CN108085310B - 一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用 - Google Patents
一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用 Download PDFInfo
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- CN108085310B CN108085310B CN201711347198.0A CN201711347198A CN108085310B CN 108085310 B CN108085310 B CN 108085310B CN 201711347198 A CN201711347198 A CN 201711347198A CN 108085310 B CN108085310 B CN 108085310B
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- protein
- pxidgf
- plutella xylostella
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Abstract
本发明公开了一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用,所述蛋白制品的氨基酸序列如SEQ ID NO:1所示。从小菜蛾四龄幼虫体中提取总RNA,逆转录合成第一链cDNA,利用设计引物从cDNA中扩增小菜蛾识别蛋白PxIDGF基因,将小菜蛾识别蛋白PxIDGF基因克隆到pCzn1载体中,然后将重组质粒转入到大肠杆菌Arctic‑Express表达菌株,诱导融合蛋白表达,通过变复性的方式,重溶目标蛋白,通过Ni柱亲和纯化获得目的蛋白。本发明制备的小菜蛾识别蛋白PxIDGF制品纯度高、活性高,具有结合和凝集大肠杆菌和金黄色葡萄球菌的能力。本发明可为深入研究小菜蛾识别蛋白PxIDGF的应用打下技术基础。
Description
技术领域
本发明属于昆虫天然免疫识别领域,特别涉及一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用。
背景技术
作为世界上农作物有害生物危害最严重的国家之一,我国的重大农业害虫有30种之多,危害严重,损失惊人。小菜蛾(Plutella xylostella)便是其中一种。作为一种危害十字花科植物的世界性农业害虫,已对拟除虫菊酯类、有机磷及氨基甲酸酯类等许多杀虫剂产生了显著抗药性。它是首个对DDT产生抗性的作物害虫,也是首个对苏云金杆菌毒素产生抗性的昆虫(Baxter等,2010)。因此迫切需要研究新的小菜蛾防治途径。而发展生物防治技术是持久高效控制小菜蛾危害的必然趋势。目前利用昆虫病原菌或病原菌产生的毒力因子来防治害虫是害虫生物防治研究的前沿之一,其成功的关键在于了解寄主昆虫的免疫防御机制。因此阐明小菜蛾对各种病原物的免疫反应机制可为新的害虫控制方法提供理论基础。
由于缺乏获得性免疫,昆虫主要依靠其自身的天然免疫来抵御各种病原物的入侵。昆虫的天然免疫包括细胞免疫和体液免疫。昆虫如失去天然免疫的保护,其生存将受到影响。有研究表明先天免疫缺陷的昆虫(基因突变所致)或人为的抑制昆虫的天然免疫,昆虫对病原物的抵抗力将减弱。体液免疫中,从病原物侵袭到最终被寄主清除一般要经过如下过程:首先是识别外来异物;随后引发激活丝氨酸蛋白酶和解除丝氨酸蛋白酶抑制剂的细胞外级联反应;然后引发信号转导途径而激活目的基因转录。这个过程需要4个重要因子,即识别分子、信号调节因子、信号转导***及效应分子。
