CN114907458A - 一种活性提高的Vip3A突变蛋白及其应用 - Google Patents
一种活性提高的Vip3A突变蛋白及其应用 Download PDFInfo
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- CN114907458A CN114907458A CN202210503231.9A CN202210503231A CN114907458A CN 114907458 A CN114907458 A CN 114907458A CN 202210503231 A CN202210503231 A CN 202210503231A CN 114907458 A CN114907458 A CN 114907458A
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- vip3a
- protein
- protease
- amino acid
- enzyme cutting
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
本发明提供了一种活性提高的Vip3A突变蛋白,所述突变蛋白与亲本Vip3A蛋白相比,在亲本Vip3A蛋白的结构域I和结构域II之间的氨基酸区域引入蛋白酶酶切位点,所述蛋白酶酶切位点选自R、A、D、E中的一种或任意几种氨基酸组合。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种提高苏云金杆菌Vip3A类杀虫蛋白杀虫活性的改造方法以及由此得到的活性提高的Vip3A突变蛋白及其应用。
背景技术
以微生物杀虫蛋白为基础的生物杀虫剂因具有高效和特异的杀虫效果,兼具易降解、污染小等环境友好性特点,是相比于化学合成农药更加可持续的农业害虫防治策略,并且已成为害虫防治措施中的重要组成部分。细菌杀虫蛋白中最具代表性的是苏云金杆菌所产生的Cry类杀虫晶体蛋白;它是目前世界范围内应用最成功的微生物杀虫蛋白,主要以杀虫制剂或者作为抗虫基因以转基因抗虫作物的方式被广泛地应用于多种重大农作物害虫的防治中,如转基因抗虫棉花。目前世界范围内转Cry类蛋白基因抗虫作物的种植面积已有约1亿公顷。但是,Cry类杀虫蛋白也面临着成分相对单一,很多害虫对其并不敏感;并且随着长期的应用,越来越多害虫对其产生抗性等紧迫状况。因此,开发新的可应用的高效微生物杀虫蛋白来充实微生物杀虫蛋白资源库已迫在眉睫。
营养期杀虫蛋白Vip3(vegetative insecticidal proteins)是由苏云金杆菌在其营养生长期产生并分泌的具有杀虫活性的蛋白。Vip3类蛋白由于与Cry类蛋白没有序列相似性,杀虫机制不同,并且同时具有高效的杀虫效果而被认为是继Cry蛋白之后第二类具有应用前景的微生物杀虫蛋白。Vip3类蛋白(约111种)根据其序列相似性可分为三大类:Vip3A、Vip3B和Vip3C三类,其中以Vip3A类蛋白(约101种)占绝大多数(Vip3Aa类约有67种)。目前,Vip3Aa类杀虫蛋白是Vip3类蛋白研究最为深入的蛋白,并已联合Cry类杀虫蛋白在转基因抗虫作物中得到了初步的应用。Vip3Aa类蛋白之间的序列相似性在95%以上,约由789个氨基酸组成,分子量约为89kDa。
研究显示,Vip3A类蛋白由五个结构域组成,天然以四聚体的形式存在。当Vip3A进入害虫中肠中后会被中肠内的蛋白酶在其结构域I和结构域II之间进行酶切,每个单体被酶切成约66kDa和20kDa的两条蛋白片段;酶切之后,通过构象变化,Vip3A变为有杀虫活性的激活状态。蛋白酶在其结构域I和结构域II之间的酶切的过程被认为是Vip3类蛋白的激活过程。但是相比于Cry类蛋白,Vip3A类蛋白的研究和应用都还处于初级阶段。在实际生产应用的需求下,需加速推进Vip3A类蛋白的研究以促进其科学合理的开发应用。
Vip3A类蛋白对多种鳞翅目农作物害虫(约15种)有杀虫效果,但是其对每种害虫的杀虫活性有着明显的不同。为了提高它的应用价值,研究人员为提高它的杀虫活性或杀虫谱等方面进行了相关的探索工作,主要是通过将不同Vip3A类蛋白分段组合成不同的嵌合体蛋白和定点突变两种方法。但是,目前这两种方法主要是根据不同Vip3A类蛋白之间的序列不同进行氨基酸之间或蛋白片段之间的互换来增加其杀虫活性,存在着随机性以及仅对某种害虫的杀虫活性有所增加的局限性。如何根据Vip3A类蛋白自身的蛋白性质和分子作用机制,普遍地提高它的杀虫活性需要进一步深入研究。
发明内容
本发明提供了一种活性提高的Vip3A突变蛋白以及应用。
本领域知晓,Vip3A蛋白包括五个结构域,结构域I、结构域II、结构域III、结构域IV以及结构域V,如现有技术(Vegetative Insecticidal Protein(Vip):A PotentialContender From Bacillusthuringiensis for EfficientManagement of VariousDetrimentalAgricultural Pests,Mamta Gupta等,《Frontiers in Microbiology》,20210531)所描述的,当Vip3A进入害虫中肠中后会被中肠内的蛋白酶在其结构域I和结构域II之间进行酶切,每个单体被酶切成约66kDa和20kDa的两条蛋白片段;酶切之后,通过构象变化,Vip3A变为有杀虫活性的激活状态。蛋白酶在其结构域I和结构域II之间的酶切的过程被认为是Vip3类蛋白的激活过程。
