CN107446030A - 一种伊维菌素偶联的Bt杀虫毒素及其应用 - Google Patents
一种伊维菌素偶联的Bt杀虫毒素及其应用 Download PDFInfo
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Abstract
本发明提供一种伊维菌素偶联的Bt杀虫毒素，其是在Bt杀虫蛋白Cry2Ab和4”,5‑O‑succinoyl伊维菌素在偶联剂EDC、NHS的共同作用下键合形成的一种偶联型杀虫毒素，该Bt杀虫毒素具有较强的毒力，其对小菜蛾具有更好的杀虫效果，即对防治农业害虫具有更好的应用前景，能够作为针对小菜蛾的农业杀虫剂；且该Bt杀虫毒素的制备方法过程简单便捷、可操作性强。
Description
【技术领域】
本发明属于农业害虫防治领域，具体涉及一种伊维菌素偶联的Bt杀虫毒素及其应用。
【背景技术】
近些年，农业科学的快速发展，农药防治起一定程度的促进作用。农药可以有效的防治农业病、虫、草、鼠等有害生物，保证了农业增产丰收这一重要环节。但病害抗药性的产生，是无论化学农药还是生物农药都不能避免的难题；害虫对农药抗药性就是克服农药位点作用，使之失效、钝化。在实际应用中，农药轮换施用、农药混配施用、增加农药对害虫的接触面积、培育害虫的敏感品系、增加生态多样性以增加害虫的多样性等等，实质上是使农药作用位点保持足够的敏感性，寻求克服抗性的手段。生物偶联是利用生物偶联技术将两种毒素偶联形成新的毒素，对保持农药位点的敏感性等具有重要的意义。
苏云金杆菌(Bacilus thuringiensis，简称Bt)是目前世界上生产量最大、应用最为广泛的一类微生物杀虫剂，其晶体蛋白对570余种鳞翅目害虫及双翅目、膜翅目和鞘翅目等数十种害虫有杀虫毒力。一般认为，昆虫在取食晶体蛋白后，通过自身的中肠蛋白酶将其酶解为活性的毒素蛋白，毒素蛋白再和中肠上皮细胞刷状缘膜(BBMVs)上的受体结合，并进一步插入膜内，形成孔洞或离子通道，引起离子渗漏，破坏肠壁上皮细胞，使肠道内溶物渗入血腔，引起败血症，致使昆虫死亡；但昆虫的抗药性使得现有的Bt毒素的防效急剧下降，因此，对Bt毒素进行改造，提供一种具有较强毒力的Bt毒素是从业人员所迫切希望地。
【发明内容】
本发明所要解决的技术问题在于提供一种伊维菌素偶联的Bt杀虫毒素及其应用。
本发明是通过以下技术方案解决上述技术问题的：一种伊维菌素偶联的Bt杀虫毒素，其制备方法的具体操作如下：按1:1.5:1.5的摩尔比分别称取4”,5-O-succinoyl伊维菌素、EDC和NHS，且溶于DMSO溶剂内，以对4”,5-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”,5-O-succinoyl伊维菌素，待用；取Bt Cry2Ab杀虫蛋白配置含5mmol/L BtCry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即Bt Cry2Ab杀虫蛋白溶液，待用；分别量取配制好的活化后的4”,5-O-succinoyl伊维菌素与所述Bt Cry2Ab杀虫蛋白溶液，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
进一步地，所述Bt Cry2Ab杀虫蛋白的制备步骤如下：
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将其接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，并先采用Ni50缓冲液除去杂蛋白，后采用Ni500缓冲液洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到BtCry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
进一步地，所述Ni50缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑50mM；Ni500缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑500mM。
进一步地，所述4”,5-O-succinoyl伊维菌素制备过程为：取伊维菌素溶解于二氯甲烷，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且伊维菌素、4-二甲氨基吡啶、三乙胺和琥珀酸酐的摩尔比为1:4:8:16，避光水浴回流3小时，接着采用乙醚萃取产物，柱层析分离即得到4”,5-O-succinoyl伊维菌素。
本发明还揭露了所述Bt杀虫毒素作为农业杀虫剂的应用。