病原识别是启动昆虫体液免疫的关键第一步,主要是由不同识别分子来完成。识别分子,又称模式识别蛋白,是昆虫免疫应答的关键因子,可以识别病原物的细胞壁成分(即病原分子相关模式pathogen associated molecular patterns,PAMPs),诸如革兰氏阴性细菌的脂多糖,***的肽聚糖、真菌的β-1,3-葡聚糖和病毒的双链RNA。不同的识别分子具有不同的结构和功能,结合不同的PAMPs。模式昆虫双翅目果蝇目前已经鉴定出的识别分子超过8种,主要有肽聚糖识别蛋白(PGRPs)、硫脂蛋白、革兰氏阴性细菌结合蛋白(GNBPs)、β-1,3-葡聚糖识别蛋白(βGRPs)、清道夫受体、C型凝集素、半乳糖凝集素和免疫球蛋白等。正是由于天然免疫对于昆虫的生存具有极其重要的影响,其已经成为害虫控制的一个重要靶标。目前昆虫天然免疫中识别分子的研究已成为免疫学研究的重点和热点。
为探讨小菜蛾对病原真菌入侵的免疫防御机制,我们对病原生物侵染小菜蛾幼虫的转录组进行了测序,并对免疫相关基因进行了筛选。鉴定出一些可能参与到小菜蛾免疫病原生物侵染的免疫基因,包括:识别分子、信号调节因子、抗菌效应因子和其它一些可能参与免疫的基因等。其中识别分子包括PxIDGF、CTL(C-type lectins)和PGRP(peptidoglycan recognition protein)等。IDGF,即器官芽生长因子,隶属于糖基水解酶第18家族,由昆虫脂肪体表达分泌到昆虫的血淋巴中,其主要功能是调节昆虫发育和生长。由于具有1个结合几丁质的凝集素结构域和1个结合几丁质的乳酸结构域而被推测可能在无脊椎动物的病原识别上起作用。关于此功能目前并未有实验数据证明。
为了明确在小菜蛾体液免疫病原微生物的过程中小菜蛾识别蛋白PxIDGF是否具有免疫识别作用,获得有活性的该蛋白非常必要。目前并未有小菜蛾识别蛋白PxIDGF的制品及制备方法方面的发明。
发明内容
本发明提供了一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用。
本发明是通过以下技术方案予以实现:
本发明提供了一种小菜蛾识别蛋白PxIDGF制品,是从小菜蛾幼虫中通过基因克隆、蛋白表达、纯化得到的一种蛋白,理论分子量为47.904KD,等电点为7.67;所述蛋白制品的氨基酸全序列为MNHKVHHHHHHMNQVVSTKKVICYYDSKSYVRESNARLLPPDLEPALPYCTHLVYGYAGVQPDTYKMISTNQNLDIDSAHANYRTITSFKTKYPQLKMLLAVGGDADLEDPQKYNALLESQQARTAFVNSGVVLAEQHGFDGIDLAWQFPKVKPKKIRSGWGNFWHGVKKTFKTTPVDEKESEHREGFTALVRELKAALSLKPHLELGVTILPNVNSTIYCDVPAIINFVDYVNLLAFDYFTPERNEKEADYTAPIYAPQNRHPEQNVDAAVKYWRNAGAPPTKIVVGIATYARTWKLDSDSEVAGVPPIHTDGAGEPGPYTKTEGLLSYPEVCMKLIAPPAGLRANIRKVTDPSKRFGTYAFRLPDSDGNGGIWVSYEDADTAGQKADYVKKNNLGGISIVDLSMDDFRELCTGNKYPILRAAKYRL。
本发明同时提供了该小菜蛾识别蛋白PxIDGF制品的制备方法:本发明选用小菜蛾(Plutella xylostella)作为材料,从小菜蛾四龄幼虫体中提取总RNA,逆转录合成第一链cDNA,利用设计的引物从cDNA中扩增小菜蛾识别蛋白PxIDGF基因,通过常规的分子克隆方法将小菜蛾识别蛋白PxIDGF基因连接到pCzn1载体中,然后将质粒转入到大肠杆菌Arctic-Express表达菌株,37℃振摇过夜,次日按1:100比例稀释菌液摇菌至OD600值为0.6-0.8,加入终浓度0.5mMIPTG,15℃振摇过夜,诱导融合蛋白表达,通过变复性的方式,重溶目标蛋白,通过Ni柱亲和纯化获得目的蛋白PxIDGF。
该制备方法包括以下步骤:
(1)pCzn1-IDGF质粒构建;
(2)pCzn 1-IDGF载体转化至大肠杆菌Arctic-Express;
(3)pCzn 1-IDGF载体融合蛋白的表达;
(4)包涵体蛋白的变复性;
(5)融合蛋白的Ni柱亲和纯化。