本发明发现了通过在Vip3A的结构域I和结构域II引入额外的蛋白酶切位点,可以提高Vip3A被蛋白酶酶切的效率,从而增加该蛋白对鳞翅目害虫的杀虫活性。
基于此,一方面,本发明提供了一种活性提高的Vip3A突变蛋白,所述突变蛋白相对于亲本Vip3A蛋白,在亲本Vip3A蛋白的结构域I和结构域II之间引入蛋白酶酶切位点。
所述引入蛋白酶酶切位点包括,将结构域I和结构域II之间的任一或任意几个氨基酸残基突变为蛋白酶酶切位点,或者,在结构域I和结构域II之间的任一或任意几个氨基酸的两端添加蛋白酶酶切位点。
在一个实施方式中,所述Vip3A蛋白的结构域I为对应于SEQ ID No.1所示序列的第1位到第191位氨基酸,所述Vip3A蛋白的结构域II为对应于SEQ ID No.1所示序列的第201位到第327位氨基酸。
在一个实施方式中,所述亲本Vip3A蛋白的结构域I和结构域II之间的区域为对应于SEQ ID No.1所示序列的第192位到第200位氨基酸的区域。
在一个实施方式中,所述引入蛋白酶酶切位点为引入R、A、D、E中的一种或任意几种氨基酸。在具体的实施方式中,所述引入蛋白酶酶切位点为引入RA和/或DE。
在一个实施方式中,在对应于SEQ ID No.1所示的第192位至第200位氨基酸的区域引入至少一种蛋白酶酶切位点,例如,RA或DE。
本发明中,所述蛋白酶酶切位点可以被来源于鳞翅目害虫的中肠蛋白酶酶切。
在一个实施方式中,所述引入至少一种蛋白酶酶切位点包括以下i-iii任一所述的方式:
i、将对应于SEQ ID No.1所示的第192位至第200位氨基酸区域的任一或任意几种氨基酸残基突变为蛋白酶酶切位点;
ii、在对应于SEQ ID No.1所示的第192位至第200位氨基酸区域的任意两个氨基酸之间添加蛋白酶酶切位点,或者在第192位氨基酸的N端添加蛋白酶酶切位点,或者在第200位氨基酸的C端添加蛋白酶酶切位点;
iii、采用i和ii组合的方式。
在一个具体的实施方式中,将对应于SEQ ID No.1所示的第192位、第193位、第194位、第195位、第196位、第197位、第198位、第199位或第200位氨基酸的任一或任意几种氨基酸残基突变为蛋白酶酶切位点;优选的,将对应于SEQ ID No.1所示的第195位、第196位、第197位、第198位或第199位氨基酸的任一或任意几种氨基酸残基突变为蛋白酶酶切位点。
在一个具体的实施方式中,在对应于SEQ ID No.1所示的第192位和第193位氨基酸之间、第193位和第194位氨基酸之间、第194位和第195位氨基酸之间、第195位和第196位氨基酸之间、第196位和第197位氨基酸之间、第197位和第198位氨基酸之间、第198位和第199位氨基酸之间、第199位和第200位氨基酸之间的任意位置添加蛋白酶酶切位点,或者在第192位氨基酸的N端添加蛋白酶酶切位点,或者在第200位氨基酸的C端添加蛋白酶酶切位点;优选的,在第194位和第195位氨基酸之间、第195位和第196位氨基酸之间、第196位和第197位氨基酸之间、第197位和第198位氨基酸之间、第198位和第199位氨基酸之间、第199位和第200位氨基酸之间的任意位置添加蛋白酶酶切位点。
所述添加蛋白酶酶切位点是指在上述合适的位置添加了酶切位点,例如,添加R、A、D、E中的一种或任意几种氨基酸;优选,RA和/或DE的氨基酸组合。
所述鳞翅目害虫选自草地贪夜蛾、棉铃虫、甜菜夜蛾中的一种或任意几种。
本发明中,氨基酸残基可以用单字母表示,也可以用三字母表示,例如:丙氨酸(Ala,A),缬氨酸(Val,V),甘氨酸(Gly,G),亮氨酸(Leu,L),谷酰胺酸(Gln,Q),苯丙氨酸(Phe,F),色氨酸(Trp,W),酪氨酸(Tyr,Y),天冬氨酸(Asp,D),天冬酰胺(Asn,N),谷氨酸(Glu,E),赖氨酸(Lys,K),甲硫氨酸(Met,M),丝氨酸(Ser,S),苏氨酸(Thr,T),半胱氨酸(Cys,C),脯氨酸(Pro,P),异亮氨酸(Ile,I),组氨酸(His,H),精氨酸(Arg,R)。
在一个实施方式中,所述亲本Vip3A蛋白的氨基酸序列与SEQ ID No.1相比,具有至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、至少99.1%、至少99.2%、至少99.3%、至少99.4%、至少99.5%、至少99.6%、至少99.7%、至少99.8%、或至少99.9%的序列同一性。
在一个实施方式中,所述亲本Vip3A蛋白为Vip3Aa类杀虫蛋白。
