CN103969448B - Cyp6aa9作为蚊对溴氰菊酯抗性检测靶标的应用 - Google Patents

Cyp6aa9作为蚊对溴氰菊酯抗性检测靶标的应用 Download PDF

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CN103969448B
CN103969448B CN201410217550.9A CN201410217550A CN103969448B CN 103969448 B CN103969448 B CN 103969448B CN 201410217550 A CN201410217550 A CN 201410217550A CN 103969448 B CN103969448 B CN 103969448B
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cyp6aa9
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CN103969448A (zh
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沈波
王卫杰
吕园
方福瑾
过琴
孙立新
杨庆贵
周丹
张东辉
孙艳
马磊
朱昌亮
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Nanjing Medical University
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Abstract

本发明公开了CYP6AA9作为蚊对溴氰菊酯抗性检测靶标的应用。采用双抗体夹心ELISA法测定待测品系CYP6AA9蛋白的OD值大于敏感品系OD值的1.18倍时，可认为待测品系对溴氰菊酯具有抗性。本发明发现了一个蚊对溴氰菊酯抗性检测的靶标，对于蚊溴氰菊酯抗性的早期发现和治理具有重要价值。

Description

CYP6AA9作为蚊对溴氰菊酯抗性检测靶标的应用
技术领域
本发明涉及CYP6AA9作为蚊对溴氰菊酯抗性检测靶标的应用。
背景技术
蚊可以传播疟疾、登革热、黄热病、丝虫病等多种蚊媒病，严重危害人类健康。控制蚊媒对于预防蚊媒病具有重要意义。化学防治一直作为蚊媒控制策略中的主要方法。但随之产生的抗药性成为蚊媒病控制的最大障碍。早期发现并实时监测蚊现场种群对不同杀虫剂的抗性水平，是蚊抗药性治理的前提。寻找有效的抗药性检测靶标对于抗药性治理具有非常重要意义。目前，拟除虫菊酯类杀虫剂，如溴氰菊酯，是应用最为广泛的一类杀虫剂，但由于缺乏相关的抗药性检测靶标，所以相应的分子生物学和免疫学测定方法至今尚未建立。因此，蚊拟除虫菊酯类抗性检测方法的建立一直是媒介防治研究的热点课题。
细胞色素P450超家族(CytochromeP450proteins,CYP)主要参与生物体内源性物质的代谢与转化及外源性化合物的活化与降解等重要生理过程。有研究证实昆虫体内CYP参与杀虫剂的代谢，是昆虫对杀虫剂(尤其是拟除虫菊酯类杀虫剂)产生高水平抗性的主要原因。昆虫体内CYP种类呈现多样性，根据氨基酸同源性分析，可分为CYP4、CYP6、CYP9、CYP12等67个家族，其中CYP6家族被证实与外源性物质代谢有关，因此与杀虫剂抗性的关系也最为密切。本发明中的抗药性检测靶标CYP6AA9蛋白属于CYP6家族，在本实验室溴氰菊酯抗性品系中高表达，可能参与了蚊抗药性的产生。
发明内容
本发明的目的是提供CYP6AA9蛋白的新用途。
本发明的技术方案是：CYP6AA9蛋白作为蚊对溴氰菊酯抗性检测靶标的应用。
发明者根据CYP6AA9基因的核苷酸序列，设计合成实时定量PCR引物，并利用实时定量PCR方法比较了CYP6AA9基因在敏感品系DS和抗性品系DR2中的表达，结果发现CYP6AA9基因在DR2品系中高表达，为DS品系中的23.65倍，提示CYP6AA9基因与蚊拟除虫菊酯抗性相关。
针对CYP6AA9蛋白的氨基酸序列，设计合成了2个肽段，经多次免疫新西兰大白兔，成功制备CYP6AA9蛋白的多克隆抗体。发明者进一步通过Westernblot方法显示CYP6AA9蛋白在DR2品系中的表达量高于DS品系，证实CYP6AA9蛋白与蚊拟除虫菊酯抗性相关。
基于CYP6AA9蛋白，发明者采用双抗体夹心ELISA法测定了DS品系、DR1品系和DR2品系中CYP6AA9蛋白的表达量。结果发现DR1品系和DR2品系的OD值分别是DS品系OD值的1.18倍和1.79倍。
本发明公开了测定淡色库蚊CYP6AA9蛋白的表达量来判断蚊对溴氰菊酯的抗性。具体是：选取国内通用的淡色库蚊敏感品系(LC50为0.02mg/L)作为参照，通过双抗体夹心ELISA法测定待测品系和敏感品系中CYP6AA9的表达量，当待测品系OD值大于敏感品系OD值的1.18倍以上时，即可认定待测品系对溴氰菊酯具有抗性。
本发明提供了一种新的淡色库蚊拟除虫菊酯抗性检测的靶标，对于检测现场种群的抗性水平及抗药性治理具有重要价值。
附图说明
图1实时定量PCR方法比较CYP6AA9基因在DS品系和DR2品系雌蚊中的表达。