步骤(1)所述pCzn1-IDGF质粒构建方法如下:从小菜蛾四龄幼虫体中提取总RNA,逆转录合成第一链cDNA,设计用于扩增小菜蛾识别蛋白PxIDGF基因的一对上、下游引物,设计的引物中引入酶切位点,利用设计的引物从cDNA中扩增小菜蛾识别蛋白PxIDGF基因,利用常规分子克隆的方法将获得的线性化目的基因小菜蛾识别蛋白PxIDGF与目标载体pCzn1连接,连接产物转化TOP10克隆菌株,挑取阳性克隆子测序,筛选获得测序正确的阳性克隆,提取质粒;
其中,所述上游引物序列为5’-GGAATTCCATATGAACCAGGTCGTCTCC-3’,下划线序列为Nde I酶切位点序列,核苷酸序列如SEQ ID NO:2所示。
下游引物序列为5’-GCTCTAGATTACAGACGGTACTTGG-3’,下划线序列为Xba I酶切位点序列,核苷酸序列如SEQ ID NO:3所示。
步骤(2)所述pCzn 1-IDGF载体转化至大肠杆菌Arctic-Express的方法为:将测序正确的阳性克隆提取质粒,将1μL质粒加入100μL感受态细菌中,置冰上20min;42℃热激90s迅速置冰中5min;加入600μL LB培养液;37℃,220rpm振摇1h,离心后全部涂布于含50μg/mlAmp的LB平板,37℃倒置培养过夜。
步骤(3)所述pCzn 1-IDGF载体融合蛋白的表达方法为:挑取转化平板上的单克隆接种于含50μg/ml AMP的3ml LB培养液的试管中,37℃,220rpm振摇过夜;次日按1:100接种于50μg/ml AMP的30ml LB培养液中,37℃220rpm振摇至菌体OD600为0.6-0.8;加入IPTG至终浓度为0.5mM,15℃,220rpm振摇过夜,诱导融合蛋白表达;在4000g条件下,离心10min,弃上清,收集沉淀。
步骤(4)所述包涵体蛋白的变复性方法为:将经过pCzn 1-IDGF载体融合蛋白的表达后的沉淀重悬于20ml裂解缓冲液中,超声破碎;将超声破碎的细胞裂解液在4℃,10000g条件下离心20min,收集沉淀;使用包涵体洗涤液洗涤沉淀包涵体3次;再用溶解缓冲液按一定比例溶解包涵体,4℃放置过夜;然后室温15000rpm条件下离心15min;上清滴加20mMTris-HCL 5mM EDTA Buffer PH7.8缓冲液中,逐步成倍梯度稀释缓慢搅拌,将蛋白溶液装入透析袋于PBS pH7.4溶液中透析过夜,收集透析袋内蛋白溶液;
其中,所述裂解缓冲液为20mM Tris-HCl,含有1mM PMSF和细菌蛋白酶抑制剂混合物,pH 8.0;
所述超声破碎条件为:功率400W,工作4s,间歇8s,共20min;
所述包涵体洗涤液为20mM Tris,1mM EDTA,2M尿素,1M NaCl,1%Triton X-100,pH8.0;
所述溶解缓冲液为20mM Tris,5mM DTT,8M尿素,pH8.0。
步骤(5)所述融合蛋白的Ni柱亲和纯化方法为:利用低压层析***,将经过包涵体蛋白的变复性方法得到的蛋白溶液以0.5ml/min流速上样至Ni-IDA结合缓冲液预平衡的Ni-IDA-Sepharose CL-6B亲和层析柱;用Ni-IDA结合缓冲液以0.5ml/min流速冲洗,至流出液OD280值到达基线,再用Ni-IDA洗涤缓冲液以1ml/min流速冲洗,至流出液OD280值到达基线,以除去杂蛋白;用Ni-IDA洗脱缓冲液以1ml/min流速洗脱目的蛋白,收集蛋白流出液;蛋白流出液加入透析袋中,使用pH7.4的PBS进行透析过夜,收集透析袋内蛋白溶液,此蛋白溶液冷干后即得小菜蛾识别蛋白PxIDGF制品;
其中,所述Ni-IDA洗涤缓冲液为20mM Tris-HCl,20mM咪唑,0.15M NaCl,pH8.0;
所述Ni-IDA洗脱缓冲液为20mM Tris-HCl,250mM咪唑,0.15M NaCl,pH8.0。
本发明所述制备的识别蛋白PxIDGF制品具有结合微生物、凝集微生物的能力。
本发明有益效果如下:
