CN112778420A - 一种哒螨灵单克隆抗体及其应用 - Google Patents
一种哒螨灵单克隆抗体及其应用 Download PDFInfo
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- CN112778420A CN112778420A CN202011344736.2A CN202011344736A CN112778420A CN 112778420 A CN112778420 A CN 112778420A CN 202011344736 A CN202011344736 A CN 202011344736A CN 112778420 A CN112778420 A CN 112778420A
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Abstract
本发明公开了一种哒螨灵单克隆抗体及其应用，该抗体包括轻链可变区和重链可变区，轻链可变区的CDR1序列如SEQ ID NO：1所示、CDR2序列如SEQ ID NO：2所示、CDR3序列如SEQ ID NO：3所示；重链可变区的CDR1序列如SEQ ID NO：4所示、CDR2序列如SEQ ID NO：5所示、CDR3序列如SEQ ID NO：6所示。该抗体对哒螨灵具有较好的检测灵敏度和添加回收率，可实现对农产品中哒螨灵残留量的检测，为哒螨灵残留量的免疫检测提供了原料。并且首次测得该抗体的可变区基因序列，为后续抗体试剂应用于检测试剂盒时采用规模化表达生产提供了序列基础。
Description
技术领域
本发明属于农药残留免疫分析技术领域，具体涉及一种哒螨灵单克隆抗体及其应用。
背景技术
哒螨灵 (Pyridaben)为广谱、触杀性杀螨剂，可用于防治多种植食性害螨，对螨的整个生 长期即卵、幼螨、若螨和成螨都有很好的效果，对移动期的成螨同样有明显 的速杀作用。然而，哒螨灵的残留也会使一些不是预定目标的生物遭到毒害。
目前，对哒螨灵残留的检测方法主要是仪器分析方法，包括气相色谱法(Gaschromatography，GC)、气相色谱-质谱串联法(GC-MS)等。这些方法结果可靠，灵敏度高，已有相关的技术标准可供参考。但由于需要昂贵的仪器、专门的操作人员，以及样品前处理复杂、成本高、时间长，因此不能更好地满足快速简便的现场检测要求。
与仪器分析方法相比，免疫检测方法因其简便、经济、快速等优点而在农药残留检测领域中受到越来越多的关注。ELISA (Enzyme Linked Immunosorbent Assay)方法是常用的免疫检测方法，但是其却有操作步骤相对较多，检测时间较长，检测结果不够直观，不能在线进行检测等缺陷。因而ELISA在哒螨灵的快速检测上的应用受到很大的限制。胶体金试纸条无需专业操作人员及任何辅助类仪器，根据反应所显现的条带几分钟即可判定结果，不仅操作简便、 快速，且结果直观准确、成本低。因此，得到高特异性和高灵敏性的单克隆单体是免疫学检测的前提。
发明内容
本发明的目的在于提供一种哒螨灵单克隆抗体及其应用，该抗体对哒螨灵具有较好特异性和检测灵敏度，可以用来建立哒螨灵的免疫学检测方法。
为了实现上述目的，本发明提供以下技术方案：
一种抗哒螨灵的单克隆抗体，包括轻链可变区和重链可变区，其中，
所述轻链可变区的CDR1序列如SEQ ID NO：1所示；
所述轻链可变区的CDR2序列如SEQ ID NO：2所示；
所述轻链可变区的CDR3序列如SEQ ID NO：3所示；
所述重链可变区的CDR1序列如SEQ ID NO：4所示；
所述重链可变区的CDR2序列如SEQ ID NO：5所示；
所述重链可变区的CDR3序列如SEQ ID NO：6所示。
以上所述轻链可变区的氨基酸序列如SEQ ID NO：7所示，重链可变区的氨基酸序列如SEQ ID NO：8所示。
本发明所述的单克隆抗体在制备哒螨灵免疫检测产品中的应用。
抗哒螨灵单克隆抗体的制备步骤为：
（1）免疫原的制备：以哒螨灵为原料，将哒螨灵分子式中的酮基经羟胺类化合物处理变成肟类化合物，再将肟类化合物上的羟基衍变成羧基化合物，再通过碳化二亚胺法与载体蛋白的氨基偶联，合成免疫原，包被原是采用碳化二亚胺法与OVA 偶联，反应结束后，通过透析分离完全抗原和未偶联的小分子半抗原，通过紫外吸收扫描方法鉴定；
（2）小鼠的免疫：将免疫原与福氏佐剂乳化完全后，通过皮下多点注射免疫BALB/c小鼠；首次免疫采用福氏完全佐剂，加强免疫使用福氏不完全佐剂，冲刺免疫时免疫剂量为前一次免疫剂量的一半，与生理盐水混合均匀后直接进行腹腔注射；各次免疫间隔为三周；第三次免疫后，间隔一周采血检测血清效价和抑制；