图2Westernblot方法比较CYP6AA9蛋白在DS品系和DR2品系雌蚊中的表达。
图3双抗体夹心ELISA法检测CYP6AA9蛋白在DS品系、DR1品系和DR2品系雌蚊中的表达。
除非特别说明，本发明中所使用的术语，一般为本领域普通技术人员通常理解的含义。
下面结合具体的实施例，并参照数据进一步详细地描述本发明。这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下的实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。
实施例1CYP6AA9基因在DR2品系雌蚊中表达量高于DS品系。
本发明中使用的淡色库蚊敏感品系(DS品系)经实验室长期饲养，期间未接触任何杀虫剂，经幼虫浸渍法测定其对溴氰菊酯的半数致死浓度(LC50)为0.02mg/L，符合国内通用的敏感品系标准，且该品系使用情况已经在国际杂志上公开发表(SunY,ZouP,YuXY,ChenC,YuJ,etal.(2012)FunctionalcharacterizationofanarrestingeneoninsecticideresistanceofCulexpipienspallens.ParasitVectors5:134)(HongS,ZhouD,ChenC,WangW,LvY,etal.(2013)Ribose-phosphatepyrophosphokinase1(PRPS1)associatedwithdeltamethrinresistanceinCulexpipienspallens.ParasitolRes112:847-854.)。抗性品系DR1和DR2是由DS品系在本实验室经一定浓度溴氰菊酯筛选至第18代和32代，测定其对溴氰菊酯的LC50分别为0.18mg/L和0.85mg/L。
发明者根据CYP6AA9基因的核苷酸序列(NCBIID：XM_001847353)，设计合成实时定量PCR引物，序列如下：F5'GCTGGCAGTTCATGGTGGT3'(SEQIDNO.1)和R5'AGACGATAGCTGATAGGGTTGGT3'(SEQIDNO.2)；并以淡色库蚊β-actin基因(NCBIID：AY100005)作为内参，其引物序列如下：F5'AGCGTGAACTGACGGCTCTTG3'(SEQIDNO.3)和R5'ACTCGTCGTACTCCTGCTTGG3'(SEQIDNO.4)。分别提取DS品系和DR2品系雌蚊(羽化后2～3d)阶段的总RNA。通过实时定量PCR方法比较CYP6AA9基因在DS品系和DR2品系中的表达。结果发现CYP6AA9基因在DR2品系中高表达，表达量为DS品系中的23.65倍(P<0.05)(图1)。
实施例2CYP6AA9蛋白在DR2品系雌蚊中表达量高于DS品系。
发明者针对CYP6AA9蛋白的氨基酸序列(NCBIID：XP_001847405)，设计合成2个肽段，序列如下：肽段1NH2-DPDIYPNPSQFDPDRC-CONH2(SEQIDNO.5)和肽段2NH2-RNVPHEPGQFPMGSLC-CONH2(SEQIDNO.6)。根据获得的2个肽段，分别经多次免疫新西兰大白兔，成功制备了2种CYP6AA9多克隆抗体，分别命名为：CYP6AA9-1和CYP6AA9-2抗体。发明者分别提取了DS品系和DR2品系雌蚊(羽化后2～3d)阶段的总蛋白，并通过Westernblot方法进一步比较了CYP6AA9蛋白在DS品系和DR2品系雌蚊中的表达。结果发现CYP6AA9蛋白在DR2品系中明显高表达(图2)。
实施例3CYP6AA9蛋白可作为蚊抗药性检测的靶抗原。
发明者分别提取了DS品系、DR1品系和DR2品系雌蚊(羽化后2～3d)阶段的总蛋白，并稀释至相同蛋白质浓度4μg/μl。对实施例2中获得的CYP6AA9-1抗体进行辣根过氧化物酶标记(用作酶标抗体)。通过双抗体夹心ELISA法测定DS品系、DR1品系和DR2品系中CYP6AA9蛋白的表达量。结果发现CYP6AA9蛋白在DR1品系和DR2品系中的读值为DS品系中的1.18倍(P<0.05)和1.79倍(P<0.05)(图3)。

Claims (3)

1.CYP6AA9作为淡色库蚊对溴氰菊酯抗性检测靶标的应用，其特征在于，通过检测淡色库蚊CYP6AA9表达量来判断淡色库蚊对溴氰菊酯的抗性。
2.根据权利要求1所述的应用，其特征在于，具体方法是：通过检测雌蚊中CYP6AA9表达量来判断淡色库蚊对溴氰菊酯的抗性。
3.根据权利要求2所述的应用，其特征在于具体是：采用双抗体夹心ELISA法检测待测品系CYP6AA9蛋白的OD值大于敏感品系OD值的1.18倍时，待测品系对溴氰菊酯具有抗性。
CN201410217550.9A 2014-05-21 2014-05-21 Cyp6aa9作为蚊对溴氰菊酯抗性检测靶标的应用 Expired - Fee Related CN103969448B (zh)

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