本发明具有原料来源丰富、简便快速等优点,表达***具有增加蛋白翻译效率、增加蛋白表达水平等特点,可在低温条件下诱导外源蛋白高水平表达及增加表达蛋白的活性几率。本发明制备的小菜蛾识别蛋白PxIDGF制品纯度高、活性高,具有结合微生物的能力及凝集微生物的能力,免除了从小菜蛾中提取识别蛋白的繁琐步骤,极大的节约实验成本和时间。本发明制备方法可大规模工厂化生产,生产周期短,生产成本较低,且不受外在环境的影响。本发明可为深入研究小菜蛾识别蛋白PxIDGF的应用打下技术基础。更重要的是,本发明首次通过微生物结合和凝集试验证实了小菜蛾识别蛋白PxIDGF具有微生物病原识别的能力,并且该结合能力在试验数据的支撑下具有现有技术中未曾报道过的微生物结合比例和凝集能力。
附图说明
图1为重组质粒酶切鉴定的结果;
图中:M为DNA分子量Marker(从下到上分别为100bp,250bp,500bp,750bp,1000bp,1500bp,2000bp,3000bp和5000bp);1为酶切前质粒;2为酶切后质粒;
图2为融合蛋白表达鉴定结果;
图中:M为蛋白分子量Marker;1为未诱导菌液;2为IPTG诱导菌液;3为15℃IPTG诱导菌体超声破碎后上清;4为15℃IPTG诱导菌体超声破碎后沉淀;
图3为纯化的目的蛋白SDS-PAGE结果;
图中:M为蛋白分子量Marker;1为未纯化样品;2为Ni-IDA洗涤缓冲液洗涤样品;3为Ni-IDA洗脱缓冲液洗脱纯蛋白;
图4为小菜蛾识别蛋白PxIDGF与微生物结合测定结果;
图中:1为孵育后上清;2-5为孵育后沉淀分别用TBS洗涤四次;6为孵育后沉淀用7%SDS洗脱后上清;7为孵育后沉淀用7%SDS洗脱后沉淀;8为纯化的小菜蛾识别蛋白PxIDGF;
图5为小菜蛾识别蛋白PxIDGF凝集微生物结果;
Control为1mM CaCl 2;+BSA为1mM CaCl 2和75ng BSA的TBS;+IDGF为1mM CaCl 2和75ng PxIDGF的TBS;+IDGF+EDTA为1mM CaCl 2、75ng PxIDGF和1mM EDTA的TBS。
具体实施方式
下面结合附图和具体实施例来进一步详细说明本发明,但本发明的实施方式不限于此。
实施例1
1、pCzn1-IDGF质粒构建
利用Trizol试剂(Takara)从小菜蛾四龄幼虫体中提取总RNA,使用cDNA合成试剂盒(Takara)逆转录合成第一链cDNA,设计用于扩增小菜蛾识别蛋白PxIDGF基因的一对上、下游引物,上游引物序列为5’-GGAATTCCATATGAACCAGGTCGTCTCC-3’(下划线序列为Nde I酶切位点序列),下游引物序列为5’-GCTCTAGATTACAGACGGTACTTGG-3’(下划线序列为Xba I酶切位点序列)。设计的引物可中引入酶切位点。利用设计的引物从cDNA中扩增小菜蛾识别蛋白PxIDGF基因,PCR体系:模板DNA2.0μL,上游引物1.0μL,下游引物1.0μL,10X buffer 5.0μL,dNTP Mixture 4.0ul,Pfu DNA聚合酶1.0μL,用ddH2O补至50μL。PCR程序:94℃5min;94℃30s,65℃30s,72℃45s,共30循环;最后72℃5min。利用常规分子克隆的方法将获得的线性化目的基因小菜蛾识别蛋白PxIDGF与目标载体pCzn1连接,连接产物转化TOP10克隆菌株,挑取阳性克隆子测序,筛选获得测序正确的阳性克隆。并利用酶切鉴定。酶切体系:质粒3ul,内切酶Nde I 0.25μL,内切酶Xba I 0.25μL,10×Buffer 1.0μL,用ddH2O补至10μL。酶切鉴定结果如图1所示。
2、pCzn 1-IDGF载体转化至大肠杆菌Arctic-Express
将测序正确的阳性克隆提取质粒,将1μL质粒加入100μL感受态细菌中,置冰上20min;42℃热激90sec,迅速置冰中5min;加入600μL LB培养液;37℃,220rpm振摇1h,离心后全部涂布于含50μg/ml Amp的LB平板,37℃倒置培养过夜。
3、IPTG诱导pCzn 1-IDGF载体融合蛋白的表达
(1)挑取转化平板上的单克隆接种于含50μg/ml AMP的3ml LB培养液的试管中,37℃220rpm振摇过夜;
(2)次日按1:100接种于50μg/ml AMP的30ml LB培养液中,37℃220rpm振摇至菌体OD600为0.6-0.8;
