CN112480219A - Sip1Aa高效可溶性杀虫蛋白突变体 - Google Patents
Sip1Aa高效可溶性杀虫蛋白突变体 Download PDFInfo
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- CN112480219A CN112480219A CN202011376272.3A CN202011376272A CN112480219A CN 112480219 A CN112480219 A CN 112480219A CN 202011376272 A CN202011376272 A CN 202011376272A CN 112480219 A CN112480219 A CN 112480219A
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Abstract
本发明涉及获得了Sip1Aa高效可溶性杀虫蛋白突变体,命名为Sip153‑248和Sip158‑243蛋白,属于生物技术领域。本发明将Sip1Aa蛋白氨基酸序列分别改变了两个氨基酸,获得的突变体蛋白Sip153‑248的杀虫活性提高了2.76倍,Sip158‑243的杀虫活性提高了2.26倍,且可溶性和稳定性均有一定的提高,有效的克服寻找苏云金芽胞杆菌表达蛋白对大猿叶甲有高毒力基因的难题、延缓害虫对工程菌和转基因植物的抗药性产生。
Description
技术领域
本发明属于生物防治技术领域,具体涉及对鞘翅目农业害虫具有高毒力的Bt杀虫基因的定点突变及由该基因所编码的蛋白质。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种革兰氏阳性细菌,广泛存在于自然界中,其外形一般呈短杆状,单生或形成短链状。1911年,德国生物学家贝尔奈(Berliner)从德国苏云金的一种地中海粉螟的患病幼虫上分离得到同一种细菌,并命名为苏云金芽胞杆菌。苏云金芽胞杆菌已有100多年的研究历史,有关该菌的报道涉及生物分子学、细胞学、分类命名、Bt的有效成分以及其杀虫机理等方方面面,研究报告已有数万篇之多。
目前,国内外对Bt Sip蛋白的报道较少。Donovan等研究Bt菌株对鞘翅目昆虫的杀虫活性的过程中,发现一些菌株的培养上清液在致死中浓度(LC50)为0.12(0.09~0.15)μg/mL时对科罗拉多马铃薯甲虫(CPB)幼虫具有杀虫活性,该基因包含1104bp,编码367个氨基酸,被命名为Sip1A,与Mtx3杀蚊蛋白的相似性为46%(Donovan W P,Engleman J T,Donovan J C,et al.Discovery and characterization of Sip1A:a novel secretedprotein from Bacillus thuringiensis,with activity against coleopteran larvae[J].Appl Microbiol Biotechnol,2006,72(4):713-719)。2012年,本实验室从Bt菌株QZL26中克隆并鉴定了包含1038bp,编码345个氨基酸序列的sip基因,但其杀虫活性未见报道(刘艳杰,李海涛,刘荣梅,等.Bt新型基因sip的克隆、表达和生物信息学分析[J].生物技术通报,2012(12):101-105)。2013年,Murawska等人应用基因组测序技术,发现IS5056菌株中含有sip基因,但未进行深入研究。2015年,张金波从Bt菌株DQ89中克隆表达了1188bp,编码395个氨基酸的Sip蛋白,显示出对大猿叶甲具有较高毒杀作用,其LC50值为1.542μg/mL(张金波,李海涛,刘荣梅,等.Bt菌株DQ89的sip基因的克隆、表达及杀虫活性分析[J].中国生物防治学报,2015,31(4):598-602)。此外,沙君雪从Bt菌株QZL38中克隆sip1Aa基因,进而构建其截短突变体,去除前90bp信号肽,可以产生37.6kDa的可溶性蛋白,并测试其对大猿叶甲的LC50值为1.051μg/mL(Sha J,Zhang J,Chi B,et al.Sip1Ab gene from a nativeBacillus thuringiensis strain QZL38and its insecticidal activity againstColaphellus bowringi Baly[J].Biocontrol Science&Technology,2018,28(5):459-467.)。
