CN117402254B - 一种识别氟苯尼考的基因工程抗体及其应用 - Google Patents
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Abstract
本发明公开了一种识别氟苯尼考的基因工程抗体及其应用,属于兽药残留分析和免疫学技术领域。所述识别氟苯尼考的基因工程抗体,其氨基酸序列如SEQ ID NO.1~7任一项所示。本发明通过建立噬菌体单链抗体库,利用噬菌体展示技术筛选获得和高亲和力的抗氟苯尼考基因工程抗体。该抗体具有优异的特异性,能够提高氟苯尼考检测的灵敏度和准确性。利用该抗体在牛奶样品检测中的回收率达到98.52%至101.22%;相对标准偏差(RSD)低于5.33%,具有极高的准确性和可靠性,对保障动物源性食品以及公共环境的安全性具有重要意义。
Description
技术领域
本发明涉及兽药残留分析和免疫学技术领域,特别是涉及一种识别氟苯尼考的基因工程抗体及其应用。
背景技术
近年来,抗生素作为一种基本的治疗用剂,广泛用于农业和畜牧业的预防措施中。农场广泛使用抗生素导致了抗生素在环境和食物链的残留,加重了抗生素耐药性感染的负担。目前,抗生素耐药性是全球健康面临的最大挑战,准确快速的检测和识别抗生素是非常有必要的。氟苯尼考(Florfenicol,FF)是一种具有抗菌谱广、毒副作用小、不易产生伤害和耐药性的兽医专用酰胺醇类的广谱抗菌抗生素,通过抑制易感细菌中的多种蛋白质的合成起作用,被广泛应用于畜禽业及各种水产动物养殖业。由于其在农业畜禽养殖生产过程中的不规范推广应用,给人体健康和公共卫生安全带来严重威胁,其在农业动物性食品生产过程中的残留问题也被广泛关注。因而,亟待建立快速、灵敏、准确的酰胺醇类药物残留检测方法,确保动物性食品以及水产养殖环境的安全。
免疫学检测是较为常用的氟苯尼考检测方法,具有灵敏性好、特异性高、成本低、检测快速等优势。应用免疫学检测方法需要亲和力高的单克隆抗体,目前,现有的氟苯尼考单克隆抗体多是利用传统杂交瘤技术制备获得,但传统杂交瘤技术制备单克隆抗体的方法耗时长、价格昂贵、且具有安全隐患。因此,提供一种亲和力高、结构稳定并且制备方法更加安全、便捷和高效的氟苯尼考单克隆抗体具有重要意义。
发明内容
本发明的目的是提供一种识别氟苯尼考的基因工程抗体及其应用,以解决上述现有技术存在的问题。本发明提供的检测氟苯尼考的基因工程抗体,通过建立噬菌体单链抗体库,利用噬菌体展示技术筛选获得,亲和力高、特异性好,能够提高氟苯尼考检测的灵敏度和准确性。
为实现上述目的,本发明提供了如下方案:
本发明提供一种识别氟苯尼考的基因工程抗体,其氨基酸序列如SEQ ID NO.1~7任一项所示。
进一步地,其氨基酸序列如SEQ ID NO.3所示。
本发明还提供所述的基因工程抗体的制备方法,包括建立氟苯尼考噬菌体单链抗体库,利用噬菌体展示技术筛选出具备高亲和力的氟苯尼考抗体的噬菌体,再将其转化到HEK293哺乳动物细胞中表达,获得所述基因工程抗体。
进一步地,所述大肠杆菌为大肠杆菌XL1-Blue感受态。
进一步地,所述氟苯尼考噬菌体单链抗体库是通过将氟苯尼考单链抗体基因片段连接到pComb3X载体中实现的。
本发明还提供所述的基因工程抗体在制备检测氟苯尼考的酶联免疫试剂盒中的应用。
本发明还提供一种检测氟苯尼考的酶联免疫试剂盒,包括所述的基因工程抗体。
本发明还提供所述的基因工程抗体或所述的酶联免疫试剂盒在检测氟苯尼考药物残留中的应用。
进一步地,所述氟苯尼考药物残留包括动物源性食品中的药物残留。
进一步地,所述动物源性食品包括牛奶。
本发明公开了以下技术效果:
本发明通过建立噬菌体单链抗体库,利用噬菌体展示技术筛选获得和高亲和力的抗氟苯尼考基因工程抗体。该抗体具有优异的特异性,能够提高氟苯尼考检测的灵敏度和准确性。