本发明的有益效果在于：提供一种伊维菌素偶联的Bt杀虫毒素，该Bt杀虫毒素具有较强的毒力，其对小菜蛾具有更好的杀虫效果，能够作为农业杀虫剂，即对防治农业害虫具有更好的应用前景；且同时揭露了该Bt杀虫毒素的制备方法，该制备方法过程简单便捷、可操作性。
【具体实施方式】
为了更好地理解本发明，下面结合实施例和应用例进一步地阐述说明本发明的内容，但这些实施例与应用例仅是示例性的，并不用于限制本发明的范围。且需要说明的是，本发明中所涉及的培养基在无特殊说明的情况下均为现有常规性培养基。
实施例1
4”,5-O-succinoyl伊维菌素的制备
取伊维菌素溶解于二氯甲烷，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且伊维菌素、4-二甲氨基吡啶、三乙胺和琥珀酸酐的摩尔比为1:4:8:16，之后避光水浴回流3小时，接着采用乙醚萃取产物，柱层析分离即得到4”,5-O-succinoyl伊维菌素。
通过MS和NMR技术鉴定4”,5-O-succinoyl伊维菌素的结构：
1H NMR(400MHz,CDCl3)δ5.86(d,J＝10.0Hz,1H),5.74(dd,J＝7.6,4.9Hz,2H),5.56(s,2H),5.40(s,1H),5.30(d,J＝9.0Hz,1H),5.13(s,0H),5.01(d,J＝10.4Hz,1H),4.79(s,1H),4.65(dt,J＝31.6,11.8Hz,3H),4.06(d,J＝5.8Hz,1H),3.95(s,1H),3.85(dt,J＝12.5,6.3Hz,2H),3.69–3.60(m,3H),3.44(s,3H),3.40–3.31(m,4H),3.28–3.17(m,2H),2.79–2.61(m,9H),2.28(ddd,J＝46.5,19.4,13.1Hz,5H),2.11–1.96(m,4H),1.78–1.60(m,9H),1.59–1.47(m,10H),1.30–1.22(m,11H),1.16(dd,J＝15.0,6.5Hz,7H),1.02–0.75(m,13H)；
13C NMR(101MHz,CDCl3)δ177.19(d,J＝11.3Hz,1H),173.42(s,0H),171.73(d,J＝37.2Hz,0H),139.75–132.46(m,-7H),128.74–117.10(m,-12H),96.40(t,J＝168.8Hz,-8H),80.66(dd,J＝156.4,125.3Hz,-5H),75.29(d,J＝69.6Hz,-3H),71.51–63.78(m,-7H),58.02–39.40(m,-23H),31.00(dddd,J＝143.1,138.5,133.7,125.6Hz,-1H),24.48–10.49(m,-10H)；
ESI-MS(m/z):1097.45[M+Na]+。
从而确定了该4”,5-O-succinoyl伊维菌素的化学结构，其化学结构如下：
实施例2
苏云金芽胞杆菌的杀虫蛋白即Bt Cry2Ab杀虫蛋白的制备
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将该含有cry2Ab基因的大肠杆菌工程菌接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，先采用Ni50缓冲液(NaCl 300mM、NaH2PO4 50mM、咪唑50mM)除去杂蛋白；后采用Ni500缓冲液(NaCl 300mM、NaH2PO4 50mM、咪唑500mM)洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到Bt Cry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
实施例3
Bt杀虫毒素的制备
取实施例1制备所得的4”,5-O-succinoyl伊维菌素(0.5mmol)，用1mL DMSO溶解，并加入EDC(0.75mmol)和NHS(0.75mmol)，室温搅拌2h，对4”,5-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”,5-O-succinoyl伊维菌素，待用；取实施例2制备所得的BtCry2Ab杀虫蛋白配置含5mmol/L Bt Cry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即Bt Cry2Ab杀虫蛋白溶液，待用；之后在4℃条件下，将100μL活化后的4”,5-O-succinoyl伊维菌素加入所述Bt Cry2Ab杀虫蛋白溶液1mL中，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
