摘要
以抗体为核心建立的血清学方法用于植物病毒的检测具有操作方便、快捷、灵敏以及高通量等优点,但国内制备的植物病毒抗体并不多,大多数重要植物病毒的检测得依赖进口的抗体或者试剂盒。因此,制备重要植物病毒的特异性抗体、建立相应的血清学检测方法对植物病毒的检测及口岸检验检疫和防治具有重要意义。番茄黄化曲叶病毒(Tomato yellow leaf curl virus, TYLCV)是严重危害番茄生产的双生病毒之一,而黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)是我国近年来葫芦科作物上发生的又一重要的检疫性病毒。本论文分别制备了抗TYLCV和CGMMV的特异性单克隆抗体(Monoclonal antibodies, MAbs),并建立了相应的的血清学检测方法。
(1)抗TYLCV单克隆抗体制备及其检测应用：用PCR方法从感染TYLCV上海分离物(TYLCV-SH2)的番茄基因组中克隆了该病毒的外壳蛋白(Coat protein, CP)基因,将其插入到原核表达载体pET-32a中构建成重组原核表达载体。重组表达载体pET-32a-CP转化大肠杆菌BL21DE3),经IPTG诱导,Ni+-NTA亲和柱纯化获得分子量约为48 kDa的融合蛋白。以纯化的重组蛋白为抗原免疫BALB/c小鼠,获得3株能稳定传代并分泌抗TYLCV的单克隆抗体杂交瘤细胞。单克隆抗体的特异性检测表明3株单抗中3E10能与4种侵染番茄的双生病毒反应,而另外2株(2B2和2E3)能特异性识别TYLCV-SH2。利用单抗3E10建立了三抗夹心ELISA检测TYLCV的方法,该方法检测病叶的灵敏度可达1：2 560(w/v, g/mL),对田间样品的检测表明,TYLCV在田间番茄植物上发病普遍。
(2)抗CGMMV单抗制备及其检测应用：用提纯的CGMMV病毒粒子免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经细胞筛选与克隆,获得6株能稳定传代并分泌抗CGMMV单克隆抗体的杂交瘤细胞,并分别制备其单抗腹水。其中单抗5D11可以与CGMMV、烟草花叶病毒(TMV)和番茄花叶病毒(ToMV) 3种烟草花叶病毒属的病毒反应,单抗8E3和11B12除与CGMMV有强阳性反应外,与TMV和ToMV存在较弱的反应,而单抗4H1、5B10和11A4仅与CGMMV有特异性反应。利用最灵敏的4H1单抗为核心建立了检测CGMMV的抗原包被间接ELISA方法(Antigen-coated plate enzyme-linked immunosorbent assay, ACP-ELISA、免疫斑点法(Dot-blot ELISA)、组织印迹法(Tissue-blotELISA)和免疫捕获RT-PCR(Immunocapture reverse transcriptase polymerase chain reaction, IC-RT-PCR)。建立的ACP-ELISA和Dot-blot ELISA方法检测感染CGMMV的黄瓜植株组织的灵敏度分别高达1:40960和1:20480 (w/v, g/mL)。Tissue-blot ELISA操作简便,尤其适合大规模的田间样品检测和诊断。IC-RT-PCR具有最高的检测灵敏度和特异性,对提纯的病毒检出量达到了0.1 pg,对感病植物组织的检测灵敏度为1：102400(w/v,g/mL)。
The antibody-based serological method had much success in plant virus detection, because it is sensitive, specific, rapid and simple to conduct, can be conducted on a large-scale. Antibodies of a few plant viruses had been produced in China, but detection of most plant viruses was relied on imported commercial antibodies or ELISA kits. Thus, it is necessary to produce antibodies against important plant viruses, and develop effective serological methods for virus detection, quarantine and prevention. Tomato yellow leaf curl virus (TYLCV) is a species of the family Geminiviridae, causing serious yield losses in tomato production, and cucumber green mottle mosaic virus (CGMMV) is a serious alien invasive pathogen of cucurbit crops. In this study, monoclonal antibodies (MAbs) against TYLCV and CGMMV were producted, and several serological methods were set up for rapid, reliable and sensitive detection.
(1) MAbs against TYLCV and their application:The coat protein (CP) gene of TYLCV isolate SH2 (TYLCV-SH2) was amplified by PCR and inserted into a prokaryotic expression vector pET-32a to produce pET-32a-CP, which was used to transform Escherichia coli BL21 (DE3). After induced by IPTG, an about 48 kDa fusion protein was obtained and purified through Ni+ -NTA affinity column. The purified recombinant CP was used to immunize BALB/c mice for producing MAbs. Three hybridoma cell lines secreting MAbs aganint TYLCV were obtained. The results of TAS-ELISA detection showed that the MAb 3E10 could react with four begomoviruses infecting tomato, while the others MAbs (2B2 and 2E3) mainly reacted with TYLCV. A TAS-ELISA method based on MAb 3E10 was set up for TYLCV detection, and this method could successfully detect virus in plant sap at 1:2 560 (w/v, g/mL). The resultes of TAS-ELISA detection of field samples showed begomoviruses were common in tomato plants.
(2) MAbs against CGMMV and their application:After selected and cloned, six hybridoma cell lines secreting MAbs against CGMMV were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by purified CGMMV particles. MAb 5D11 could react strongly with CGMMV, TMV and ORSV, not with ToMV. MAbs 8E3 and 11B12 could react strongly with CGMMV and weakly with TMV and ToMV, while MAbs 4H1,5B10 and 11A4 could react strongly only with CGMMV, but not with the other three tobamoviruses. Based on the most sensitive MAb 4H1, the indirect antigen-coated plate (ACP)-ELISA, Dot-blot ELISA, Tissue-blot ELISA and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) for CGMMV detection were set up. Both ACP-ELISA and Dot-blot ELISA could specifically detect CGMMV in infected cucumber leaf tissue extracts diluted 1:40 960 andl:20 480 (w/v, g/mL), separately. Tissue-blot ELISA is more practical for routine detection of large-scale samples in the field survey. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus and could successfully detect virus in plant sap at 1:102 400 (w/v, g/mL).
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