Preparation and Application of Polyclonal Antibodies Against Nucleocapsid Protein N and Envelope Membrane Glycoprotein Gn of Tomato Spotted Wilt Orthotospovirus
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摘要: 由番茄斑萎病毒Tomato spotted wilt orthotospovirus(TSWV)侵染引起的番茄斑萎病毒病在我国蔬菜、花卉等作物上造成了严重的经济损失。本研究克隆了TSWV核衣蛋白N基因及膜多糖蛋白Gn基因，利用Gateway系统获得N和Gn基因的原核表达载体。将其转化到大肠杆菌表达菌株Rosetta (DE3)细胞内，IPTG诱导表达N蛋白和Gn蛋白，以此为抗原分别免疫新西兰大白兔，制备N蛋白和Gn蛋白的多克隆抗体。Western blot检测结果表明，N蛋白的多克隆抗体能够与N蛋白发生特异性结合反应，间接酶联免疫吸附(indirect enzyme-linked immunosorbent assay，in-ELISA)分析检测表明抗体滴度为1：12 800，而Gn蛋白的多克隆抗体特异性差，效价未检出。本研究制备的N蛋白多克隆抗体可用于田间病株的病害诊断，同时有助于开展病毒在介体昆虫内定位、病毒和介体昆虫互作及功能分析的研究。Abstract: Tomato spotted wilt disease caused by tomato spotted wilt orthotospovirus (TSWV) has resulted seriously yield and quality losses in vegetables and flowers in China. In this study, nucleocapsid protein N gene and membrane polysaccharide protein Gn gene of TSWV were cloned into prokaryotic expression vectors, respectively. The plasmids were transformed into Rosetta(DE3) cells for expression of N or Gn proteins, which were used as immunogens to immunize New Zealand white rabbits, then the polyclonal antibodies against N and Gn proteins were successfully prepared. Western blot analysis showed that the polyclonal antibody against N protein could bind to the N protein specifically. Indirect ELISA showed that the titer of antiserum against N protein was about 1:12 800. However, the specificity of antibody against Gn protein was poor, and the titer was not detected. The polyclonal antibody against N protein has been prepared successfully, and it can be a useful reagent for disease diagnosis in field, the research of virus localization in insect body and the interaction of virus and insect vector.
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Key words:
- tomato spotted wilt Orthotospovirus /
- N protein /
- Gn protein /
- polyclonal antibody
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图 1 TSWV N(A)和Gn(B)的RT-PCR扩增结果
注：M为DNA分子标记Trans2k Plus；A为TSWV N基因RT-PCR扩增产物；B为TSWVGn基因RT-PCR扩增产物。
Figure 1. RT-PCR amplifications of TSWV N(A) and Gn (B)
图 2 SDS-PAGE分析TSWV N和Gn蛋白的原核表达
注: M为10~180 kDa蛋白分子标记; A为TSWV N蛋白的原核表达, 1和2为经IPTG诱导的N蛋白, 3和4为未经IPTG诱导的N蛋白; B为TSWB Gn蛋白的原核表达, 1为经IPTG诱导的Gn蛋白, 2为未经IPTG诱导的Gn蛋白。
Figure 2. SDS-PAGE analysis of TSWV N and Gn protein expression
图 3 Western blot检测N蛋白多克隆抗体的特异性
注: M为10~180 kDa蛋白分子标记; 1为感染TSWV的辣椒; 2为健康辣椒。
Figure 3. Specificity of N polyclonal antibodies detected by western blot
图 5 利用N(A)和Gn(B)抗体Dot-ELISA检测辣椒中的TSWV
注: A和B分别是N和Gn抗体建立的Dot-ELISA检测感病辣椒; 1~3为TSWV感病辣椒; 4~6为健康辣椒。
Figure 5. Dot-ELISA analysis of TSWV in pepper by using N(A) or Gn(B) antibody
表 1 PCR引物
Table 1. PCR primers
引物名称 引物序列5'-3' 引物用途 N-F GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GTC TAA GGT TAA GCT CAC N的扩增 N-R GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA AGC AAG TTC TGC AAG TTT TG N gene cloning Gn-F GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGA TCA TCC TGA GGT TTA TG Gn的扩增 Gn-R GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC ACC AGG TTT TTT TAT CAA ATA AC Gn gene cloning 
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