应用例1
Bt杀虫毒素的杀虫活性分析
(1)小菜蛾的准备
采集室内种群的小菜蛾蛹，羽化，收集成虫产卵，每24小时收集一次小菜蛾卵，收集的同批的小菜蛾卵在人工气候箱内以同样条件饲养以备LC50测定，该人工气候箱的条件为：温度25℃、相对湿度70％、光周期16：8(L：D)。
(2)验证方法与结果
采用24孔平板饲喂法测定Bt Cry2Ab杀虫蛋白、实施例3制备所得的Bt杀虫毒素对小菜蛾二龄幼虫的死亡率，同时设置实验组和对照组。具体实验方法为，实验组：将Bt杀虫毒素进行稀释，得到不同浓度的Bt杀虫毒素溶液；接着在无菌环境下，吸取1mL的饲料分装于24孔板中并自然晾干，之后分别吸取100μL配置好的不同浓度的Bt杀虫毒素溶液并均匀的涂布于饲料表面，以100μL Na2CO3/NaHCO3缓冲液作为空白对照，各组做好标记、自然状态下晾干；二龄小菜蛾接种到24孔板(每孔5-7只)，每个浓度梯度4孔重复，48小时测定后小菜蛾的死亡率(虫的死亡以用毛笔轻触小菜蛾不动为准)，得出LC50。在对照组，与实验组的唯一区别在于以Bt Cry2Ab杀虫蛋白作为效应物。试验结果见下表1。
表1 Bt杀虫毒素的杀虫效果
经由表1并利用SPSS软件的回归模型进行分析，得出：对照组即Bt Cry2Ab杀虫蛋白对二龄小菜蛾的LC50为0.922μg/cm2，实验组即本发明Bt杀虫毒素对二龄小菜蛾的LC50为0.401μg/cm2，杀虫毒力大概提高了2.30倍这个结果表明本发明Bt杀虫毒素确实能够提高对小菜蛾的杀虫毒力。
综上所述，本发明通过偶联而形成的Bt杀虫毒素具有较强的毒力，本发明Bt杀虫毒素对小菜蛾具有更好的杀虫效果，换而言之，其能够作为农业杀虫剂用于杀害小菜蛾，对防治农业害虫具有更好的应用前景。
Claims (5)
1.一种伊维菌素偶联的Bt杀虫毒素，其特征在于：其制备方法的具体操作如下：按1:1.5:1.5的摩尔比分别称取4”,5-O-succinoyl伊维菌素、EDC和NHS，且溶于DMSO溶剂内，以对4”,5-O-succinoyl伊维菌素的羧基进行活化，得活化后的4”,5-O-succinoyl伊维菌素，待用；取Bt Cry2Ab杀虫蛋白配置含5mmol/L Bt Cry2Ab杀虫蛋白的Na2CO3/NaHCO3缓冲液，即BtCry2Ab杀虫蛋白溶液，待用；分别量取配制好的活化后的4”,5-O-succinoyl伊维菌素与所述Bt Cry2Ab杀虫蛋白溶液，搅拌1小时进行偶联反应，获得Bt杀虫毒素。
2.根据权利要求2一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述Bt Cry2Ab杀虫蛋白的制备步骤如下：
(1)将cry2Ab基因通过热击法转化至大肠杆菌工程菌以获得含有cry2Ab基因的大肠杆菌工程菌，之后将其接种至LB固体培养基上，将LB固体培养基置于30℃下恒温培养24h进行菌种活化；
(2)将活化后的大肠杆菌工程菌菌种接种于LB液体培养基中，并置于摇瓶中37℃发酵培养，转速设定为180r/min，待菌液OD600到达0.5后，将温度调整为25℃，180r/min继续培养24h；接着将发酵培养所得发酵液置于4℃、10000r/min条件下离心10min，离心结束后取沉淀重悬于Na2CO3裂解液，于4℃下超声裂解30min，裂解后的溶液4℃、10000r/min离心30min，取上清液；
(3)将步骤(2)所得上清液过IDA-Ni亲和层析柱，并先采用Ni50缓冲液除去杂蛋白，后采用Ni500缓冲液洗脱目的蛋白；洗脱所得目的蛋白经脱盐柱PD-10脱盐则得到Bt Cry2Ab杀虫蛋白，将Bt Cry2Ab杀虫蛋白置于-20℃下保存，备用。
3.根据权利要求2所述的一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述Ni50缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑50mM；Ni500缓冲液的配方为NaCl 300mM、NaH2PO4 50mM、咪唑500mM。
4.根据权利要求1所述的一种伊维菌素偶联的Bt杀虫毒素，其特征在于：所述4”,5-O-succinoyl伊维菌素制备过程为：取伊维菌素溶解于二氯甲烷，并加入4-二甲氨基吡啶、三乙胺和琥珀酸酐，且伊维菌素、4-二甲氨基吡啶、三乙胺和琥珀酸酐的摩尔比为1:4:8:16，之后避光水浴回流3小时，接着采用乙醚萃取产物，柱层析分离即得到4”,5-O-succinoyl伊维菌素。
5.一种权利要求1所述伊维菌素偶联的Bt杀虫毒素的应用，其特征在于：所述Bt杀虫毒素作为农业杀虫剂的应用。
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