本发明所述蛋白质内的特定氨基酸位置(编号)是利用标准序列比对工具通过将目标蛋白质的氨基酸序列与SEQ ID No.1进行比对而确定的,譬如用Smith-Waterman运算法则或用CLUSTALW2运算法则比对两个序列,其中当比对得分最高时认为所述序列是对准的。比对得分可依照Wilbur,W.J.and Lipman,D.J.(1983)Rapid similarity searchesofnucleic acid and protein data banks.Proc.Natl.Acad.Sci.USA,80:726-730中所述的方法进行计算。在ClustalW2(1.82)运算法则中优选使用默认参数:蛋白质缺口开放罚分=10.0;蛋白质缺口延伸罚分=0.2;蛋白质矩阵=Gonnet;蛋白质/DNA端隙=-1;蛋白质/DNAGAPDIST=4。优选采用AlignX程序(vectorNTI组中的一部分),以适于多重比对的默认参数(缺口开放罚分:10og缺口延伸罚分0.05)通过将蛋白质的氨基酸序列与SEQ ID No.1进行比来确定本发明所述蛋白质内特定氨基酸的位置。
在一个实施方式中,所述引入蛋白酶酶切位点包括采用定点突变或同源重组或基因编辑的方式引入所述蛋白酶酶切位点。
本发明中,在Vip3A蛋白中引入蛋白酶酶切位点,例如,引入R、A、D、E中的一种或任意几种氨基酸;或者,引入RA和/或DE的氨基酸组合;能够提高鳞翅目害虫的中肠蛋白酶对Vip3A蛋白的酶切效率,从而提高Vip3A蛋白对鳞翅目害虫的杀虫活性。
另一方面,本发明还提供了编码上述Vip3A突变蛋白的基因;包含所述基因的载体;包含所述基因或所述载体的宿主细胞。
另一方面,本发明还提供了上述突变蛋白、基因、载体或宿主细胞的如下应用:
(1)在抑制/杀灭鳞翅目害虫中的应用;
(2)在制备抗鳞翅目害虫的转基因植物中的应用;
在一个实施方式中,本发明的突变蛋白在制备抗鳞翅目害虫的转基因植物时,可以与其他的杀虫蛋白,例如Cry蛋白联用。
另一方面,本发明还提供了一种提高鳞翅目害虫中肠蛋白酶对Vip3A蛋白的酶切效率的方法,所述方法包括对Vip3A蛋白进行突变得到Vip3A突变蛋白的步骤;所述方法还包括将Vip3A突变蛋白与鳞翅目害虫中肠蛋白酶进行接触的步骤。
所述Vip3A突变蛋白与亲本Vip3A蛋白相比,在亲本Vip3A蛋白的结构域I和结构域II之间引入蛋白酶酶切位点。所述引入蛋白酶酶切位点包括,将结构域I和结构域II之间的任一或任意几个氨基酸残基突变为蛋白酶酶切位点,或者,在结构域I和结构域II之间的任一或任意几个氨基酸的两端添加蛋白酶酶切位点。在一个实施方式中,所述引入蛋白酶酶切位点为引入R、A、D、E中的一种或任意几种氨基酸。在具体的实施方式中,所述引入蛋白酶酶切位点为引入RA和/或DE。
所述鳞翅目害虫中肠蛋白酶为来源于鳞翅目害虫中肠内的蛋白酶,在一个具体的实施方式中,将所述中肠组织样品固液(例如,采用离心的方式)分离,得到的上清液即为中肠蛋白酶;例如,文献(Characterization of the resistance to Vip3Aa inHelicoverpaarmigera from Australia and the role of midgut processing andreceptor binding)中所提到的解剖棉铃虫幼虫获得中肠组织,将中肠组织离心后获得上清液即为中肠蛋白酶,或者,文献(Midgut juice of Plutellaxylostella highlyresistant to Bacillus thuringiensis Cry1Ac contains a three times largeramount of glucosinolate sulfatase which binds to Cry1Ac compared to that ofsusceptible strain)中所提到的将解剖获得的小菜蛾的中肠组织离心后所获得的上清液即为中肠蛋白酶。
附图说明
图1.Vip3Aa类蛋白氨基酸在结构域I和结构域II之间的氨基酸序列比对结果,图中Vip3Aa11/1-789是实施例中具体采用的Vip3A蛋白。
图2.Vip3A类蛋白氨基酸在结构域I和结构域II之间的氨基酸序列比对结果,图中Vip3Aa/1-789是实施例中具体采用的Vip3A蛋白。
图3.Vip3A及其突变体蛋白经凝胶过滤层析纯化后所收集的样品进行SDS-PAGE电泳后的结果;图中,12.5,13和13.5分别代表经凝胶过滤层析纯化后在12.5,13和13.5毫升所收集的目标蛋白。
图4.Vip3A蛋白及其突变体蛋白经草地贪夜蛾中肠蛋白酶酶切后进行SDS-PAGE电泳后的结果。C表示未经酶切的对照蛋白;1,2,3和4分别表示经酶切1,2,3和4小时后的蛋白样品电泳后的结果。
图5.对图5中Vip3A蛋白及其各突变体蛋白经草地贪夜蛾中肠蛋白酶酶切3小时后所剩余的未被酶切的蛋白与对照蛋白的比例的统计结果。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、Vip3Aa蛋白基因突变体的构建
