CN111149772A - 一种利用人工寄主提取寄生蜂毒液的方法 - Google Patents
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Abstract
本发明公开了一种利用人工寄主提取寄生蜂毒液的方法;首先制作人工寄主,再接种寄生蜂寄生,最后收集寄生蜂毒液,即可。本发明不仅高效,而且避免了解剖带来的寄生蜂其他组织的污染;且可以提取到大量、高纯度的寄生蜂毒液。
Description
技术领域
本发明属于生物技术领域,具体涉及一种利用人工寄主提取寄生蜂毒液的方法。
背景技术
寄生蜂是寄生性膜翅目昆虫,是重要的害虫天敌资源,在害虫生物防治中起到显著的生态和经济效益(严智超等, 寄生蜂毒液蛋白组成、功能及进化的研究进展. 中国生物防治学报, 2017. 33(1): 1-10.)。毒液是寄生蜂所保守共有,起到抑制寄主免疫、影响寄主发育、调节寄主代谢等功能,最终帮助寄生蜂后代在受害寄主中成功发育(Asgari Sand Rivers DB, Venom proteins from endoparasitoid wasps and their role inhost-parasite interactions. Annual Review of Entomology, 2011. 56: 313-335.)。寄生蜂种类繁多,已报道有10万余种,其多样性远超其他带毒节肢动物,如蜘蛛、蝎子、蜜蜂等。并且,在与寄主长期的对抗进化中,寄生蜂毒液在分子结构、靶标和功能上都获得了丰富的多样性。因而,寄生蜂毒液是宝贵的基因资源宝库,在新型杀虫基因筛选和药物开发中,均具有广阔的应用前景(Moreau SJM and Guillot S, Advances and prospects onbiosynthesis, structures and functions of venom proteins from parasiticwasps. Insect Biochemistry and Molecular Biology, 2005. 35(11): 1209-1223.)。
不像蜜蜂、蜘蛛、蝎子等毒液(主要起捕食或防御功能),寄生蜂毒液无法通过直接刺激或电击刺激等方法来收集。寄生蜂毒液的现有提取方法,仍是通过解剖寄生蜂毒囊并破裂来获取(Yan ZC et al., A venom serpin splicing isoform of theendoparasitoid wasp Pteromalus puparum suppresses host prophenoloxidasecascade by forming complexes with host hemolymph proteinases. JournalBiological Chemistry, 2017. 292(3): 1038-1051.),存在诸多的问题。如,寄生蜂个体微小,解剖所需的专业技术要求高,且耗时费力。同时,解剖过程中难免会引入来自毒囊等寄生蜂组织破裂所造成的污染。
前期研究报道,部分寄生蜂可在包裹特定溶液的人工寄主里产卵。报道的成功案例中,所采用的人工寄主材质有蜡质(Nettles WC et al., Synergistic action ofpotassium chloride and magnesium sulfate on parasitoid wasp oviposition.Science, 1982. 218(4568): 164-6.)