Sip1Aa蛋白的结构信息尚不清楚,但Sip1Aa蛋白的结构分析可以通过生物信息学研究方法,应用同源建模技术在理论上完成。蛋白质稳定性,溶解性是其发挥生物活性的关键,其中共价交联的二硫键是维持空间结构的重要力量。有研究表明,新引入的二硫键会影响蛋白质的折叠过程,1987年,Pantoniano等人通过程序辅助选择适合引入二硫键的枯草杆菌蛋白酶上的位点,获得了稳定性比天然蛋白质更好的突变体(Pantoliano,M.W;Ladner,R.C;Bryan,P.N;Rollence,M.L;Wood,J.F;Poulos,T.L,Protein engineering ofsubtilisin BPN:enhanced stabilization through the introduction of twocysteines to forma disulfide bonds.Biochemistry 1987,26(8),2077-2082.)。BinleyJ M等人在gp120的C末端和gp41的免疫显性片段之间引入了二硫键,增加了分子间二硫键的稳定(Binley J M,Sanders R W,Clas B,et al.A Recombinant HumanImmunodeficiency Virus Type 1Envelope Glycoprotein Complex Stabilized by anIntermolecular Disulfide Bond between the gp120and gp41Subunits Is anAntigenic Mimic of the Trimeric Virion-Associated Structure[J].J.Virol.2000,74(2):627-643.),Hai-Xiao Liu等人在A15C Ngb突变体中增加额外的二硫键与只含有单一二硫键的WT Ngb的相比,其化学稳定性和pH稳定性显著增强(Hai-Xiao Liu,LianzhiLi,Xin-Zhi Yang,et al.Enhancement of protein stability by an additionaldisulfide bond designed in human neuroglobin[J].RSC Advances,2019,9(8):4172-4179.),二硫键可以帮助蛋白质折叠,从而影响蛋白质的稳定性,因此人们希望通过增加二硫键来提升蛋白质的作用的效果。有研究表明,即使蛋白质终产物不含二硫键,二硫键也会在蛋白质形成过程中发挥作用。Xiaoxiao Xu等人通过消除PIIIA结构中的二硫键,发现蛋白结合的亲和力降低(Xu X,Xu Q,Chen F,et al.Role of the disulfide bonds on thestructure and activity ofμ-conotoxin PIIIA in the inhibition of Na V 1.4[J].RSC Advances,2019,9(2):668-674.),二硫键的消除对信号通路的影响都从反面印证了二硫键对蛋白质的重要性。。
发明内容
本申请通过在线服务器DisulfidebyDesignTM预测可能形成二硫键的一对氨基酸位点。通过增加二硫键来增强Sip1Aa蛋白在大肠杆菌中稳定性,增加蛋白的可溶性,进一步提高蛋白杀虫活性,通过蛋白质工程技术的设计可以满足人们对新的蛋白质分子的需要,并为今后研究提供相应工程菌。为了初步探究Sip1Aa蛋白杀虫活性关键位点,对Sip1Aa蛋白的三级结构进行预测,并以此指导定点突变试验,期望获得高活性的突变体蛋白,为阐明Sip1Aa蛋白的结构与功能关系以及杀虫机理的研究提供了理论依据。