Vip3Aa类蛋白之间的序列相似性在95%以上,主要由789个氨基酸组成(图1),分子量约为89kDa。Vip3Aa被苏云金杆菌产生并分泌后以原毒素的形式存在,在其进入害虫中肠后会被中肠内蛋白酶在其结构域I和结构域II之间酶切,变构成有杀虫活性的激活毒素。我们发现Vip3Aa被中肠蛋白酶酶切的效率与其杀虫活性成正比。因此我们尝试在Vip3Aa的结构域I和结构域II之间的蛋白酶的初始酶切位点附近增加其他高效的酶切位点是否能增加它的酶切激活效率,并进一步增加它的杀虫活性。本实施方式中,所采用的亲本Vip3Aa氨基酸序列如下所示(SEQ ID No.1)。MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELITKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILKSQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYQGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKDVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK
我们前期的研究发现精氨酸(R)和丙氨酸(A)的组合是Vip3Aa信号肽的酶切位点,因此,我们首先将RA插入到Vip3Aa的初始酶切位点附近,构建了表1中的M1-M8的Vip3Aa的突变体。本实施方式中,选择Vip3Aa蛋白的第192位至第200位的区域(T192S193S194K195V196K19 7K198D199G200)尝试添加或改进酶切位点。
除了将RA插入之外,我们还将Vip3Aa蛋白的第193位和194位的丝氨酸直接置换成了RA,构建了表1中M9的突变体。在M9的基础上,我们进一步将RA插入到Vip3Aa的初始酶切位点附近,构建了表1中的M10-M15的Vip3Aa的突变体。另外,在M9的基础上,我们还将第196位的缬氨酸置换成了潜在的酶切位点天冬氨酸(D)和谷氨酸(E),构建了表1中M16的突变体。
表1:Vip3Aa突变体蛋白相对于野生型Vip3Aa的突变位点列表
本实施方式中采用SEQ ID No.1所示的Vip3Aa中进行上述酶切位点(RA或DE)的添加或引入。本领域知晓,不同的Vip3Aa中,上述区域(T192S193S194K195V196K197K198D199G200)是非常保守的,如图1所示;甚至是,在所有的Vip3A蛋白中,上述区域(T192S193S194K195V196K197K19 8D199G200)中,尤其是(K195V196K197K198D199)也是非常保守的,如图2所示。因此,在本发明的教导下,本领域技术人员也可以在其他的Vip3A或者Vip3Aa对应于SEQ ID No.1所示序列的上述区域(T192S193S194K195V196K197K198D199G200)中添加或引入酶切位点(RA或DE)从而得到不同的突变体。
本实施方式中,表1中的Vip3Aa蛋白所有的突变体都采用相似的构建方法。具体的构建方法是将要突变的位点设计到Vip3Aa蛋白基因片段PCR的引物中,对Vip3Aa基因进行PCR,获得两段在其突变位点有重叠的DNA片段。根据同源重组的原理利用北京全式金-Uni Seamless Cloning and Assembly Kit产品将包含突变位点的Vip3Aa的DNA片段同源重组到含有SUMO(small ubiquitin-like modifier)蛋白标签的pET28a载体的SUMO的DNA之后。反应体系为10μL(1μL利用PCR线性化的pET28a-His-SUMO载体,2μL突变位点前的PCR片段,2ul突变位点后的PCR片段,5μL 2×Assembly Mix),吹打混匀后,放于50℃反应30min,将重组后的产物在冰上冷却。将重组产物与大肠杆菌E.coli Bl21(DE3)感受态混合,在冰上孵育30min,42℃热激90s,冰浴3min,加入1mL的LB培养基之后,37℃培养30min,离心后将菌体涂布在50ug/ml卡那抗性的LB琼脂培养板上。37℃培养过夜,挑取单菌落进行PCR验证;将PCR正确的转化子扩大培养,保存菌液并提取质粒;将质粒送至测序公司进行测序验证,将测序正确的质粒及菌种放于-80℃冰箱保存。
实施例2、Vip3Aa突变体蛋白的诱导表达
表1中的Vip3Aa蛋白及其所有的突变体都采用相同的诱导表达方法。将菌种在50ug/mL的卡那抗性的LB固体培养板划线,37℃培养活化;挑取单菌落至10mL的LB液体培养基中,37℃,250rpm/min,振荡培养12h。将培养后的菌液全部转接进1L的LB液体培养基中,37℃,220rpm/min,振荡培养约4h。待菌液的浓度到达OD600=1左右后,加入终浓度为0.2mM的IPTG,20℃,220rpm/min,振荡培养12h,诱导蛋白的表达。诱导蛋白表达后将菌体转移至离心杯中,4500rpm/min,离心15min,倒去培养基,将菌体用40ml的缓冲液lysis buffer(20mM Tris-Hcl pH 8.0,300mM NaCl)重悬菌体,放于冰上准备蛋白纯化。