（3）细胞融合与单克隆抗体的制备：通过聚乙二醇（PEG 2000）法，使小鼠脾细胞和小鼠骨
髓瘤细胞融合，通过HAT培养基培养，利用间接ELISA检测阳性细胞孔，并进一步利用间接竞争ELISA法测定阳性细胞孔的抑制效果，通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆，最终筛选获得杂交瘤细胞株；使用特异性的细胞株制备腹水并用Protein A进行抗体纯化，之后用超滤离心管去盐，冷冻抽干获得干粉状抗体，并于-20 ℃冻存备用。
有益效果：本发明提供了一种哒螨灵单克隆抗体及其应用，该抗体对哒螨灵具有较好的检测灵敏度（IC50值为0.21ng/mL），添加回收率为90-108%，可实现对农产品中哒螨灵残留量的检测，为哒螨灵残留量的免疫检测提供了原料，具有重要的实际应用价值。并且首次测得该抗体的可变区基因序列，为后续抗体试剂应用于检测试剂盒时采用规模化表达生产提供了序列基础。
附图说明
图1 为哒螨灵抗原合成的化学反应式。
图2 为哒螨灵检测的抑制曲线图。
具体实施方式
下面结合具体实施例来进一步描述本发明，但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 杂交瘤细胞株的制备
（1）完全抗原的制备
以哒螨灵原料加入5mL乙醇溶液中，在搅拌条件下加入O-（羧甲基）羟胺半盐酸盐，使得哒螨灵和（羧甲基）羟胺的浓度分别为5mM和10mM，加热回流反应2h，反应完成后，将反应液降至室温，通过旋转蒸发去掉有机溶剂，然后加入20mL水，使用乙醚抽提，得到乙醚抽提液，然后再进行旋转蒸发去除溶剂，得到化合物1；
室温下将得到的化合物1溶于5ml无水二甲基亚砜溶液中，然后加入KOH（终浓度为15mmo1），在搅拌的条件下加入100 mg溴乙酸，继续搅拌反应2 h，然后加入冰水混合物终止反应，再用乙酸乙酯萃取，除去未反应的化合物1，水相用3mol/L HCl酸化，出现白色沉淀；将反应物用漏斗过滤后再用去离子水洗涤并真空干燥，得到产物化合物2；
称取7mg化合物2、4mg EDC（1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐）和2mgNHS（N-羟基琥珀酰亚胺），加入DMF溶液中溶解，室温下搅拌反应5h；另取10mg BSA溶解于5mL、0.05M pH9.6的碳酸盐缓冲液中配置成BSA溶液，然后将上述反应液逐滴加入到BSA溶液中，室温搅拌反应过夜后，得到完全抗原，4℃透析三天，备用。将化合物2采用同样的方法与OVA进行偶联反应，制备包被抗原。
（2）动物免疫
选择的6～8周龄的BALB/c小鼠进行免疫，将以上制得的1mg/mL的完全抗原与等量福氏佐剂乳化均匀后，通过皮下多点注射免疫BALB/c小鼠，每只100μL。首次免疫采用福氏完全佐剂，加强免疫使用福氏不完全佐剂，冲刺免疫时免疫剂量为前一次免疫剂量的一半，与生理盐水混合均匀后直接进行腹腔注射；各次免疫间隔为三周。第三次免疫后，间隔一周采血检测血清效价和抑制；选择抑制最好的小鼠，在五免后21天冲刺免疫，准备融合。
（3）细胞融合
在冲刺免疫三天后，按照常规PEG（分子量为2000）方法进行细胞融合，具体步骤如下：
a、小鼠摘眼球取血，颈椎脱臼法处死小鼠后，立即放入75％酒精中消毒，浸泡5min左右，无菌操作取出小鼠的脾脏，用注射器胶头适度研磨并通过200目细胞筛网得到脾细胞悬液，收集，离心（1200rpm，8min），用RPMI-1640培养基洗涤脾细胞三次，最后一次离心后，
将脾细胞稀释到一定体积，计数，备用；
b、收集SP2/0细胞：融合前7-10天，将SP2/0瘤细胞用含10％FBS(胎牛血清)RPMI-1640培养基在5％CO2培养箱中培养。融合前要求SP2/0瘤细胞数量达到1-4*107，保证融合前SP2/0瘤细胞处于对数生长期。融合时，收集瘤细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
c、将脾细胞和SP2/0细胞按照计数比1：10 的比例混合，离心后用50% PEG融合，时间1 min，之后按照从慢到快，加入RPMI-1640基础培养液，离心后悬浮于含20%胎牛血清、2%的50×HAT的RPMI-1640筛选培养液中，加到96孔细胞培养板，置于37℃、5% CO2的培养箱中培养。
（4）细胞筛选与细胞株建立
在细胞融合的第3天对融合细胞进行RPMI-1640筛选培养液半换液，第5天进行用含20% 胎牛血清、1%的100×HT的RPMI-1640过渡培养液进行全换液，在第7天取细胞上清进行筛选。