本发明采用点突变的方法,将Sip1Aa蛋白氨基酸序列上的T149、G153、T158、K178、K243、H248、R251和G314位点引入二硫键,成功构建sip149-251、sip153-248、sip158-243、sip178-314突变体。通过紫外线照射发现4株突变体蛋白相比于Sip1Aa对紫外的稳定性均有提升,其中Sip153-248和Sip158-243稳定性最好,Sip149-251和Sip178-314次之。通过测定杀虫活性发现相比于Sip1Aa,Sip153-248和Sip158-243的杀虫活性提高超过两倍,突变体Sip153-248的杀虫活性提高了2.76倍,Sip 158-243的杀虫活性提高了2.26倍。突变体Sip149-251和Sip178-314的杀虫活性无明显变化。本申请获得了高效可溶稳定的Sip1Aa杀虫蛋白,有效的克服寻找苏云金芽胞杆菌表达蛋白对大猿叶甲有高毒力基因的难题、延缓害虫对工程菌和转基因植物的抗药性产生。
突变体Sip153-248和Sip158-243,其氨基酸序列如SEQ ID No.3,SEQ ID No.5所示。
编码上述突变体sip153-248和sip158-243的基因。
所述的基因序列如SEQ ID No.4,SEQ ID No.6所示。
上述突变体SEQ ID No.3,SEQ ID No.5所示在杀灭大猿叶甲害虫中的应用。
所述应用为将所述的突变体SEQ ID No.3,SEQ ID No.5制成杀虫剂杀灭大猿叶甲。
本发明采用定点突变技术将Sip1Aa蛋白的T149、G153、T158、K178、K243、H248、R251和G314位点引入二硫键,成功构建sip149-251、sip153-248、sip158-243、sip178-314突变体。实验表明,突变体Sip153-248和Sip158-243可溶性、稳定性大大提高,并且对大猿叶甲的杀虫活性提高2倍左右。
附图说明
图1突变体蛋白的表达,其中1-6泳道分别对应lane M:DNA marker;lane 1:Sip149-251;lane 2:Sip153-248;lane 3:Sip158-243;lane 4:Sip178-314;lane 5:Sip1Aa;lane 6:pET21b
图2突变体三维结构图,
图3Sip1Aa突变体蛋白纯化结果,其中1-7泳道分别对应lane M:DNA marker;1:pET21b;2:粗蛋白;3:纯化后Sip1Aa;4:纯化后Sip149-251;5:纯化后Sip153-248;6:纯化后Sip158-243;7:纯化后Sip178-314,
图4蛋白质稳定性测试结果。
具体实施方式
下面结合实施例对本发明做进一步的详细说明。
1.材料与试剂
1.1菌株与质粒
实验所用菌株与质粒详见表1,可以对公众发放。
表1 菌株与质粒
1.2引物
参考GenBank公布的已知sip1Aa基因序列,设计全长引物一对Sipa-F-重组/Sipa-R-重组。构建单点突变体引物。以下引物均由生工生物工程(上海)股份有限公司合成。
表2 PCR鉴定引物及突变引物序列
1.3培养基和抗生素
液体LB:胰蛋白胨1%,NaCl 1%,酵母提取物0.5%;氨苄青霉素(Ampicillin):称取100mg氨苄青霉素(Ampicillin)溶于1ml无菌水,过0.22μm滤膜除菌使用时稀释1000倍。
1.4溶液与缓冲液:
PBS缓冲(pH 7.4):KH2PO4 2mmol/L、Na2HPO4 8mmol/L、NaCl 136mmol/L和KCL2.6mmol/L溶于1L去离子水中;结合缓冲液(Binding buffer)(PH 7.4):20mmol/L磷酸钠,0.5mol/L氯化钠,10mmol/L咪唑;洗脱缓冲液Elution Buffer(PH 7.4):20mmol/L磷酸钠,0.5mol/L氯化钠,设置咪唑浓度梯度为40mmol/L、250mmol/L和500mmol/L;半胱氨酸标准溶液(1mmol/L):准确称取0.017563g L-半胱氨酸,用1mL甲醇溶解,ddH2O定容至100mL;DTNB标准溶液(10mmol/L):准确称量0.0198175g DTNB用50mmol/L Na2HPO4(PH 7.0)配制成50mL溶液,存放于棕色瓶中;DTNB分析溶液(0.1mmol/L):由1体积DTNB标准溶液加99体积0.25mmol/L Tris-HCl(PH 8.3)缓冲液配制而成,现用现配。
1.5酶和生化试剂
蛋白质Marker购自TaKaRa公司;2×Taq Mix DNA聚合酶购于CWBIO公司;DNA凝胶回收试剂盒购于Axygen公司;定点突变试剂盒购自南京诺唯赞;其他试剂均为国产分析纯试剂。
1.6供试昆虫