、琼脂(Tilden RL and Ferkovich SM, Kairomonalstimulation of oviposition into an artificial substrate by the endoparasitoidMicroplitis croceipes (Hymenoptera: Braconidae). Annals of the EntomologicalSociety of America. 81(1): 152-156.)、Parafilm膜(谢中能和李理, 麦蛾茧蜂的离体培养. 生物防治通报, 1989. 5(2): 49-51.)、塑料薄膜(李丽英等, 十二种赤眼蜂的产卵行为及其与体外培育成功率的关系. 环境昆虫学报, 1989(1): 31-35.)等;其中包裹的溶液既有支持寄生蜂后代完全发育的培养基(李丽英等, 十二种赤眼蜂的产卵行为及其与体外培育成功率的关系. 环境昆虫学报, 1989(1): 31-35.),也有仅诱导产卵但不支持发育的溶液,如盐离子溶液、氨基酸溶液等。
检索后未发现有利用人工寄主提取寄生蜂毒液的报道。
发明内容
针对现有技术提取寄生蜂毒液产量低、纯度低、易污染的不足,本发明的目的是提供一种利用人工寄主提取寄生蜂毒液的方法;利用本发明的方法能够提取到大量、高纯度的寄生蜂毒液,满足科学研究以及农业、医学领域的用途。
本发明解决其技术问题采用的技术方案是:
一种利用人工寄主提取寄生蜂毒液的方法,首先制作人工寄主,再接种寄生蜂寄生,最后收集寄生蜂毒液。
上述利用人工寄主提取寄生蜂毒液的方法,主要包括如下具体步骤:
步骤1,制作人工寄主:配制诱导寄生蜂产卵的配方溶液,采用厚度为1-100 μm的可塑穿透性材料将诱导寄生蜂产卵的配方溶液包裹,形成诱导寄生蜂产卵的人工寄主;
步骤2,接种寄生蜂寄生:按10:1-100:1的蜂卵比,将寄生蜂雌蜂和其人工寄主置于同一密闭容器内,在黑暗条件下,让寄生蜂寄生4-24 h;
步骤3,收集寄生蜂毒液:将步骤2所得溶液过滤,采用孔径小于寄生蜂胚胎的滤芯,目的是使寄生后的溶液通过滤网,以过滤去除寄生蜂胚胎;接着收集过滤后的人工寄主溶液,即为寄生蜂毒液。
本申请的可塑穿透性材料需控制在1-100μm之间,材料厚度过薄,强度不够,材料厚度过厚,穿透不了;厚度控制在20-50μm,更优选的,厚度为30-40μm。
本申请将寄生时间控制在4-24h,低于4h,收集的毒液量不够;高于24h,胚胎死亡或孵化成幼虫,破裂或分泌出物质影响纯度。
作为本申请的一种优选技术方案,所述步骤1中,可诱导寄生蜂产卵的配方溶液为包含昆虫来源物质的培养液、盐离子溶液、氨基酸溶液中的一种,或上述多种的混合。
优选的,所述包含昆虫来源物质的培养液,其对应组分及对应质量百分比如下:40-45%柞蚕蛹血淋巴、10-20%鸡蛋黄、30-40%麦乳精溶液和10-20%尼氏盐溶液;
更优选的,所述尼氏盐溶液的各组分由NaCl、KCl、CaCl2、NaHCO3和H2O组成,其对应质量比为7.5:0.1:0.2:0.2:1000。
更优选的,所述包含昆虫来源物质的培养液,其对应组分及对应质量百分比如下:40%柞蚕蛹血淋巴、20%鸡蛋黄、30%麦乳精液和10%尼氏盐溶液。
优选的,所述盐离子溶液为KCl、NaCl、MgSO4、CaCl2、NaHCO3、MgCl2中的一种或多种的混合;优选的,所述盐离子溶液为KCl、MgSO4的混合,其中KCl终浓度为124.7 mM;MgSO4终浓度为36.5 mM。
优选的,所述氨基酸溶液为亮氨酸、苯丙氨酸、组氨酸的混合;其中亮氨酸终浓度900 mg/mL,苯丙氨酸终浓度600 mg/mL,组氨酸终浓度700 mg/mL。
优选的,所述盐离子-氨基酸混合溶液,其组成及对应质量百分比是:KCl 0.62%、MgSO4·H2O 0.60%、氨基丁酸0.2%、亮氨酸0.2%、异亮氨酸0.2%和鸟氨酸0.2%。
作为本申请的一种优选技术方案,所述步骤1中,可塑穿透性材料为蜡、Parafilm膜或塑料薄膜。