实施例3、Vip3Aa突变体蛋白的分离纯化
表1中的Vip3Aa蛋白及其所有的突变体都采用相同的分离纯化方法。
细菌破碎:将高压破碎匀浆仪提前开机预冷,将菌体重悬液转移至高压破碎仪中,缓慢调节压力至800pa,循环破碎细菌约3min,肉眼可见菌液变的透明。细菌破碎后,将破碎液用18000g,4℃离心50min,将离心后的上清转移至50mL离心管中。
Ni柱亲和纯化:由于pET28a载体上带有6×His亲和标签,采用Ni亲和纯化的原理,将细菌裂解后的上清液加入提前用lysis buffer平衡好的Ni柱,控制流速约2ml/min,把流穿液重复上Ni柱2-3次以使目的蛋白充分与Ni柱结合。用50ml的lysis buffer分4次流穿Ni柱,洗去未结合的杂蛋白。准备2ml的0.2mg/ml的Ulp1蛋白酶加入到结合有目的蛋白的Ni柱中,室温酶切3h,将Vip3Aa的突变体蛋白从SUMO蛋白标签后酶切下来,使其不带有任何蛋白标签,以免蛋白标签影响其后续的功能检测。酶切之后,用8ml的lysis buffer分四次将酶切下来的Vip3Aa突变体蛋白从Ni柱上洗脱下来。
凝胶过滤层析纯化:取Superdex 200Increase分子筛层析柱连接到AKTApure层析仪上,用lysis buffer平衡分子筛。将洗脱下来的蛋白溶液用浓缩管浓缩至1ml左右,转移至离心管之后,16000g,4℃,离心5min,去除沉淀。将蛋白浓缩液注射至上样环中,通过AKTApure层析仪利用分子筛进行凝胶过滤层析。设置流速为0.5ml/min,运行25ml,在第7ml开始收集样品,每0.5ml收集一管。收集UV吸收峰对应的蛋白样品,并进行SDS-PAGE验证蛋白的纯化效果。将纯化后的蛋白用液氮速冻后保存于-80℃冰箱。凝胶过滤层析纯化后的SDS-PAGE跑胶结果显示(如图3所示):每一个Vip3Aa的突变体都可以正常的分离纯化,可以获得较为纯净的Vip3Aa蛋白及其突变体蛋白。
实施例4、Vip3Aa突变体蛋白的酶切检测
中肠蛋白酶提取:挑选20只健硕的草地贪夜蛾四龄幼虫,放置在冰上10min将其冻僵麻痹。用解剖镊子将幼虫的头尾去除,然后将其中肠组织从前部拖拽出来。将中肠用镊子撕开露出包裹着食物的围食膜,将围食膜轻轻拖拽出来。然后,将中肠组织收集到离心管中,16000g,4℃离心10min,收集上清液即为中肠蛋白酶粗提液,分装后放置于-80℃冰箱中备用。
Vip3Aa突变体蛋白的酶切检测:Vip3Aa蛋白及其所有的突变体都采用相同的酶切检测方法。将Vip3Aa蛋白平均分成5份,每份含有5μg蛋白,将其中的4份与中肠蛋白酶提取液以15:1的比例混合,剩余一份不加入蛋白酶作为对照。用lysis buffer缓冲液将每份补足至20μl,混匀后放置在27℃进行酶切。分别在1h,2h,3h和4h的时候取出一份混合液,加入终浓度为2mM的蛋白酶抑制剂AEBSF并混匀。在每份样品中加入4μl的6×上样缓冲液,99℃煮样3min后,用SDS-PAGE跑胶检测每个蛋白的酶切情况。每个蛋白连续重复三次,比较不同Vip3Aa突变体蛋白被中肠蛋白酶酶切的效率。中肠蛋白酶的酶切结果显示(图4-图5):Vip3Aa所有的突变体被草地贪夜蛾中肠蛋白酶酶切的效率相比于Vip3Aa都有明显的增加,表明他们的酶切激活效率都有明显的增加。
实施例5、Vip3Aa突变体蛋白的草地贪夜蛾幼虫生测
Vip3Aa蛋白及其所有的突变体都采用相同的生测方法。将草地贪夜蛾虫卵转移至孵化盒中,放置在27℃,约40%的湿度的培养箱中孵化培养。待虫卵孵化后,放入少量固体培养基继续培养,待幼虫长至约2龄时准备Vip3Aa蛋白及突变体的生测实验。将预先配置好的草地贪夜蛾固体培养基用微波炉加热融化,并分装于无菌的24孔板中,每个孔中培养基的厚度约为5-8mm。待培养基凝固后,配置20ng/cm2,40ng/cm2,60ng/cm2,80ng/cm2和100ng/cm2的Vip3Aa蛋白并均匀涂布到培养基表面,每个浓度24个孔。待培养基完全晾干之后,将大小约2龄的草地贪夜蛾转移到涂有Vip3Aa蛋白的培养基上,每个孔放1只幼虫。继续培养7天之后,观测虫子的生存状况(没有生长的幼虫为功能性死亡记为死亡)。每个蛋白连续重复三次,比较不同Vip3Aa突变体蛋白的杀虫活性。草地贪夜蛾幼虫生测结果显示(表2),Vip3Aa所有的突变体蛋白的杀虫活性相比于Vip3Aa在不同程度上都有明显的增加。
表2.Vip3Aa及其突变体蛋白对草地贪夜蛾幼虫(二龄)的半致死浓度
序号 | 蛋白名称 | LC50(ng/cm<sup>2</sup>) | 95%置信区间(ng/cm2) |
WT | Vip3Aa-WT | 61.09 | 47.60-89.57 |
M1 | Vip3A-193RA | 31.57 | 28.42-34.90 |
M2 | Vip3A-194RA | 40.71 | 28.15-56.18 |
M3 | Vip3A-195RA | 42.12 | 31.25-58.44 |
M4 | Vip3A-196RA | 37.36 | 26.55-52.61 |