实验所用标准化大猿叶甲试虫由中国农业科学院植物保护研究惠赠。
2实验方法
2.1二硫键预测
通过在线分析服务器Disulfide by DesignTM(http://cptweb.cpt.wayne.edu/DbD2/),分析在sip1Aa基因序列中可能形成的二硫键位点,选择位于保守结构域内且两个氨基酸位点的间距大于50bp的一对氨基酸位点,将其均突变为半胱氨酸从而使其形成二硫键。
2.2突变体构建
以转入大肠杆菌JM109感受态进行甲基化修饰后的pET21b-sip1Aa为模板。PCR反应体系为50μL,包含1μL模板,25μL 2xMax Buffer,1μL dNTP Mix(10mmol/L),1μL PhantaMax Super-Fidelity DNA Polymerase,以构建突变体sip149-251为例,用T149C-F和R251C-R扩增片段A,用R251C-F和T149C-R扩增片段B,PCR程序:95℃预变性30s;95℃变性15s,退火30s,72℃60s/kb;30个循环,72℃终止延伸5min,加入1μL DpnI在37℃反应2h进行消化。再用同源重组试剂盒将片段A和B进行重组,完成两个半胱氨酸突变。重组产物转化大肠杆菌BL21感受态,37℃培养12h。用引物Sipa-f/Sipa-R鉴定阳性克隆。由吉林省库美生物科技有限公司测序,利用DNAMAN对DNA序列进行分析。
2.3突变体蛋白表达和提取
挑取单菌落接种于含氨苄抗性的LB试管中,37℃,220r/min培养12h,按1%接种量接种至100mL LB液体培养基中,37℃,220rmp/min培养至OD600为0.6左右。加入梯度iptg16℃,160rmp诱导12h,(0μL、1μL、10μL、30μL、50μL和100μL)。8000rmp/min,4℃,15min,收集菌体。用PBS缓冲液清洗菌体,重复三次。超声波破碎(86%,3s,3s,10min),12000rpm,4℃离心收集上清。通过SDS-PAGE检测蛋白质表达情况。
2.4蛋白质纯化
由于载体带有His标签,可以用镍柱纯化。固定好镍柱,加入5倍柱体积的无菌水,加入8倍体积的结合液,加入粗蛋白,再加入8体积结合液,然后梯度洗脱,咪唑浓度分别为40mmol/L、250mmol/L和500mmol/L。通过SDS-PAGE检测蛋白纯化情况。
2.5 DTNB法测定游离半胱氨酸含量
25℃条件下,用Tris-HCl缓冲液将半胱氨酸标准溶液梯度稀释,浓度分别为0mmol/L、0.025mmol/L、0.05mmol/L、0.1mmol/L、0.15mmol/L和0.2mmol/L。取上述各浓度溶液1mL分别加入5mL的DTNB分析液摇匀,静止10min,在波长412nm处测定吸光值,绘制标准曲线。待测样品取1mL加入5mL的DTNB分析液摇匀,静止10min,在波长412nm处测定吸光值,根据标准曲线确定游离半胱氨酸浓度。
2.6杀虫活性测定
将待测蛋白稀释成不同浓度,同时以pET21b在大肠杆菌中表达的蛋白作为阴性对照,将蛋白涂抹到新鲜的小白菜上对大猿叶甲进行毒力测定。每个条件接种16只大猿叶甲幼虫,每个处理重复3次。在27℃,相对湿度55%±5%,光暗周期为14/10的条件下培养48h后统计死亡率,通过Probit分析计算LC50。
2.7紫外条件下蛋白质稳定性
将蛋白质定量到同一浓度,放置在紫外灯下直照,在30min,1h,3h,6h,14h,17h,20h和24h时分别取样进行SDS-PAGE电泳检测。通过Image J对目标蛋白质进行定量分析。本研究中生物活性测定所用标准化大猿叶甲试虫为中国农业科学院植物保护研究所惠赠,孵化于东北农业大学。
2.8数据统计分析
在对大猿叶甲生物活性测定48h后分别记录活虫数和死虫数,计算校正死亡率以及功能死亡率(计算功能死亡率时生长受到严重抑制的昆虫即视为死亡),使用SPSS软件对生物活性测定的数据进行分析,计算LC50和95%置信区间。
3试验结果
3.1二硫键位点预测
通过在线分析服务器(http://swissmodel.expasy.org/)预测出20对可能形成二硫键的位点,结合sip1Aa保守结构区、Swiss-PdbViewer和拉氏构象图筛选出7对位点,排除间距小于50bp的一对氨基酸位点,最终确定8个半胱氨酸突变位点:T149-R251,G153-H248,T158-K243和K178-G314。同源重组后,菌落PCR,挑单菌落,同时将突变体送去吉林省库美生物科技有限公司测序,结果表明8个位点均成功突变为半胱氨酸。
