CN112111011B - 一种特异性抗噻虫嗪抗体的可变区序列及其重组全长抗体 - Google Patents
一种特异性抗噻虫嗪抗体的可变区序列及其重组全长抗体 Download PDFInfo
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Abstract
本发明公开了一种特异性抗噻虫嗪抗体的可变区序列,重链可变区编码基因的氨基酸序列为SEQ ID NO:2所示。本发明还公开了一种抗噻虫嗪重组全长抗体,包括重链恒定区、重链可变区、轻链恒定区和轻链可变区,重链可变区编码基因的氨基酸序列为如SEQ ID NO:2所示。本发明又公开了一种抗体表达质粒。将本发明得到的序列基因分别连接到包含了重链恒定区基因和轻链恒定区基因的表达载体,采用双质粒系统的哺乳动物细胞表达获得了重组全长抗体,保证了鼠源亲本抗体的识别活性,并使得抗噻虫嗪抗体可大批量稳定生产,为噻虫嗪残留检测的各种免疫分析方法提供了可靠的核心试剂。
Description
技术领域
本发明属于基因工程技术领域,尤其是涉及一种特异性抗噻虫嗪抗体的可变区序列及其重组全长抗体的制备。
背景技术
新烟碱杀虫剂已经成为全球市场增长最快的杀虫剂,广泛应用于全球120多个国家。该类杀虫剂通过抑制害虫中枢神经系统的乙酰胆碱酯酶受体,阻断害虫中枢神经系统的正常传导,引起害虫麻痹而死,对刺吸式害虫比如飞風、叶蝉、蚜虫、蓟马等具有良好的防治效果。噻虫嗪(3-(2-氯-1,3-噻唑-5-基甲基)-5-甲基-1,3,5-恶二嗪-4-基叉(硝基)胺)是一种全新结构的第二代烟碱类杀虫剂,有较强的内吸和渗透作用,因此会分布在作物各个部位,且因其具有水溶性强和半衰期长等特点,很容易通过降雨和灌溉等途径进入到地表水和土壤中。研究表明,地表水、沉积物和土壤中存在着较高水平的噻虫嗪残留,这些农药残留对生态健康和非靶标生物如传粉昆虫、水生生物和哺乳动物有巨大威胁。欧盟现已禁止噻虫嗪在室外环境下使用,在欧盟标准中(2017/671),不同的水果、蔬菜、游籽、谷物、茶叶、咖啡、草药、动物源产品和蜂蜜中噻虫嗪的最大残留限量(MRLs)为0.01-20mg/kg。为防止消费者和有益生物受到噻虫嗪残留的危害,对环境和农产品中噻虫嗪残留的监测具有重要意义。
目前,针对噻虫嗪残留的检测方法主要有基于仪器的检测方法和基于免疫分析的检测方法。仪器分析方法有HPLC、GC和MS等,这些仪器分析方法均具有很高精密度、准确度和灵敏度,能够满足噻虫嗪的精准定性定量的检测要求,但仪器分析方法需要昂贵的分析仪器、专业的实验人员和复杂的样品前处理程序,因此很难实现大规模的现场快速分析,而免疫分析快速检测方法能满足农药残留现场检测以及大批量样本筛查的需求。
农药免疫分析快速检测技术是基于农药抗原和抗体的特异性结合而实现的,故抗体的制备是农药免疫快速检测的核心关键。传统的抗体有多克隆抗体和单克隆抗体,均是通过免疫动物而制备的,且后者因只有一个抗原识别表位特异性强而被广泛应用于农药免疫分析快速检测。有众多研究者报道了基于单克隆抗体建立的酶联免疫吸附分析(ELISA)方法用于噻虫嗪检测。Ma等人报道的方法检测灵敏度(IC50)为0.57μg/L,最低检测限(IC10)为0.05μg/L(Ma H.,Xu Y.,Li Q.X.,Xu T.,Wang X.,Li J.Application of enzyme-linked immunosorbent assay for quantification of the insecticidesimidacloprid and thiamethoxam in honey samples[J].Food Additives andContaminants.2009,26,713–718.)。Xu等人的报道其检测灵敏度(IC50)为0.50μg/L,最低检测限(IC10)为0.05μg/L(Xu T.,Wei K.Y.,Wang J.,Ma H.X.,Li J.,Xu Y.J.,LiQ.X.Quantitative analysis of the neonicotinoid insecticides imidacloprid andthiamethoxam in fruit juices by enzyme-linked immunosorbent assays[J].Journalof AOAC International.2010,93,12–18.)。Watanabe等人报道的检测灵敏度(IC50)为0.72μg/L,最低检测限(IC10)为0.10μg/L(Watanabe E.,Miyake S.QuantitativeDetermination of Neonicotinoid Insecticide Thiamethoxam in AgriculturalSamples:a Comparative Verification Between High-Performance LiquidChromatography and Monoclonal Antibody-Based Immunoassay[J].Food AnalyticalMethods.2013,6,658–666.)。但是,依赖杂交瘤细胞制备单克隆抗体这一技术生产成本高、耗时,且需要杂交瘤细胞的长期低温保存和定期驯化,但在这些过程中杂交瘤细胞会因保存条件不当,有效基因丢失或突变而无法产生有效的抗体。