利用本发明提供的基因工程抗体结合传统定量ELISA的检测方法,能够准确检测出动物源性食品或水产动物养殖水体中的氟苯尼考残留量,样品回收率在98.52%至101.22%之间;相对标准偏差(RSD)低于5.33%,具有极高的准确性和可靠性,对保障动物源性食品以及公共环境的安全性具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为载体蛋白、半抗原、免疫原和包被原的紫外可见光谱,其中,BSA和KLH分别为载体蛋白,KLH-半抗原为免疫原,BSA-半抗原为包被原;
图2为间接ELISA特异性评估免疫过程中的抗体效价;
图3为间接ELISA特异性评估免疫过程中的抗体结合能力;
图4为间接ELISA特异性评估免疫过程中的抗体特异性;
图5为重链30对(编号2-25,27-32)、轻链26对(编号33-35,38-60)可变区引物PCR琼脂糖凝胶电泳结果,编号1,26,36,37和61为maker;
图6为拼接后scFv片段电泳结果;
图7为生物淘洗中噬菌体-抗体的富集;
图8为识别与氟苯尼考结合的阳性克隆的ELISA;
图9为间接ELISA分析氟苯尼考的7种单链抗体;
图10为SEQ ID NO.3(E04)抗体与不同抗生素的特异性检测。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1免疫原、包被原的制备以及动物免疫
1.免疫原制备
取30g氟苯尼考琥珀酸钠,溶于500μL二甲基甲酰胺(DMF),以此合成半抗原。随后,加入15mgN-羟基琥珀酰亚胺(NHS)和30mg 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC),混合搅拌6个小时。在一个独立容器中,将20mg BSA/KLH溶于4mL的0.13M NaHCO3。在冰浴条件下,将半抗原溶液加入BSA/KLH溶液,4℃过夜搅拌。随后,将所得产物用PBS缓冲液透析3天。最后,获得免疫原KLH-FF(KLH-半抗原)和包被原BSA-FF(BSA-半抗原)。
载体蛋白BSA和KLH、半抗原、免疫原KLH-FF以及包被原BSA-FF的紫外可见光谱如图1,由图1可知,氟苯尼考半抗原Hapten有三个特征吸收峰(262nm、267nm和274nm),BSA和KLH的特征峰在280nm。半抗原和载体蛋白结合后,BSA-FF和KLH-FF显示出蛋白和半抗原结合的特征吸收峰,表明成功产生抗原。
2.动物免疫
以免疫原KLH-FF对三只雌性Balb/c小鼠(购自北京华阜康生物)进行皮下注射。具体为:用等体积的完全弗氏佐剂乳化的免疫原KLH-FF对小鼠进行皮下注射,之后以两周的间隔进行加强免疫,加强免疫时换用等体积的不完全弗氏佐剂乳化免疫原KLH-FF。在第二次免疫后和每次免疫后一周收集血清样品,通过ELISA评估血清抗体的特异性。
当KLH-FF作为免疫原时,五轮免疫后,2号鼠较其他两只免疫的小鼠(1号鼠和3号鼠)表现出较高的效价(1:32000)和较好的结合能力(IC50=4.23ng/mL)(图2-3)。
此外,取五轮免疫后2号鼠的血清样品,通过ELISA评估血清抗体对氟苯尼考、氯霉素、氟苯尼考胺、头孢噻呋、四环素和氧氟沙星的特异性,结果如图4所示,相较于其他五种抗生素及其类似物,五轮免疫后2号鼠的血清样品对氟苯尼考还表现出了较高的特异性。
实施例2噬菌体展示筛选单克隆抗体
1.构建噬菌体文库
在第五次免疫后从2号小鼠脾脏中分离淋巴细胞,提取总RNA并逆转录成cDNA,用作模板,通过PCR扩增重链VH和轻链VL。重链和轻链成功从2号鼠脾脏中扩增,大小约450bp(图5)。通过SOE-PCR扩增,并按1:1比例拼接成完整的单链抗体基因,获得约850bp大小单链抗体(scFv)片段(图6)。
用sfiI酶对上述所得scFv片段以及pComb3X载体(购自艾柏森生物)进行酶切,随后用T4连接酶连接。将纯化的抗体基因克隆到pComb3X载体中,电穿孔到大肠杆菌XL1-Blue感受态细胞中。通过在2YT-ATG琼脂板(购自艾柏森生物)上铺板细胞来评估文库大小。通过这种方法,可以构建噬菌体展示抗体库。