3.2突变体蛋白的表达与纯化
通过对突变体进行梯度诱导表达,当加入50μl诱导剂时,发现4个突变体均能正常表达,如图1,证明半光氨酸突变不影响菌体表达。4个突变体比Sip1Aa的白质溶解度有所提高。
3.3新引入二硫键的检测
通过DTNB,用于比色法测定样品中游离巯基含量。如果体系中存在巯基化合物,DTNB转变成黄色的5-巯基-2-硝基苯甲酸,由于5-巯基-2-硝基苯甲酸在412nm处具有最大吸收,通过测定412nm的吸光度来测定样品中是否含有自由巯基。DTNB的吸收光谱并不干扰巯基的测定。样品与DTNB混合后,以Sip1Aa作为阳性对照,测定412nm的吸光度,实验结果吸光度为零,说明样品中不含游离的半胱氨酸,证明半胱氨酸两两配对形成二硫键。蛋白质三维结构(图2)A.sip149-251、B.sip153-248、C.sip158-243和D.sip178-314。
3.4蛋白质纯化检测
由于载体带有His标签,可以用镍柱纯化。固定好镍柱,加入5倍柱体积的无菌水,加入8倍体积的结合液,加入粗蛋白,再加入8体积结合液,然后梯度洗脱,咪唑浓度分别为40mmol/L、250mmol/L和500mmol/L。通过SDS-PAGE检测蛋白纯化情况(图3所示),显示Sip1Aa蛋白可溶性增加。
3.5杀虫活性测定
将4种纯化后的突变体蛋白设定为50、20、10、5、1和0.1μg/mL六个浓度梯度对大猿叶甲进行定量杀虫活性测定,培养48h后统计死、活虫数,计算致死中浓度LC50。定量杀虫活性测定结果见表3。突变体Sip 149-251、Sip 178-314的杀虫活性无明显变化,突变体Sip153-248的杀虫活性提高了2.76倍,Sip 158-243的杀虫活性提高了2.26倍。
表3 杀虫活性测定
3.6蛋白质稳定性测试
在254nm波长下紫外照射蛋白质。取不同照射时间的蛋白进行SDS-PAGE,通过BSA蛋白定量实验绘制成图4。可见在紫外线影响下突变体蛋白和Sip1Aa中均有所下降,但二硫键突变株的蛋白质相比于Sip1Aa蛋白下降趋势相对较缓,其中Sip153-248和Sip158-243蛋白可以更好地抗紫外线对其的影响,Sip 149-251和Sip 178-314次之。24h时Sip1Aa、Sip149-251和Sip 178-314均下降至原始含量的90%,Sip153-248下降至原始含量的92.6%,Sip158-243下降至原始含量的92%。
4结论
本发明采用定点突变技术将Sip1Aa蛋白进行改造,突变体Sip153-248和Sip158-243的杀虫活性分别增强了2.76、2.26倍,Sip149-251和Sip178-314杀虫活性无明显变化。Sip153-248和Sip158-243蛋白质溶解性得到了提高,并且在紫外下也的具有良好的稳定性,在紫外灯下连续照射24h还保持原蛋白含量的92.6%和92%。
序列表
<110> 东北农业大学
<120> Sip1Aa高效可溶性杀虫蛋白突变体
<141> 2020-11-30
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Claims (10)
1.突变体Sip153-248,其氨基酸序列如SEQ ID No.3所示。
2.编码权利要求1所述的突变体Sip153-248的基因。
3.根据权利要求2所述的基因,其核苷酸序列如SEQ ID No.4所示。
4.权利要求1所述突变体Sip153-248在杀灭大猿叶甲害虫中的应用。
5.根据权利要求4所述应用,为将权利要求1所述的突变体Sip153-248制成杀虫剂杀灭大猿叶甲。
6.突变体Sip158-243,其氨基酸序列如SEQ ID No.5所示。
7.编码权利要求6所述的突变体Sip158-243的基因。
8.根据权利要求7所述的基因,其核苷酸序列如SEQ ID No.6所示。
9.权利要求6所述突变体Sip158-243在杀灭大猿叶甲害虫中的应用。
10.根据权利要求9所述应用,为将权利要求6所述的突变体Sip158-243制成杀虫剂杀灭大猿叶甲。
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