(3)取出1ml培养物,10000g室温离心2min,弃上清,用100μl 1×上样缓冲液重悬菌体沉淀;
(4)向剩余的培养物中加入IPTG至终浓度为0.5mM,15℃220rpm振摇过夜,诱导融合蛋白表达;
(5)取出1ml培养物,10000g室温离心2min,弃上清,用100μl 1×上样缓冲液重悬菌体沉淀。剩余培养物4000g,离心10min,弃上清,用PBS重悬菌体沉淀;重悬液进行超声波破碎后,分别取上清液与沉淀液加入上样缓冲液重悬。
(6)进行12%SDS-PAGE检测分析,考马斯亮蓝染色显带,结果如图2所示。
4、包涵体蛋白的变复性
将经过pCzn 1-IDGF载体融合蛋白的表达后的沉淀重悬于20ml裂解缓冲液(20mMTris-HCl,含有1mM PMSF和细菌蛋白酶抑制剂混合物,pH 8.0),超声破碎(功率400W,工作4sec,间歇8sec,共20min);将超声破碎的细胞裂解液4℃10000g离心20min,收集沉淀;使用包涵体洗涤液(20mM Tris,1mM EDTA,2M尿素,1M NaCl,1%Triton X-100,pH8.0)洗涤沉淀包涵体3次;再用溶解缓冲液(20mM Tris,5mM DTT,8M尿素pH8.0),按一定比例溶解包涵体,4℃放置过夜;然后室温15000rpm条件下离心15min;上清滴加20mM Tris-HCL 5mMEDTABuffer PH7.8缓冲液中,逐步成倍梯度稀释缓慢搅拌,将蛋白溶液装入透析袋于pH7.4的PBS溶液中透析过夜,收集透析袋内蛋白溶液。
5、融合蛋白的Ni柱亲和纯化
利用低压层析***,将经过包涵体蛋白的变复性方法得到的蛋白溶液以0.5ml/min流速上样至Ni-IDA结合缓冲液预平衡的Ni-IDA-Sepharose CL-6B亲和层析柱;用Ni-IDA结合缓冲液以0.5ml/min流速冲洗,至流出液OD280值到达基线,再用Ni-IDA洗涤缓冲液(20mM Tris-HCl,20mM咪唑,0.15M NaCl,pH8.0)以1ml/min流速冲洗,至流出液OD280值到达基线,以除去杂蛋白;用Ni-IDA洗脱缓冲液(20mM Tris-HCl,250mM咪唑,0.15M NaCl,pH8.0)以1ml/min流速洗脱目的蛋白,收集蛋白流出液;蛋白流出液加入透析袋中,使用PBS(PH7.4)进行透析过夜,收集透析袋内蛋白溶液,此蛋白溶液冷干后即为所述的小菜蛾识别蛋白PxIDGF制品。
对采用实施例1方法得到的小菜蛾识别蛋白PxIDGF制品样品进行分析:
1、12%SDS-PAGE分析
取100μL样品加入20μL 5×SDS Loading Buffer煮沸5min,12000rpm离心5min,取20μL上样。进行12%SDS-PAGE,用考马斯亮蓝染色液染色2h,用考马斯亮蓝脱色液脱色过夜。进行12%SDS-PAGE分析结果如图3所示。
2、蛋白印迹分析
样本进行12%SDS-PAGE,并转移到硝酸纤维素膜上。随后,用封闭缓冲液(5%BSA)在室温下封闭膜室1.5小时,并与PxIDGF多克隆抗体(封闭缓冲液中1:2500稀释)一起在4℃温育过夜并用IgG在室温下共孵育2小时。用DAB染料试剂(Boster Biotech)观察条带。
3、微生物结合测定
取处于对数中期的细菌(大肠杆菌和金黄色葡萄球菌),以6000rpm离心5min,取沉淀用Tris-缓冲盐水(TBS;50mM Tris-HCl,150mM NaCl,pH7.5)洗涤三次,并悬浮于TBS中,配成浓度为2×108cells/ml的细菌悬液。将细菌悬液(200μl)与200μlPxIDGF(150μg/ml)混合。将混合物在室温下温育1小时,温和旋转。将细菌沉淀,用TBS洗涤四次,并用7%SDS洗脱。洗涤和洗脱溶液使用蛋白质印迹进行分析。制备的的小菜蛾识别蛋白PxIDGF制品具有结合大肠杆菌和金黄色葡萄球菌的能力,结果见图4所示。由图可知小菜蛾识别蛋白PxIDGF在与大肠杆菌孵育后沉淀中的分配比例达到90%,与金黄色葡萄球菌孵育后沉淀中的分配比例达到95%。