随着基因工程技术的发展,序列明确的重组抗体逐渐变成研究热点,包括单链抗体、Fab抗体、重组全长抗体等,且开发具有优异性能的新型候选抗体一直是免疫测定的恒定目标。在众多重组抗体表达系统中,哺乳动物细胞表达系统能够指导重组抗体的正确折叠,提供准确的多种翻译后的加工和修饰功能,因此可以稳定产生与亲本抗体活性一致的重组抗体,不仅证实了亲本抗体可变区序列的准确性,还可以解决常规抗体制备面临的许多问题,如传统抗体的批次间差异、细胞株传代保存过程中容易丢失有效基因而导致细胞株无法产生有效抗体。只要获得正确的抗体序列基因,就可以在体外大量制备多种形式的重组抗体(包括全长IgG完整抗体、单链抗体scfv、抗原识别片段Fab等),而无需牺牲小鼠来制备腹水抗体,操作简便,制备周期短,有利于大规模产生抗体,并有助于灵敏、可靠的农药残留快速检测技术的发展和应用。
发明内容
为了克服现有技术的不足,本发明提供一种灵敏度高、特异性强的抗噻虫嗪重组全长抗体的制备方法和稳定生产,以及该抗体的应用。
本发明解决其技术问题所采用的技术方案是:一种特异性抗噻虫嗪抗体的可变区序列,重链可变区编码基因的氨基酸序列为SEQ ID NO:2所示。
作为优选,重链可变区编码基因的核苷酸序列如SEQ ID NO:1所示。
作为优选,轻链可变区编码基因的氨基酸序列为SEQ ID NO:4所示。
作为优选,轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
本发明还公开了一种抗噻虫嗪重组全长抗体,包括重链恒定区、重链可变区、轻链恒定区和轻链可变区,所述重链可变区编码基因的氨基酸序列为如SEQ ID NO:2所示。
作为优选,所述重链可变区编码基因的核苷酸序列为如SEQ ID NO:1所示。
作为优选,所述轻链可变区编码基因的氨基酸序列如SEQ ID NO:4所示。
作为优选,所述轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
本发明又公开了一种抗体表达质粒,含有上述任一项所述的重链可变区和小鼠重链IgG1恒定区的核苷酸序列,可以表达抗噻虫嗪重组全长抗体的重链蛋白;或者,含有如权利要求3-4中任一项所述的轻链可变区和小鼠轻链Kappa恒定区的核苷酸序列,可以表达抗噻虫嗪重组全长抗体的轻链蛋白。
本发明将一株能稳定分泌抗噻虫嗪高特异、高灵敏单抗的杂交瘤细胞株的重链和轻链可变区基因成功扩增出来、测序和合成,采用同源重组技术分别构建了重链和轻链表达载体,并共转染至哺乳动物细胞HEK 293(F)中,最终获得抗噻虫嗪的重组全长抗体。经ELISA技术验证,该重组全长抗体与传统的抗噻虫嗪小鼠腹水单抗相比,有一致的高灵敏度和高选择性,可以代替腹水单抗,应用于噻虫嗪农药残留检测。
与现有技术相比,本发明具有以下优点:本发明公开的抗噻虫嗪重组全长抗体的重链可变区、轻链可变区序列来源于经多次免疫小鼠、细胞融合及筛选得到的一株高特异性的抗噻虫嗪的单克隆细胞株。将此序列基因分别连接到包含了重链恒定区基因和轻链恒定区基因的表达载体,采用双质粒系统的哺乳动物细胞表达获得了重组全长抗体,保证了鼠源亲本抗体的识别活性,并使得抗噻虫嗪抗体可大批量稳定生产,为噻虫嗪残留检测的各种免疫分析方法提供了可靠的核心试剂。
附图说明
图1为本发明以反转录获得的cDNA为模板,噻虫嗪抗体重链可变区基因、轻链可变区基因PCR扩增琼脂糖凝胶电泳结果。
图2为本发明SDS-PAGE验证全长抗体在哺乳动物细胞HEK293F中的表达。
图3为本发明抗噻虫嗪重组全长抗体和小鼠单克隆抗体对噻虫嗪的ELISA竞争曲线
(单克隆抗体:噻虫嗪浓度从低到高依次为0.006、0.024、0.098、0.39、1.56、6.25、25.00μg/L;重组全长抗体:噻虫嗪浓度从低到高依次为0.012、0.049、0.195、0.78、3.13、12.50、50.00μg/L)。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对发明实施例中的技术方案进行清楚、完整的描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围,如无特殊说明,实施例中的实验方法均为常规方法。