优选的,所述塑料薄膜为聚乙烯塑料薄膜或聚丙烯塑料薄膜。
作为本申请的一种优选技术方案,所述步骤3中,将步骤2所得溶液过滤包含两种转移方式,一是将人工寄主破裂或打开,连同其中溶液转移到过滤装置过滤;二是直接移取寄生后的人工寄主内的溶液至过滤装置过滤。
优选的,所述步骤3中,滤芯为纤维膜或尼龙滤网。
优选的,所述步骤3中,过滤采用方法为离心法、负压方法或注射器加压法等任意一种。
有益效果
本发明提供的利用人工寄主提取寄生蜂毒液的方法,与现有技术相比,具有以下有益效果:
(1)本发明省时省力,并且避免了解剖带来的寄生蜂其他组织的污染;
(2)本发明的方法可以提取到大量、高纯度的寄生蜂毒液。
附图说明
图1是松毛虫赤眼蜂寄生人工卵卡装置的示意图;
图2是松毛虫赤眼蜂毒液收集装置的示意图;
图3是实施例2松毛虫赤眼蜂毒液的SDS-PAGE电泳图;
附图标记说明:1-氨基酸溶液;2-卡子;3-平整的聚乙烯塑料薄膜;4-人工卵卡;5-松毛虫赤眼蜂雌蜂;6-收集盒;7-寄生后的氨基酸溶液;8-尼龙滤网。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的,均视为可以通过市场购买的常规产品。
实施例1
本实施例是利用包裹KCl-MgSO4溶液的人工蜡卵,获取短管赤眼蜂毒液,方法如下:
(1)制作人工蜡卵
取直径2 cm、高7 cm的玻璃管,加入13 mL KCl-MgSO4溶液(124.7 mM KCl,36.5 mMMgSO4),并置于50 ℃水浴锅中;将熔点为48 ℃的石蜡和凡士林按3:1混合;将0.5 g该石蜡-凡士林混合物加热融化,加入玻璃管,覆盖在KCl-MgSO4溶液面,形成约2 mm厚的蜡液覆盖层;将内径1 mm的毛细管,插入液面吸取氨基酸溶液及蜡液覆盖层,然后迅速移出接触Parafilm膜表面,形成包裹氨基酸溶液的人工蜡卵(直径为2–3 mm、高1.5–2 mm)。
(2)接入短管赤眼蜂寄生
取滴有5个蜡卵的Parafilm膜,置于玻璃管中,接入50头羽化一天内的短管赤眼蜂雌蜂;置于室温黑暗环境下,让短管赤眼蜂自由寄生4 h。
(3)收集短管赤眼蜂毒液
寄生后,取出步骤(2)人工蜡卵的Parafilm膜,用细毛笔去除攀附在Parafilm膜上的短管赤眼蜂;采用塑料薄片将人工蜡卵底部翘起,剥离Parafilm膜并挑入过滤离心管Corning™ Costar™ Spin-X™ Centrifuge Tube Filters(孔径为0.45 µm),100 g离心2 min,收集过滤后的溶液,即为短管赤眼蜂毒液。
实施例2
本实施例是利用含氨基酸溶液的聚乙烯薄膜卵卡,获取松毛虫赤眼蜂毒液,方法如下:
(1)制作聚乙烯塑料薄膜卵卡
取长16 cm、宽12 cm、厚度为40 µm的聚乙烯塑料薄膜,采用玻璃研磨棒,按标准的PCR板96孔的排布,按压出48个直径2–3 mm、高约3 mm的半圆形突起;制成聚乙烯塑料薄膜压制的人工卵卡4,将压制好的人工卵卡4放在紫外灯下,正反面各照射灭菌1 h:尔后,在每个半圆内加入4 µL诱导松毛虫赤眼蜂产卵的氨基酸溶液1(900 mg/mL亮氨酸,600 mg/mL苯丙氨酸,700 mg/mL组氨酸)。
(2)接入松毛虫赤眼蜂寄生
将CO2麻醉后的松毛虫赤眼蜂雌蜂5接入收集盒6内,使其蜂卵比约为10:1;再将薄膜卵卡4的凸起面朝向收集盒6,再盖上一张长16 cm、宽12 cm的平整聚乙烯塑料薄膜;采用卡子2,将两张薄膜绷紧覆盖在收集盒6上;待松毛虫赤眼蜂苏醒,置于室温黑暗环境下,让其自由寄生12 h。
(3)收集松毛虫赤眼蜂毒液