筛选分两步：第一步先用间接ELISA筛选出阳性细胞孔，第二步以哒螨灵作为标准品，用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对标准品均有较好抑制的细胞孔，采用有限稀释法进行亚克隆，用同样的方法进行检测。重复三次，获得抗哒螨灵单克隆抗体的杂交瘤细胞株。
实施例2 抗哒螨灵单克隆抗体的制备
取8-10周龄BALB/c小鼠，每只小鼠腹腔注射无菌石蜡油1mL；7天后每只小鼠腹腔注射1×106杂交瘤细胞，从第七天开始收集腹水，将腹水通过辛酸-饱和硫酸铵法进行抗体纯化，获得的单抗置于-20℃保存。
实施例3 抗哒螨灵单克隆抗体灵敏度的鉴定
（1）包被：将包被原用0.05M pH9.6碳酸盐缓冲液从3ng/mL开始3倍比稀释，100μL/孔，37℃反应2h；
（2）洗涤：将板内溶液倾去，并用洗涤液洗涤3次，每次3min；
（3）封闭：拍干后，加入200μL/孔封闭液，37℃反应2h，洗涤后烘干备用；
（4）加样：将抗血清从1:1000开始倍比稀释，并加入到各稀释度的包被孔中，100μL/孔，37℃反应30min；充分洗涤后，加入1:3000稀释的HRP-羊抗鼠IgG，100μL/孔，37℃反应
30min。
（5）显色：将酶标板取出，充分洗涤后，每孔加入100μL的TMB显色液，37℃避光反应
15min；
（6）终止和测定：每孔加入50μL终止液以终止反应，然后用酶标仪测定各孔的OD450值。图1为测得的抑制曲线图，其R2＝0.997，Y=0.125+1.31/（1+（X/0.172）3.136），IC50为：0.21ng/mL，对哒螨灵的灵敏度较好，可用于哒螨灵分析检测。
实施例4 添加回收实验
以阴性水作为实验样本，向阴性样本中添加不同含量的哒螨灵标准品，按照实施例2的步骤进行标准曲线的测定和检测样本的测定，结果如表1所示，从表1中得出，哒螨灵的回收率在90％-108％，符合样本的添加回收要求。
表1 添加回收结果（n=5）
实施例5 哒螨灵单克隆抗体的氨基酸序列测序及分析
将筛选到的最佳的杂交瘤细胞株交于抗体序列测序服务公司测得抗体的氨基酸序列，所得单克隆抗体的具体序列如下：
抗体轻链可变区氨基酸序列为：
MSASPGEKVTMTCSASSSVSYMHWYQQKPGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDVATYYCHQGSGYPYTFGAGTKLEIKR（SEQ ID NO：7）；
轻链可变区的CDR1序列为：PGTSP（SEQ ID NO：1）；
轻链可变区的CDR2序列为：GRFSGS（SEQ ID NO：2）；
轻链可变区的CDR3序列为：SGYPYTFG（SEQ ID NO：3）；
抗体重链可变区氨基酸序列为：
QVQLKQSGPGLVKPSQSLSLTCSVTAYSITSGYYWNWIRQFPGNKLEWMGYISYDGNNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCTRAYYGMASYFDYWGQGTTVTVSS（SEQ ID NO：8）；
重链可变区的CDR1序列为：SITSGYY（SEQ ID NO：4）；
重链可变区的CDR2序列为：YISYDG（SEQ ID NO：5）；
重链可变区的CDR3序列为：TEDTATYYC（SEQ ID NO：6）。
序列表
<110> 苏州诚检生物科技有限公司
<120> 一种哒螨灵单克隆抗体及其应用
<141> 2020-11-26
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Claims (3)
1.一种抗哒螨灵的单克隆抗体，包括轻链可变区和重链可变区，其特征在于：
所述轻链可变区的CDR1序列如SEQ ID NO：1所示；
所述轻链可变区的CDR2序列如SEQ ID NO：2所示；
所述轻链可变区的CDR3序列如SEQ ID NO：3所示；
所述重链可变区的CDR1序列如SEQ ID NO：4所示；
所述重链可变区的CDR2序列如SEQ ID NO：5所示；
所述重链可变区的CDR3序列如SEQ ID NO：6所示。
2. 根据权利要求1所述的一种抗哒螨灵的单克隆抗体，其特征在于，所述轻链可变区的氨基酸序列如SEQ ID NO：7所示，重链可变区的氨基酸序列如SEQ ID NO：8所示。
3.权利要求1或2所述的单克隆抗体在制备哒螨灵免疫检测产品中的应用。
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