CN1384195A - 棉铃虫颗粒体病毒增效蛋白应用 - Google Patents

棉铃虫颗粒体病毒增效蛋白应用 Download PDF

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CN1384195A
CN1384195A CN01114674A CN01114674A CN1384195A CN 1384195 A CN1384195 A CN 1384195A CN 01114674 A CN01114674 A CN 01114674A CN 01114674 A CN01114674 A CN 01114674A CN 1384195 A CN1384195 A CN 1384195A
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孟小林
徐进平
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WANDEFU GENE ENGINEERING Co Ltd SHENZHEN
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Abstract

本发明公开了利用基因工程生产的棉铃虫颗粒体病毒(HaGV)增效蛋白作为有效成分,提高病毒杀虫剂、BT杀虫剂、阿维菌素等生物杀虫剂或利用生物杀虫剂制备的复合生物杀虫剂,或单独用于转基因抗虫植物的防治虫害方法。

Description

棉铃虫颗粒体病毒增效蛋白应用
本发明涉及利用工程菌株Escherichia coli M15(pEHa)生产棉铃虫增效蛋白PHEn的方法，以及PHEn在农林业病虫生物防治中的应用。
目前，已在9种颗粒体病毒、舞毒蛾(Lymantria dispar)NPV和4种痘病毒中发现了昆虫病毒增效蛋白(Hashimoto，Y and B.G.Corsaro，etal.1991.J.Gen.Virol，72：2645-265l；Hayakawa，T.and J.H.Xu，et al.1996.Gene.177：269-270).昆虫病毒增效蛋白是病毒基因编码的一种金属蛋白酶，具有一个典型的金属蛋白酶锌-结合域HEXXH，位置十分保守，且对中肠围食膜的降解作用受到金属螯合剂的抑制。(Jongeneel，C.V.and J.Bouvier，et al.FEBS Lett.242：211-214；Lepore，L.S and P.R.Poelvink，et al.J.lnvertbr.Path-ol.68：131-140)。昆虫病毒增效蛋白可以提高昆虫的敏感性，加速核型多角体病毒NPV的感染进程，同时能提高苏云金杆菌伴孢晶体对幼虫的毒力(Corsaro B.G.and Guzen M.R.et al.1993Academic Press 127-145)。增效蛋白的作用机制有二种：一种是增效蛋白能与中肠细胞膜上的特异位点结合，介导病毒襄膜与细胞质膜之间的融合，促进杆状病毒进入宿主细胞内，从而增加病毒感染的效果。但增效蛋白与中肠细胞膜上的特异位点结合并不是增进病毒感染所必需的。另一种机制是增效蛋白通过降解肠粘蛋白IIM而破坏昆虫幼虫中肠围食膜，使病毒粒子更容易进入中肠细胞，同时提高了某些必须穿透围食膜而发生作用的生物杀虫剂的效能(Wang P andHammer AD.1997.J.Gen.Viorl.78：3018-3089.)。
棉铃虫颗粒体病毒(HaGV)增效蛋白基因的ORF大小为2706bP，编码含902个氨基酸，分子量为104.6KD的蛋白质。我们已经构建了含HaGV增效蛋白的基因3′端2.1Kb片段的工程菌株Escherichia coli M15(pEHa)。本发明的目的是提供一种利用该工程菌株生产棉铃虫颗粒体病毒增效蛋白的(PHEn)晶体方法，以及利用PHEn作为有效成分，用于病毒杀虫剂、BT杀虫剂、阿维菌素等生物杀虫剂或利用生物杀虫剂制备的复合生物杀虫剂而提高杀虫效果，或单独用于转基因抗虫植物防治虫害的方法。一、生产棉铃虫增效蛋白晶体PHEn方法：1.PHEn的表达：
挑取工程菌株Escherichia coli M15(pEHa)单菌落于含100μg/mlAmpcillin和25μg/ml Kan amycin的25ml LB培养基中，37℃，200rpm过夜培养。活化菌液以1∶20接种含100μg Ampcillin和25μg/ml Kanamycin的500ml LB培养液中，继续培养，当其浓渡OD600＝0.3时，加入IPIG至终浓渡1mmol/L，37℃诱导5h，收获菌液，4000rpm离心10min，收集菌体沉淀。2.PHEn的提取和纯化：
在细菌沉淀中加入2-5倍体积的超声波处理液，(50mmol/L Tris-HClPH8.0-0.1mol/L NaCl-2mg/ml溶菌酶)，反复冻融3次，37℃，保温1h，然后置于冰中用超声波破碎细菌。置于光学显微镜高倍物镜观察，有大量晶体。处理液上30-60％蔗糖梯度，4000rpm离心40min，取出包涵体带，用灭菌双蒸水洗3次，收获纯化的PHEn晶体，4℃保存。3.PHEn的鉴定，采用SDS-PAGE电泳分析：(1)制备样品：
收集工程菌株Escherichia coli M15(pEHa)未诱导培养物和经IPTG诱导5h后培养物各1mL，经离心沉淀，收获菌体，纯化的PHEn晶体，用适量灭菌双蒸水重悬。(2)上样和电泳：
参照Sambrook“分子克隆手册”制备10％分离胶和浓缩胶。在样品中加等体积2×SDS凝胶加样缓冲液，于100℃加热3-5min变性。样品上样。以100V电泳至溴酚兰进入分离胶，提高电压至150V，当溴酚兰到达底部前约1cm处结束电泳。(3)固定、染色和脱色：
剥胶后以5倍体积染液固定、染色过夜，收凝胶浸泡于脱色液中平缓摇动4h以上，其间更换脱色液4-5次。(4)结果分析：
脱色完全后即可观察照像。PHEn的10％ SDS-PAGE结果见图1，工程菌株Escherichia coli M15(pEHa)表达了含增效蛋白C-端702个氨基酸，共720个氨基酸的融合蛋白，分子量为78.17×103D SDS-PAGE，结果显示融合蛋白的分子量约为78KD。二、PHEn生物活性测定及其应用：1.PHEn对昆虫病毒的增效生物活性：
将AcNPV(苜蓿银纹夜蛾核型多角体病毒)、SeNPV(甜菜夜蛾核型多角体病毒)、HaNPV(棉铃虫核型多角体病毒)、DpCPV(松毛虫质型多角体病毒)和纯化的PHEn作系列稀释并用血球计数板计数，以3龄不同靶昆虫幼虫作为供试虫，设(1)NPV或CPV：2.4×105IB/ml；(2)2.4×105IB/ml(NPV或CPV多角体)+1.2×103OB/ml(OB：PHEn包涵体)；(3)2.4×105IB/ml+未诱导菌体蛋白1μg/μl；(4)105OB/ml(5)单蒸水。取3龄初幼虫室温下单头饲养于24孔Costar细胞培养皿中，每孔加适量人工饲料，并加10μl上述各种处理夜于人工饲料表面，使其充分吸收，幼虫取食48h后补加足量新鲜人工饲料，统计死亡和存活虫数(见表1)，结果表明，PHEn对幼虫不产生致死作用，可以提高AcNPV、SeNPV、HaNPV、SpltNPV、DpCPV对各自靶幼虫感染死亡率平均提高20-30％，缩短LT50平均为1.8天以上。
            表1：PHEn对昆虫病毒的增效活性
2.4×1051B/ml    2.4×105IB/ml+1.2×103OB/ml     2.4×105IB/ml+1ug固体蛋白/μl 106OB/ml 水
cNPV 供试虫数         40          40          40       40   20
感染后死亡率％         65          96          66       0   0
SeNPV 供试虫数         45          45          45       45   30
感染后死亡率％         73          92          70       0   0
HaNPV 供试虫数         35          35          35       30   30
感染后死亡率％         76          94          75       0   0
DpCPV 供试虫数         50          50          50       30   30
感染后死亡率％         82          97          84       4   2
SpltNPV 供试虫数         30          30          30       30   30
感染后死亡率％         65          86          67       0   0
2.PHEn对Bt的增效生物活性：
将Bt菌株(16000μg/ml)作1∶1000，1∶2000，1∶4000，1∶6000，1∶8000稀释成五个不同浓度，人工饲料添食感染2龄棉铃虫幼虫，每个稀释度30头，3个重复，同时设立正常对照。根据统计结果，Bt对2龄棉铃虫幼虫致死中浓渡LC50为0.652g/L，取该致死中浓度Bt稀释液与1×103OB/ml(OB PHEn包涵体)复配，人工饲料添食感染2龄棉铃虫幼虫，每个稀释度40头，3个重复，同时设立Bt对照，实验结果表明PHEn提高Bt对2龄棉铃虫幼虫死亡率为37.6％。3.PHEn对阿维菌素的增效生物活性：
将阿维菌素(1.8％乳油)作1∶3000，1∶6000，1∶9000，1∶12000稀释成4个不同浓度，人工饲料添食感染2-3龄棉铃虫幼虫，每个稀释度40头，3个重复，同时设立正常对照。根据统计结果，阿维菌素(1.8％孔油)对2-3龄棉铃虫幼虫致死中浓渡LC50为1∶5800，取致死中浓度阿维菌素稀释液与1×1030B/ml(OBPHEn包涵体)复配，人工饲料添食感染2-3龄棉铃虫幼虫，每个稀释度40头，3个重复，同时，设立阿维菌素对照，实验结果表明，PHEn提高阿维菌素(1.8％新油)对2-3龄棉铃虫幼虫死亡率为29.73％。
图面说明：图1，HaGV增效蛋白PHEn表达和纯化
 M.分子量标准
 1、2、3、E.Coli M15(pQE30)蛋白
 4.纯化晶体PHEn
 5、6、7、IPTG诱导Escherichia coli M15(pEHa)5h蛋白