4、微生物凝集试验
将2×106个大肠杆菌或金黄色葡萄球菌细胞加入到具有1mM CaCl 2(对照),具有1mM CaCl 2和75ng BSA的TBS,具有1mM CaCl 2和75ng PxIDGF的TBS以及1mM CaCl 2、75ngPxIDGF和1mM EDTA的TBS。将所有处理在室温下孵育2小时。用Eclipse Ti倒置显微镜(Nikon)检查可发现细菌聚集体,结果见图5所示。结果显示在20倍物镜视野下可观察到平均5个细菌凝集团(取50个视野平均值)。
序列表
<110> 安徽农业大学
<120> 一种小菜蛾识别蛋白PxIDGF制品及其制备方法和应用
<130> 2017
<141> 2017-12-15
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 428
<212> PRT
<213> Plutella xylostella
<400> 1
Met Asn His Lys Val His His His His His His Met Asn Gln Val Val
1 5 10 15
Ser Thr Lys Lys Val Ile Cys Tyr Tyr Asp Ser Lys Ser Tyr Val Arg
20 25 30
Glu Ser Asn Ala Arg Leu Leu Pro Pro Asp Leu Glu Pro Ala Leu Pro
35 40 45
Tyr Cys Thr His Leu Val Tyr Gly Tyr Ala Gly Val Gln Pro Asp Thr
50 55 60
Tyr Lys Met Ile Ser Thr Asn Gln Asn Leu Asp Ile Asp Ser Ala His
65 70 75 80
Ala Asn Tyr Arg Thr Ile Thr Ser Phe Lys Thr Lys Tyr Pro Gln Leu
85 90 95
Lys Met Leu Leu Ala Val Gly Gly Asp Ala Asp Leu Glu Asp Pro Gln
100 105 110
Lys Tyr Asn Ala Leu Leu Glu Ser Gln Gln Ala Arg Thr Ala Phe Val
115 120 125
Asn Ser Gly Val Val Leu Ala Glu Gln His Gly Phe Asp Gly Ile Asp
130 135 140
Leu Ala Trp Gln Phe Pro Lys Val Lys Pro Lys Lys Ile Arg Ser Gly
145 150 155 160
Trp Gly Asn Phe Trp His Gly Val Lys Lys Thr Phe Lys Thr Thr Pro
165 170 175
Val Asp Glu Lys Glu Ser Glu His Arg Glu Gly Phe Thr Ala Leu Val
180 185 190
Arg Glu Leu Lys Ala Ala Leu Ser Leu Lys Pro His Leu Glu Leu Gly
195 200 205
Val Thr Ile Leu Pro Asn Val Asn Ser Thr Ile Tyr Cys Asp Val Pro
210 215 220
Ala Ile Ile Asn Phe Val Asp Tyr Val Asn Leu Leu Ala Phe Asp Tyr
225 230 235 240
Phe Thr Pro Glu Arg Asn Glu Lys Glu Ala Asp Tyr Thr Ala Pro Ile
245 250 255
Tyr Ala Pro Gln Asn Arg His Pro Glu Gln Asn Val Asp Ala Ala Val
260 265 270
Lys Tyr Trp Arg Asn Ala Gly Ala Pro Pro Thr Lys Ile Val Val Gly