寄生后,人工卵卡4内凸起内得到寄生后的氨基酸溶液7;将寄生装置置于4 ℃ 20min,以降低松毛虫赤眼蜂的活动力;取一新的收集盒,开口处盖上长16 cm、宽12 cm的1500目尼龙滤网8,再将塑料薄膜卵卡从寄生装置取下,将其凹面朝向滤网,置于滤网上,并用卡子2将塑料薄膜卵卡和尼龙滤网绷紧卡住;采用Eppendorf 5810R台式离心机,100 g离心5min,收集过滤后的溶液,即为松毛虫赤眼蜂毒液,约200 µL。
(4)松毛虫赤眼蜂毒液SDS-PAGE
采用上海生工改良型Bradford蛋白浓度测定试剂盒,对收集的松毛虫毒液浓度进行了测定,约为50 µg/mL。将松毛虫赤眼蜂毒液转移至3 kDa Amicon® Ultra-0.5超滤管,14000 g离心1 h。尔后将浓缩的松毛虫赤眼蜂毒液加入5x SDS-PAGE电泳上样缓冲液,95℃加热2 min,10000 g离心1 min。采用金斯瑞SurePAGE 预制胶和Tris-MOPS-SDS跑胶缓冲液,200 V跑胶40 min。最后,采用金斯瑞eStain L1蛋白染色仪,对SDS-PAGE胶进行染色脱色。
实施例3
本实施例是利用含盐离子-氨基酸混合溶液的聚乙烯塑料薄膜卵卡,获取纽斯赤眼蜂毒液,方法如下:
(1)制作聚乙烯塑料薄膜卵卡
参照实施例2中方法,不同之处是其中加入的诱导产卵溶液为盐离子-氨基酸混合溶液。其组成及对应质量百分比是:KCl 0.62%、MgSO4·H2O 0.60%、氨基丁酸0.2%、亮氨酸0.2%、异亮氨酸0.2%和鸟氨酸0.2%。
(2)接入纽斯赤眼蜂寄生
将CO2麻醉后的纽斯赤眼蜂雌蜂5接入收集盒6内,使其蜂卵比约为50:1;再将薄膜卵卡4的凸起面朝向收集盒6,再盖上一张长16 cm、宽12 cm的平整聚乙烯塑料薄膜;采用卡子2,将两张薄膜绷紧覆盖在收集盒6上;待纽斯赤眼蜂苏醒,置于室温黑暗环境下,让其自由寄生24 h。
(3)收集纽斯赤眼蜂毒液
寄生后,人工卵卡4内凸起内得到寄生后的盐离子-氨基酸混合溶液;将寄生装置置于4℃ 20 min,以降低纽斯赤眼蜂的活动力;取一新的收集盒,开口处盖上长16 cm、宽12 cm的1500目尼龙滤网8,再将塑料薄膜卵卡从寄生装置取下,将其凹面朝向滤网,置于滤网上,并用卡子2将塑料薄膜卵卡和尼龙滤网绷紧卡住;采用Eppendorf 5810R台式离心机,100 g离心5 min,收集过滤后的溶液,即为纽斯赤眼蜂毒液。
实施例4
本实施例是利用包含完全培养液(含昆虫来源物质)的聚丙烯薄膜卵卡,获取平腹小蜂毒液,方法如下:
(1)制作聚丙烯塑料薄膜卵卡
参照实施例2中方法,不同之处是采用厚度为30 µm的聚丙烯塑料薄膜,其中加入的诱导产卵溶液为含昆虫来源物质的完全培养基;其组成及对应质量百分比为:40-45%柞蚕蛹血淋巴、10-20%鸡蛋黄、30-40%麦乳精溶液和10-20%尼氏盐溶液;所述尼氏盐溶液的各组分由NaCl、KCl、CaCl2、NaHCO3和H2O组成,对应质量比为7.5:0.1:0.2:0.2:1000;
其中,配方可以根据实际情况进行调整,如柞蚕蛹血淋巴44.4%、10%麦乳精溶液33.3%、鸡蛋黄11.1%和尼氏盐11.2%;还如柞蚕蛹血淋巴40%、鸡蛋黄20%、麦乳精溶液30%和尼氏盐溶液10%;再如柞蚕蛹血淋巴43.5%、鸡蛋黄11.5%、麦乳精溶液32.5%和12.5%尼氏盐溶液等。
(2)接入平腹小蜂寄生
将CO2麻醉后的平腹小蜂雌蜂接入收集盒6内,使其蜂卵比约为100:1;再将薄膜卵卡4的凸起面朝向收集盒6,再盖上一张长16 cm、宽12 cm的平整聚乙烯塑料薄膜;采用卡子2,将两张薄膜绷紧覆盖在收集盒6上;待平腹小蜂苏醒,置于室温黑暗环境下,让其自由寄生12 h。
(3)收集平腹小蜂毒液