Claims (2)

1.利用工程菌株Escherichia coli M15(pEHa)生产棉铃虫颗粒体病毒(HaGV)增效蛋白PHEn的方法。其特征在于：将工程菌株Escherichia coli M15(pEHa)单菌落挑到内含100μg/ml的氨苄青霉素和25μlg/m卡那霉素的LB培养基中，37℃，200rpm过夜培养，以1∶20比例接种到含上述双抗的500mlLB培养基中继续培养，当其溶液达到OD600＝0.3时，加入异丙基-β-D-硫代半乳糖苷至终浓渡1mM，37℃，200rpm诱导表达5h，收获菌液，4000rpm离心10min，收获菌体沉淀，再经提取、纯化获得HaGV增效蛋白晶体(PHEn)。
2.权利要求所述方法生产的HaGV增效蛋白与昆虫病毒、苏云金杆菌、阿维菌素等生物杀虫剂或利用生物杀虫剂制备的复合生物杀虫剂复合，用于农林业病虫的生物防治；或单独使用该增效蛋白于转基因抗虫植物防治虫害。
CN01114674A 2001-05-09 2001-05-09 棉铃虫颗粒体病毒增效蛋白应用 Pending CN1384195A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1791676B (zh) * 2003-05-19 2010-05-12 Enea－意大利国家新技术能源及环境局 制备特征为双粒病毒组持续抗性的转基因植物的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1791676B (zh) * 2003-05-19 2010-05-12 Enea－意大利国家新技术能源及环境局 制备特征为双粒病毒组持续抗性的转基因植物的方法

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