275 280 285
Ile Ala Thr Tyr Ala Arg Thr Trp Lys Leu Asp Ser Asp Ser Glu Val
290 295 300
Ala Gly Val Pro Pro Ile His Thr Asp Gly Ala Gly Glu Pro Gly Pro
305 310 315 320
Tyr Thr Lys Thr Glu Gly Leu Leu Ser Tyr Pro Glu Val Cys Met Lys
325 330 335
Leu Ile Ala Pro Pro Ala Gly Leu Arg Ala Asn Ile Arg Lys Val Thr
340 345 350
Asp Pro Ser Lys Arg Phe Gly Thr Tyr Ala Phe Arg Leu Pro Asp Ser
355 360 365
Asp Gly Asn Gly Gly Ile Trp Val Ser Tyr Glu Asp Ala Asp Thr Ala
370 375 380
Gly Gln Lys Ala Asp Tyr Val Lys Lys Asn Asn Leu Gly Gly Ile Ser
385 390 395 400
Ile Val Asp Leu Ser Met Asp Asp Phe Arg Glu Leu Cys Thr Gly Asn
405 410 415
Lys Tyr Pro Ile Leu Arg Ala Ala Lys Tyr Arg Leu
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Claims (9)
1.一种小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述识别蛋白PxIDGF制品具有结合微生物、凝集微生物的能力,所述微生物为大肠杆菌或金黄色葡萄球菌;
所述小菜蛾识别蛋白PxIDGF制品是从小菜蛾幼虫中通过基因克隆、蛋白表达、纯化得到的一种蛋白制品,理论分子量为 47.904KD,等电点为7.67;所述蛋白制品的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述小菜蛾识别蛋白PxIDGF制品的制备方法,包括以下步骤:
(1)pCzn1- IDGF质粒构建;
(2)pCzn 1-IDGF载体转化至大肠杆菌 Arctic-Express;
(3)IPTG 诱导 pCzn 1-IDGF载体融合蛋白的表达;
(4)包涵体蛋白的变复性;
(5)融合蛋白的Ni柱亲和纯化。
3.根据权利要求2所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述步骤(1)pCzn1- IDGF质粒构建方法如下:从小菜蛾四龄幼虫体中提取总RNA,逆转录合成第一链cDNA,设计用于扩增小菜蛾识别蛋白PxIDGF基因的一对上、下游引物,设计的引物中引入酶切位点,利用设计的引物从cDNA中扩增小菜蛾识别蛋白PxIDGF基因,利用常规分子克隆的方法将获得的线性化目的基因小菜蛾识别蛋白PxIDGF与目标载体pCzn1连接,连接产物转化TOP10克隆菌株,挑取阳性克隆子测序,筛选获得测序正确的阳性克隆,提取质粒;
所述上游引物序列为5’-GGAATTCCATATGAACCAGGTCGTCTCC -3’,核苷酸序列如SEQ IDNO:2所示,
下游引物序列为5’- GCTCTAGATTACAGACGGTACTTGG -3’,核苷酸序列如SEQ ID NO:3所示。
4.根据权利要求2所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述步骤(2)pCzn 1-IDGF载体转化至大肠杆菌 Arctic-Express的方法为:将测序正确的阳性克隆提取质粒,将1 μL质粒加入 100 μL 感受态细菌中,置冰上 20min;42℃热激 90 s迅速置冰中 5 min;加入 600μL LB 培养液; 37℃,220 rpm 振摇 1 h,离心后全部涂布于含 50 μg/ml Amp 的 LB 平板,37℃倒置培养过夜。
