGCSGGAGAGAGTGTTGATGCTG 8575～8970 nt，
396 bpChiVMV-CP-R1 CCGCTCGAGAAGAATTATTTGCATCTGGTC ChiVMV-CP-F CGGAATTCGCSGGAGAGAGTGTTGATGCTG 8575～9435 nt，
861 bpChiVMV-CP-R2 CCGCTCGAGTATTCCCCGAACGCCCAGCAGATTG 划线位置为酶切位点。
The underlined position is the enzyme cleavage site.
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表 2 多克隆抗体检测田间烟草样品
Table 2. Field detection on tobacco samples using polyclonal antibodies
多克隆抗体
Polyclonal antibodies烟草样品总数
Total number of tobacco samples阳性株数
Number of positive samples阴性株数
Number of negative samplesantiCP1-287aa 39 13 26 antiCP1-132aa 39 2 37
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