Preparation and Application of Polyclonal Antibodies Recognizing the Capsid Protein of Chilli Veinal Mottle Virus
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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:Aim Chilli veinal mottle virus (ChiVMV) is one of the most harmful viruses in the production of solanaceae palnts and a subject of quarantine inspections at ports. The aim of this study is to develop polyclonal antibodies against ChiVMV coat proteins with a high level of specificity, which can then be utilized for the field detection of infected plants. Methods The full-length (861 bp) and partial fragment (396 bp) of the coat protein gene from the ChiVMV GZ-tabacco isolate were amplified using RT-PCR, recombined into the prokaryotic expression vector pET28a, and subsequently transformed into E.coil BL21 for induced expression. After chromatographic isolation and dialysis purification of the expression products, they were used to immunize Dahl rabbits for the preparation of polyclonal antibodies. The antibody potency and specificity were assessed through enzyme-linked immunosorbent assay (ELISA) and Western blot.. Results Two polyclonal antibodies against coat proteins, namely antiCP1-287aa and antiCP1-132aa, were successfully prepared with antibody titer of 1:6400 and 1:12800, respectively. Both antibodies, antiCP1-287aa and antiCP1-132aa, effectively detected the antigen by Western blot. In indirect ELISA tests of field strains, it was observed that the ChiVMV strain was specifically detectable by antiCP1-132aa, whereas the Potato virus Y （PVY） strain was not. However, antiCP1-287aa could not differentiate between these two strains. Conclusion The polyclonal antibody, antiCP1-132aa , exhibits high specificity, making it suitable for detecting ChiVMV-infected strains in the field. Additionally, it lays the foundation for the functional study of the coat protein of ChiVMV. -
图 1 ChiVMV与PVY的外壳蛋白间氨基酸序列比较
Figure 1. Comparison of amino acid sequences of coat proteins from ChiVMV and PVY
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，DL2000 DNA Marker、DL5000 DNA Marker；1、3，ChiVMV CP1-287aa序列扩增及重组质粒双酶切验证；2、4，ChiVMV CP1-132aa序列扩增及重组质粒双酶切。
Figure 2. Amplification of the corresponding gene fragments of two antigens and their expression vector construction
M1, M2, DL2000 DNA Marker, DL5000 DNA Marker; 1, 3, Amplification of ChiVMV CP1-287aa fragment and double enzyme digestion of recombinant plasmidt; 2, 4, Amplification of ChiVMV CP1-132aa fragment and double enzyme digestion of recombinant plasmid.
图 3 抗原蛋白的纯化
M，Prestained Protein Ladder 26616；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; A for CP1-132aa protein purification; B for CP1-287aa protein purification; 1, 11: unstratified samples; 2, 12: unstratified samples over the column; 3 to 10, 13 to 21: imidazole eluates of different concentrations (30, 40, 50, 60, 70, 80, 90, 100, 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 titer of the 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，Prestained Protein Ladder 26616；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; A shows the detection of infected ChiVMV leaves using antiCP; B shows the antiCPsensitivity detection.
表 1 RT-PCR扩增引物
Table 1. The 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. Detection of field 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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