寄生后,人工卵卡4内凸起内得到寄生后的完全培养液;将寄生装置置于4 ℃ 20 min,以降低平腹小蜂的活动力;取一新的收集盒,开口处盖上长16 cm、宽12 cm的1500目尼龙滤网8,再将塑料薄膜卵卡从寄生装置取下,将其凹面朝向滤网,置于滤网上,并用卡子2将塑料薄膜卵卡和尼龙滤网绷紧卡住;采用Eppendorf 5810R台式离心机,100 g离心5 min,收集过滤后的溶液,即为平腹小蜂毒液。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求为保护范围。
Claims (10)
1.一种利用人工寄主提取寄生蜂毒液的方法,其特征在于,首先制作人工寄主,再接种寄生蜂寄生,最后收集寄生蜂毒液。
2.根据权利要求1所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,包括以下主要步骤的系列操作:
步骤1,制作人工寄主:配制诱导寄生蜂产卵的溶液,然后采用厚度为1-100 μm的可塑穿透性材料将诱导寄生蜂产卵的配方溶液包裹,形成诱导寄生蜂产卵的人工寄主;
步骤2,接种寄生蜂寄生:按10:1-100:1的蜂卵比,将寄生蜂雌蜂和人工寄主置于同一密闭容器内,黑暗条件下保持4-24 h;
步骤3,收集寄生蜂毒液:将步骤2所得人工寄主采用滤芯进行过滤,收集过滤后的人工寄主溶液,即为寄生蜂毒液。
3.根据权利要求2所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述步骤1中,可塑穿透性材料为蜡、Parafilm膜或塑料薄膜,优选的,所述塑料薄膜为聚乙烯塑料薄膜或聚丙烯塑料薄膜。
4.根据权利要求2所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述步骤1中,可诱导寄生蜂产卵的配方溶液为包含昆虫来源物质的培养液、盐离子溶液、氨基酸溶液中的一种或上述多种的混合。
5.根据权利要求3所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述包含昆虫来源物质的培养液,其对应组分及对应质量百分比如下:40-45%柞蚕蛹血淋巴、10-20%鸡蛋黄、30-40%麦乳精溶液和10-20%尼氏盐溶液;优选的,所述尼氏盐溶液的各组分由NaCl、KCl、CaCl2、NaHCO3和H2O组成,其对应质量比为7.5:0.1:0.2:0.2:1000。
6. 根据权利要求4所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述盐离子溶液为KCl、NaCl、MgSO4、CaCl2、NaHCO3、MgCl2中的一种或多种的混合;优选的,所述盐离子溶液为KCl、MgSO4的混合,其中KCl终浓度为124.7 mM;MgSO4终浓度为36.5 mM。
7. 根据权利要求4所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述氨基酸溶液为亮氨酸、苯丙氨酸、组氨酸的混合;其中亮氨酸终浓度为900 mg/mL,苯丙氨酸终浓度600 mg/mL,组氨酸终浓度700 mg/mL。
8.根据权利要求2所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述步骤3中,将步骤2所得溶液过滤包含两种转移方式,一是将人工寄主破裂或打开,连同其中溶液转移到过滤装置过滤;二是直接移取寄生后的人工寄主内的溶液至过滤装置过滤。
9.根据权利要求8所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述步骤3中,滤芯为纤维膜或尼龙滤网。
10.根据权利要求8所述的利用人工寄主提取寄生蜂毒液的方法,其特征在于,所述步骤3中,过滤采用方法为离心法、负压方法或注射器加压法中的一种。
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