人源化抗稻纵卷叶螟类钙粘蛋白单域抗体的筛选及活性鉴定

doi:10.3969/j.issn.1674-7968.2020.05.012

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摘要利用苏云金芽胞杆菌(Bacillus thuringiensis, Bt) Cry毒素基因培育转基因抗虫作物品种,为蛋白质杀虫材料的应用带来巨大示范效应。昆虫类钙粘蛋白是Cry毒素的潜在受体之一,开展受体结合筛选测定,对新型杀虫蛋白材料的筛选和创制具有重要意义。本研究以纯化的稻纵卷叶螟(Cnaphalocrocis medinalis)类钙粘蛋白毒素结合区域(toxin binding region, TBR)蛋白作为抗原,利用噬菌体展示技术从人(Homo sapiens)源化噬菌体单域抗体库中筛选抗稻纵卷叶螟类钙粘蛋白的单域抗体。采用负筛选的策略,经4轮吸附-洗脱-扩增,富集特异性识别TBR蛋白的噬菌体单域抗体。从第4轮筛选中随机挑取单菌落进行单克隆ELISA鉴定,获得4个含有完整单域抗体基因片段的阳性克隆;对阳性克隆进行测序和序列比对,最终获得3个序列不同的阳性克隆(B4 , C11和E8)。通过Western blot和ELISA结合试验进行的鉴定结果显示,筛选获得的3株阳性克隆与稻纵卷叶螟类钙粘蛋白均具有较高的结合活性。竞争抑制试验表明,100 μg/mL Cry1Ac对C11与稻纵卷叶螟TBR蛋白结合能力可产生28.6%的抑制。本研究利用噬菌体展示技术筛选获得抗稻纵卷叶螟类钙粘蛋白的噬菌体单域抗体并验证其生物活性,为进一步开拓具有Cry毒素功能活性和生物活性的模拟物提供了材料基础。

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	江礼鹏
	李懿航
	胡家萌
	胡晓丹
	刘贝贝
	刘贤金

关键词 ：类钙粘蛋白, 负筛选, 噬菌体展示, 单域抗体, 竞争抑制

Abstract：Bacillus thuringiensis (Bt) Cry toxin gene has been used to cultivate insect-resistant crop varieties, which has brought great demonstration effects to the application of protein insecticidal materials. Cadherin is one of the potential receptors of Cry toxin, carrying out target screening is of great significance for the screening and creation of novel insecticidal protein materials. In this study, Humanized Single Domain Antibody Library was screened to generate single-domain antibodies against cadherin-like protein of rice leaf roller (Cnaphalocrocis medinalis) with cadherin-like protein toxin binding region (TBR) as the antigen by phage display technique. The substructive selection strategy was used to enrich phage single domain antibodies that specifically recognized TBR protein after 4 rounds of adsorption-elution-amplification. Single colonies were randomly picked from the 4th round of screening and identified by monoclonal ELISA. Four positive clones containing the complete single domain antibody gene fragment were obtained. After sequencing and amino acid sequence alignment, 3 positive clones with different sequences were finally obtained (B4, C11 and E8). The 3 positive clones identified by Western blot and ELISA binding tests had high binding activity to cadherin-like protein from rice leaf roller. Competitive inhibition test showed that 100 μg/mL Cry1Ac could inhibit 28.6% binding of C11 to the cadherin-like protein of rice leaf roller. In this study, phage display technique was used to screen phage single domain antibodies against cadherin-like protein of rice leaf roller, which could provide material basis for further development of mimics with Cry toxin functional and biological activities.

Key words： Cadherin-like protein Substructive selection Phage displaying Single domain antibody Competitive inhibition

收稿日期: 2019-10-09

ZTFLH:

S482.3

基金资助:国家自然科学基金(31630061)

通讯作者: *jaasliu@jaas.ac.cn

引用本文:

江礼鹏, 李懿航, 胡家萌, 胡晓丹, 刘贝贝, 刘贤金. 人源化抗稻纵卷叶螟类钙粘蛋白单域抗体的筛选及活性鉴定[J]. 农业生物技术学报, 2020, 28(5): 884-891.
JIANG Li-Peng, LI Yi-Hang, HU Jia-Meng, HU Xiao-Dan, LIU Bei-Bei, LIU Xian-Jin. Screening and Activity Identification of Humanized Single Domain Antibodies Against Cadherin-like Protein of Cnaphalocrocis medinalis. 农业生物技术学报, 2020, 28(5): 884-891.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.05.012 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I5/884

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