本发明涉及的抗噻虫嗪重组全长抗体包括重链恒定区、重链可变区、轻链恒定区和轻链可变区,其重链恒定区序列为小鼠IgG1的重链恒定区序列,该抗体的轻链恒定区序列为小鼠κ抗体轻链恒定区序列。
小鼠抗噻虫嗪重组全长抗体的制备
1)抗噻虫嗪单克隆抗体可变区基因扩增
经小鼠免疫、细胞融合和杂交瘤细胞筛选,制备了一株能分泌高特异性的抗噻虫嗪抗体的杂交瘤细胞株E7E2,亚型IgG1/Kappa,采用Trizol法提取杂交瘤细胞的总RNA,经琼脂凝胶电泳鉴定RNA全长性较好可满足本实验的要求。以RNA为模板,采用Random 6mers和Oligo dT Primer混合的RT Primer Mix反转录合成cDNA。使用具有亚型特异性的引物进行单抗可变区PCR扩增,其中上游引物位于可变区前的信号肽部位,下游引物位于可变区之后的恒定区。
PCR扩增的程序为
PCR扩增产物琼脂糖凝胶电泳结果参见图1,扩增出含有VH和VL基因片段的条带。用凝胶提取试剂盒纯化,将纯化后的产物克隆至PMD 19-T(氨苄抗性)载体并转化大肠杆菌TOP10感受态细胞后挑选10个单克隆菌落送测序,序列经NCBI数据库Blast比对后,鉴定出序列完整、亚型相符和正确表达框的VH和VL基因。
抗噻虫嗪单克隆抗体E7E2重链可变区序列为:
GAGGTTCTGCTCCAGCAGTCTGGGACTGTGCTGACAAGGCCTGGGGCCTCCGTGAAGATGTCCTGTGAGGCTTCTGGCTACAGGTTTACCAGCAACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGGTGGTATTTATCCTGGAAATGGTGCAACTGGCTACAATCAGAAGTTCAGGGGCAGGGCCACACTGACTGTAGTCACATCCGCCAGCACTGCCTACATGGAACTCAACAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTTCAAGAGGGGGGCTTTACTATGATTACGACGCCTGGCTTGGTTACTGGGGTCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ IDNO:1)
功能性重链可变区全长为366个碱基,结构域从第1位碱基开始,编码122个氨基酸,该有功能的重链V-GENE与鼠源IGHV1-5*01F一致性为91.32%,J-GENE与鼠源IGHJ3*01F一致性为93.62%,D-GENE与鼠源IGHD2-4*01F一致性较高。
以IMGT法定义结构域,具体结构域划分为:
结构域 | FR1 | CDR1 | FR2 | CDR2 | FR3 | CDR3 | FR4 |
碱基序列 | 1-75 | 76-99 | 100-150 | 151-174 | 175-288 | 289-333 | 334-366 |
抗噻虫嗪单克隆抗体E7E2重链可变区氨基酸序列
EVLLQQSGTVLTRPGASVKMSCEASGYRFTSNWMHWVKQRPGQGLEWIGGIYPGNGATGYNQKFRGRATLTVVTSASTAYMELNSLTNEDSAVYYCSRGGLYYDYDAWLGYWGQGTLVTVSA(SEQ ID NO:2)
抗噻虫嗪单克隆抗体E7E2轻链可变区序列为
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAAGCAGCCTCAATCTCTTGCAAGTCAAGTCAGAGCCTCGTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCTAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGCAAATCAACAGAGTGGAGGCTGAGGATTTGGGACTTTATTACTGCGTGCAAGGTTCACATTTTCCTCACACGTTCGGCTCGGGGACAAAGTTGGAAATAAGA(SEQ ID NO:3)
功能性轻链可变区全长为个336个碱基,结构域从第1位碱基开始,编码112个氨基酸,该功能性轻链V-GENE与鼠源IGKV1-133*01F的一致性为97.28%,J-GENE与鼠源IGKJ4*01F的一致性为97.06%。
以IMGT法定义结构域,具体结构域划分为:
结构域 | FR1 | CDR1 | FR2 | CDR2 | FR3 | CDR3 | FR4 |