M5 | Vip3A-197RA | 31.24 | 21.69-43.07 |
M6 | Vip3A-198RA | 27.89 | 19.19-39.30 |
M7 | Vip3A-199RA | 40.74 | 30.25-57.35 |
M8 | Vip3A-200RA | 32.69 | 25.42-40.97 |
M9 | Vip3A<sup>SS193RA</sup> | 47.43 | 43.62-51.14 |
M10 | Vip3A<sup>SS193RA</sup>-193RA | 31.31 | 23.56-42.09 |
M11 | Vip3A<sup>SS193RA</sup>-195RA | 30.35 | 18.26-47.56 |
M12 | Vip3A<sup>SS193RA</sup>-196RA | 34.62 | 27.31-46.01 |
M13 | Vip3A<sup>SS193RA</sup>-197RA | 25.44 | 16.85-33.65 |
M14 | Vip3A<sup>SS193RA</sup>-198RA | 30.91 | 21.90-42.39 |
M15 | Vip3A<sup>SS193RA</sup>-199RA | 24.45 | 17.97-31.73 |
M16 | Vip3A<sup>SS193RA</sup>-V196DE | 38.16 | 34.7-41.66 |
以上所述仅为本申请的实施例而已,并不用于限制本申请。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的权利要求范围之内。
SEQUENCE LISTING
<110> 山东大学
<120> 一种活性提高的Vip3A突变蛋白及其应用
<130> 11
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 789
<212> PRT
<213> Artificial Sequence
<220>
<223> vip3Aa
<400> 1
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe
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Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
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Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu
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Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
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Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn
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Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln
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Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
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Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
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Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
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Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val
145 150 155 160
Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile
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Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
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Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu
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Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val
210 215 220
Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly
225 230 235 240
Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile
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Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
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Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr
275 280 285
Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
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Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val
305 310 315 320
Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala
325 330 335
Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
340 345 350
Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
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Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
370 375 380
Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu
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Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe
405 410 415
Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
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Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
435 440 445
Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
450 455 460
Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val
465 470 475 480
Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala
485 490 495
Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
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Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
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Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
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Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr
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Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His
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Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
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Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
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Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
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Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr
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Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
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Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
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Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
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Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg
705 710 715 720
Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser
725 730 735
Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val
740 745 750
Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu
755 760 765
Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr
770 775 780
Asp Val Ser Ile Lys
785
Claims (10)
1.一种活性提高的Vip3A突变蛋白,所述突变蛋白与亲本Vip3A蛋白相比,在亲本Vip3A蛋白的结构域I和结构域II之间的氨基酸区域引入蛋白酶酶切位点,所述蛋白酶酶切位点选自R、A、D、E中的一种或任意几种氨基酸组合。
2.如权利要求1所述的Vip3A突变蛋白,其特征在于,所述Vip3A蛋白的结构域I为对应于SEQ ID No.1所示序列的第1位到第191位氨基酸的区域,所述Vip3A蛋白的结构域II为对应于SEQ ID No.1所示序列的第201位到第327位氨基酸的区域。
3.如权利要求2所述的Vip3A突变蛋白,其特征在于,所述突变蛋白与亲本Vip3A蛋白相比,在对应于SEQ ID No.1所示的第192位至第200位氨基酸的区域引入至少一种蛋白酶酶切位点;
优选的,所述引入至少一种蛋白酶酶切位点包括以下i-iii任一所述的方式:
i、将对应于SEQ ID No.1所示的第192位至第200位氨基酸区域的任一或任意几种氨基酸残基突变为蛋白酶酶切位点;
ii、在对应于SEQ ID No.1所示的第192位至第200位氨基酸区域的任意两个氨基酸之间添加蛋白酶酶切位点,或者在第192位氨基酸的N端添加蛋白酶酶切位点,或者在第200位氨基酸的C端添加蛋白酶酶切位点;
iii、采用i和ii组合的方式。
4.如权利要求1-3任一所述的Vip3A突变蛋白,其特征在于,所述蛋白酶酶切位点选自RA和/或DE的氨基酸组合。
5.编码权利要求1-4任一所述Vip3A突变蛋白的基因。
6.包含权利要求5所述基因的载体。
7.包含权利要求5所述基因或权利要求6所述载体的宿主细胞。
8.权利要求1-4任一所述的Vip3A突变蛋白,或权利要求5所述的基因,或权利要求6所述的载体,或权利要求7所述的宿主细胞在抑制/杀灭鳞翅目害虫中的应用;或者,在制备抗鳞翅目害虫的转基因植物中的应用。
9.一种提高鳞翅目害虫中肠蛋白酶对Vip3A蛋白的酶切效率的方法,所述方法包括对Vip3A蛋白进行突变得到Vip3A突变蛋白,进而将Vip3A突变蛋白与鳞翅目害虫中肠蛋白酶进行接触的步骤;所述Vip3A突变蛋白与亲本Vip3A蛋白相比,在亲本Vip3A蛋白的结构域I和结构域II之间引入蛋白酶酶切位点,所述蛋白酶酶切位点选自R、A、D、E中的一种或任意几种氨基酸组合。
10.根据权利要求9所述的方法,其特征在于,所述引入蛋白酶酶切位点包括:
将结构域I和结构域II之间的任一或任意几个氨基酸残基突变为蛋白酶酶切位点,或者,在结构域I和结构域II之间的任一或任意几个氨基酸的两端添加蛋白酶酶切位点。
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