取样涂板后,剩余所有溶液进一步震荡培养1h,用M13K07辅助噬菌体(购自艾柏森生物)拯救。离心,沉淀悬浮于2YT-ATK培养基(购自艾柏森生物),在30℃、225rpm的条件下过夜表达。收集上清液,取10μL上清液,用2YT培养基稀释107倍后,再取10μL稀释液感染大肠杆菌XL1-Blue感受态细胞,随后涂板。随机挑选单克隆菌落进行测序,确定序列插入的正确性。
2.对噬菌体文库进行抗体的生物淘洗
采用高亲和力酶标板包被抗原(抗原:FF-BSA)过夜,0.1%PBST洗板后用3%脱脂奶粉在室温下封闭1h,弃封闭液洗板、待用。向上述封闭后的抗原板中加入约5×1012PFU,在37℃条件下与抗原孵育2h,0.1%PBST洗板。Glycine-HCl洗脱结合液洗脱后,用Tris-HCl调节pH为7.4。将上述洗脱液与大肠杆菌XL-Blue混合,37℃孵育30min后震荡培养。拯救噬菌体后在30℃条件下震荡培养过夜得到扩增的抗体第1轮文库。重复上述淘洗过程依次得到第2轮和第3轮文库。
经过三轮连续的固相包被抗原的生物淘洗,可得到不同的特异性噬菌体的富集。第三轮淘洗之后,随机从滴定板中选择64个克隆,随后20个克隆被噬菌体-ELISA确定为阳性克隆(图7)。从这些克隆中随机选择7个不同的克隆,通过DNA测序,在HEK293系统中表达,进一步进行验证(图8)。通过噬菌体展示淘选出的7个抗体(D01、H02、E04、C05、G07、A09和D09)的序列如SEQ ID NO.1~7所示。
SEQ ID NO.1(D01):
QAVVTQESALTTSPGETVTLTCRSSTGAVTTRNYANWVQQKPDHLFTGLMGGTSNRAPGVP
ARFSGSLIGDKAALTITGAQTEDEAIYFCALWSSDHWVFGGGTKLTVLGSSGGGGSGGGGG
GSSRSSQVQLQQSGPEVVRPGVSVKISCKGSSYTFTDYAIHWVRQSHAKSLEWIGGINTYY
GNTKYNQKFKGRATLTVDKSSSTAYMELARLTSEDSAVYYCERGNYRENVLDYWGQGTTVTVSS;
SEQ ID NO.2(H02):
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLIYLVSELDSGV
PDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSSGGGGSGGGGG
GSSRSSEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKKLEWVASISGGG
STYYLDSVKGRFTISRDNARNILYLQMISLRSEDTAMYYCARGHTYGSRRGHFGYWGQGTTLTVSS;
SEQ ID NO.3(E04):
DVVMTQTPLTLSVAIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG
VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSSGGGGSGGGG
GGSSRSSEVQLVESGGGLVKPGGSLKLSCAASGFTLSNYAMSWVRQTPDMRLEWVASICPG
VVTYYPDSVKGRFTISRDDGRNILYLQMSSLKSEDTAIYHCARGHCYGTRRGHFGYWGQGTTLTVSS;
SEQ ID NO.4(C05):
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA
RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSSGGGGSGGGGGG
SSRSSQVQLQQPGSVLVRPGVSVKISCKGSGYTFTDYAMHWMKQSHAKTLEWIGGINTYY
GNTKYSQKLKDRATMTVDKSSGTAYMELARLTSEDSALYYCVRGDLLYHAMDYWGQGTSVTVSS;
SEQ ID NO.5(G07):
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWFLQRPGQSPKRLIYLVSKLDSG
VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSSGGGGSGGGG
GGSSRSSEVKLVESGGGLVKPGGSLKLSCAASEFTFSNYAMSWVRQTPEKRLEWVASISSG
GSTYYPDSVKGRFTISRDNARNILYLQMNSLRSEDTAMYYCARGHTYGSRRGHFDSWGQGTTLTVSS;
SEQ ID NO.6(A09):
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG
VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKSSGGGGSGGGG
GGSSRSSEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWIRQISEKRLEWVASISSGGT
TYYPDSVKGRFTISRDYARNILYLQMSSLRSEDTAIYYCARGHTYGSRRGHFDYWGQGTTLTVSS;
SEQ ID NO.7(D09):
QAVVTQESALTTSPGETVTLTCRSSSGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA
RFSGSLIGDKAALTITGAQTEDEAIYFCALWSSNHWVFGGGTKLTVLGSSGGGGSGGGGGG
SSRSSEVQLQQSGPELVRPGVSVKISCKGSSYIFTDYAMHWVKQSHAQSLEWIGGINTNYG
NTKYNQKLKDKATMTVDKSSSTAYLELARLTSEDSAVYFCARGNYRENTLDYWGQGTSVTVSS。
通过ELISA检测每个抗体的结合能力和特异性。所有噬菌体去除非特异性吸附后展示出极高的响应信号(图9)。明显地,抗体E04对氟苯尼考显示出极高的灵敏度和特异性(图10)。
实施例3氟苯尼考基因工程抗体的应用
本实施例在动物性食品样品牛奶中对本发明开发的氟苯尼考抗体的实际性能进行了评估。
将FF分别添加到不含FF的牛奶样品中,浓度分别为2、40和250ng/mL。添加后,各取5mL上述样品,8000rpm离心10min。然后,去除上层脂肪层,收集剩余牛奶样品2mL。移取2mL样品于新鲜离心管中,加入4mL乙酸乙酯。再次,将混合物以8000rpm离心10min。接着,取上层乙酸乙酯层2.4mL,用氮吹仪吹干。将干燥后的残渣在PBS缓冲溶液中重新配制至最终体积为0.8mL,用于进一步分析。利用本发明开发的氟苯尼考抗体E04通过传统定量ELISA的检测方法验证其实际性能。结果如表1所示。
表1
表1表明,在添加牛奶样品回收实验中具有极好的回收率,达到98.52%至101.22%;相对标准偏差(RSD)低于5.33%。这些结果证实了本发明用于检测氟苯尼考开发的氟苯尼考抗体具有符合要求的准确性和可靠性。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (7)
1.一种识别氟苯尼考的基因工程抗体,其特征在于,其氨基酸序列如SEQ ID NO.1~7任一项所示。
2.根据权利要求1所述的基因工程抗体,其特征在于,其氨基酸序列如SEQ ID NO.3所示。
3.如权利要求1或2所述的基因工程抗体在制备检测氟苯尼考的酶联免疫试剂盒中的应用。
4.一种检测氟苯尼考的酶联免疫试剂盒,其特征在于,包括权利要求1或2所述的基因工程抗体。
5.如权利要求1或2所述的基因工程抗体或权利要求4所述的酶联免疫试剂盒在检测氟苯尼考药物残留中的应用。
6.根据权利要求5所述的应用,其特征在于,所述氟苯尼考药物残留包括动物源性食品中的药物残留。
7.根据权利要求6所述的应用,其特征在于,所述动物源性食品包括牛奶。
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