5.根据权利要求2所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述步骤(3)pCzn 1-IDGF载体融合蛋白的表达方法为:挑取转化平板上的单克隆接种于含50 μg/ml AMP的3 ml LB培养液的试管中,37℃,220 rpm振摇过夜;次日按1:100接种于50 μg/ml AMP的30 ml LB培养液中,37℃ 220 rpm振摇至菌体OD600为0.6-0.8;加入IPTG至终浓度为0.5 mM,15℃,220 rpm振摇过夜,诱导融合蛋白表达;在4000g条件下,离心10 min,弃上清,收集沉淀。
6.根据权利要求2所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述步骤(4)包涵体蛋白的变复性方法为:将经过pCzn 1-IDGF载体融合蛋白的表达后的沉淀重悬于20 ml 裂解缓冲液中,超声破碎;将超声破碎的细胞裂解液4℃,10000g离心20 min,收集沉淀;使用包涵体洗涤液洗涤沉淀包涵体3次;再用溶解缓冲液按一定比例溶解包涵体,4℃放置过夜;然后室温15000rpm条件下离心15min;上清滴加到20 mMTris-HCL 5mM EDTA Buffer PH7.8缓冲液中,逐步成倍梯度稀释缓慢搅拌,将蛋白溶液装入透析袋于PBS pH7.4溶液中透析过夜,收集透析袋内蛋白溶液;
其中,所述裂解缓冲液为20 mM Tris-HCl,含有1 mM PMSF和细菌蛋白酶抑制剂混合物,pH 8.0;
所述超声破碎条件为:功率400 W,工作4 s,间歇8 s,共20 min;
所述包涵体洗涤液为20mM Tris,1mM EDTA,2M尿素,1M NaCl,1%Triton X-100,pH8.0;
所述溶解缓冲液为20mM Tris,5mM DTT,8M尿素,pH8.0。
7.根据权利要求2所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,所述步骤(5)融合蛋白的Ni柱亲和纯化方法为:利用低压层析***,将经过包涵体蛋白的变复性方法得到的蛋白溶液以0.5 ml/min流速上样至Ni-IDA 结合缓冲液预平衡的Ni-IDA -Sepharose CL-6B亲和层析柱;用Ni-IDA 结合缓冲液以0.5 ml/min流速冲洗,至流出液OD280值到达基线,再用Ni-IDA 洗涤缓冲液以1 ml/min流速冲洗,至流出液OD280值到达基线,以除去杂蛋白;用Ni-IDA 洗脱缓冲液以1 ml/min流速洗脱目的蛋白,收集蛋白流出液;蛋白流出液加入透析袋中,使用pH7.4的PBS进行透析过夜,收集透析袋内蛋白溶液,此蛋白溶液冷干后即得小菜蛾识别蛋白PxIDGF制品;
其中,所述Ni-IDA 洗涤缓冲液为20 mM Tris-HCl,20 mM咪唑,0.15 M NaCl,pH8.0;
所述Ni-IDA 洗脱缓冲液为20 mM Tris-HCl,250 mM咪唑,0.15 M NaCl,pH8.0。
8.根据权利要求1所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,在微生物结合实验中,30μg的蛋白制品与4×107cells/ ml的大肠杆菌在室温条件下温和旋转孵育1小时使得蛋白在孵育后沉淀中的分配比例达到90%,30μg的蛋白制品与4×107cells/ ml的金黄色葡萄球菌在室温条件下温和旋转孵育1小时使得蛋白在孵育后沉淀中的分配比例达到95%。
9.根据权利要求1所述小菜蛾识别蛋白PxIDGF制品在抵御微生物侵染中的应用,其特征在于,在微生物凝集实验中,75ng蛋白制品与2×106个大肠杆菌或2×106个金黄色葡萄球菌在室温条件下孵育2小时后在20倍物镜视野下可平均观察到5个细菌凝集团。
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