碱基序列 | 1-78 | 79-111 | 112-162 | 163-171 | 172-279 | 280-306 | 307..336 |
抗噻虫嗪单克隆抗体E7E2轻链可变区氨基酸序列
DVVMTQTPLTLSVTIGQAASISCKSSQSLVYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLQINRVEAEDLGLYYCVQGSHFPHTFGSGTKLEIR(SEQ ID NO:4)
2)表达质粒的构建
在鉴定出正确的噻虫嗪E7E2的重链和轻链可变区基因后,合成正确的基因序列。采用同源重组法分别将重链和轻链可变区基因连接到经过HindIII/EcoRI双酶切线性化的表达载体pCDNA3.4-Mouse-IgG1-CH和pCDNA3.4-Mouse-Cκ上。其中,pCDNA3.4-Mouse-IgG1-CH上面含有小鼠IgG1重链恒定区域基因,pCDNA3.4-Mouse-Cκ上面含有小鼠Kappa轻链恒定区域基因,故构建的重/轻链重组质粒上面分别包含了重链可变区基因和恒定区基因/轻链可变区基因和恒定区基因。将构建的重组质粒转化大肠杆菌TOP10感受态细胞、摇菌培养并测序。
3)重组全长抗体的表达
将测序正确的质粒对应的菌液继续大体积培养扩增,提取质粒,待用。复苏HEK293(F)细胞,120rpm,8%CO2,37℃悬浮培养3代。转染前,将细胞接种至新的培养瓶,接种密度为1.5×106个/mL,悬浮培养2h后,将重链质粒、轻链质粒和转染试剂按照一定的比例37℃静置孵育15min后,转染HEK293(F)细胞。转染后的细胞悬浮培养至活率低于70%后,4000rpm离心5min,收集上清培养液,采用Protein A亲和层析柱纯化细胞上清中重组全长抗体,纯化过程中液体流速均为1mL/min。纯化后的抗体用0.01M PBS溶液进行透析过夜,透析结束后,测定抗体浓度为3mg/mL,采用SDS-PAGE(图2)进行验证。
重组全长抗体的活性评价
抗噻虫嗪重组全长抗体和抗噻虫嗪天然小鼠腹水单抗用ELISA方法进行对比
将噻虫嗪-OVA(混合酸酐法制备)作为包被原包被96孔板,然后用兔抗鼠IgG-HRP作为检测抗体,TMB作为反应底物,噻虫嗪和其他新烟碱类农药标准品作为检测物,进行ELISA检测。结果(图3)如下:
a)检测范围
抗噻虫嗪重组全长抗体用于ELISA法检测范围(IC20-IC80)为:0.10-1.66μg/L。
抗噻虫嗪小鼠腹水单抗用于ELISA法检测范围(IC20-IC80)为:0.13-1.80μg/L。
b)灵敏度(IC50)
抗噻虫嗪重组全长抗体IC50为:0.41μg/L。
抗噻虫嗪小鼠单克隆抗体IC50为:0.48μg/L。
c)ELISA法特异性
本发明中通过检测与其他七种常见的新烟碱类杀虫剂的交叉反应来评价抗体的特异性。ELISA数据显示抗噻虫嗪的鼠单抗和重组全长抗体与噻虫胺有0.04%的交叉反应率,与剩余的6种杀虫剂则没有交叉反应。
因此,抗噻虫嗪重组全长抗体和鼠源单克隆抗体只对噻虫嗪特异性识别,表现出高亲和力结合作用。
上述具体实施方式用来解释说明本发明,而不是对本发明进行限制,在本发明的精神和权利要求的保护范围内,对本发明作出的任何修改和改变,都落入本发明的保护范围。
SEQUENCE LISTING
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<120> 一种特异性抗噻虫嗪抗体的可变区序列及其重组全长抗体
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Claims (4)
1.一种抗噻虫嗪重组全长抗体,包括重链恒定区、重链可变区、轻链恒定区和轻链可变区,其特征在于:所述重链可变区的氨基酸序列如SEQ ID NO:2所示;所述轻链可变区的氨基酸序列如SEQ ID NO:4所示。
2.根据权利要求1所述的抗噻虫嗪重组全长抗体,其特征在于:所述重链可变区编码基因的核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述的抗噻虫嗪重组全长抗体,其特征在于:所述轻链可变区编码基因的核苷酸序列如SEQ ID NO:3所示。
4.一种抗体表达质粒,其特征在于:含有如权利要求1-2中任一项所述的重链可变区和小鼠重链IgG1恒定区的核苷酸序列,可以表达抗噻虫嗪重组全长抗体的重链蛋白;且,含有如权利要求1、权利要求3中任一项所述的轻链可变区和小鼠轻链Kappa恒定区的核苷酸序列,可以表达抗噻虫嗪重组全长抗体的轻链蛋白。
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