Production of Polyclonal and Monoclonal Antibodies Against the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3Aa16
Production of Polyclonal and Monoclonal Antibodies Against the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3Aa16
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DOI：
10.1007/s12010-014-1426-9
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发表时间：
2015-03-01
影响因子：
3
通讯作者：
Stephan, Dietrich
中科院分区：
文献类型：
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作者：
Ben Hamadou-Charfi, Dorra;Sauer, Annette Juliane;Stephan, Dietrich
本研究的目的是建立一种定量测定苏云金芽孢杆菌培养上清液中营养活性杀虫蛋白Vip3A的方法。用苏云金芽孢杆菌标准菌株BUPM95在3L中发酵产生Vip3A蛋白，Western印迹检测到BUPM95在指数生长期培养上清液中含有Vip3Aa16毒素。然而，免疫印迹检测显示，除毒素外，突尼斯斯法克斯生物技术中心制备的抗Vip3Aa16多克隆抗体还识别另外两条条带(62和180 kDa)。出于这个原因，为了开发一种可靠的毒素定量技术，我们考虑在德国Julius Kuhn研究所生产多克隆抗体。这些抗体是制备针对Vip3Aa16重组菌株Escherichia coliBL21(DE3)产生的蛋白的单抗的基础。用平板捕捉抗原(PTA)和三抗体夹心酶联免疫吸附试验(TAS-EL ISA)检测这些单抗。对杂交瘤细胞培养上清液进行筛选，得到4个产生单抗的阳性克隆。
The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kuhn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.