CN114703166B - 一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法 - Google Patents
一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法 Download PDFInfo
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- CN114703166B CN114703166B CN202210474152.XA CN202210474152A CN114703166B CN 114703166 B CN114703166 B CN 114703166B CN 202210474152 A CN202210474152 A CN 202210474152A CN 114703166 B CN114703166 B CN 114703166B
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Abstract
本发明公开了一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法,所述蛋白的氨基酸序列由SEQ ID NO:1所示,或在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或多个氨基酸序列获得具有同等活性的蛋白。本发明还提供了所述抗真菌效应蛋白的编码基因、表达载体或工程菌,本发明还提供了假结核耶尔森氏菌抗真菌蛋白的分离纯化方法。所述蛋白在抗真菌药剂中的应用以及所属蛋白的编码基因在转化植物中的应用。本发明涉及的YPK_D305抗真菌基因属于一个几丁质酶,该基因具有更高的抗真菌活性,靶向作用真菌范围也更广泛,为病原真菌防治提供策略和途径,具有较大的学术价值和潜在的应用前景。
Description
技术领域
本发明属于基因工程技术领域,涉及一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法,具体的说,涉及来源于假结核耶尔森氏菌的抗真菌蛋白YPK_D305,抗真菌的应用,蛋白的分离纯化方法。
背景技术
假结核耶尔森氏菌(Yersinia pseudotuberculosis)是一种重要的人兽共患肠道致病菌,具备四套VI型分泌系统(Type VI Secretion System,T6SS),可以将不同类型的效应蛋白投递至宿主细胞中,从而在与宿主的相互作用中发挥重要作用。
病原真菌可对诸多农作物造成破坏性的损害,并且对牲畜和人类产生许多不利的健康后果。每年真菌感染直接造成水稻、小麦、玉米、土豆和大豆等五大粮食农作物的产量损失高达1亿吨以上。真菌感染的植物病害有很多,例如稻瘟病、小麦赤霉病、玉米黑穗病、大豆锈病、辣椒疫病、苹果轮纹病等。现今,植物病害的防治主要依赖于化学农药,其弊端在于农药使用不仅受到严格的监管和限制,同时会对环境造成不可逆的破坏。因此,寻找全新的、安全可靠的环境友好型抗真菌基因或抗真菌型效应蛋白则被认为是实践中用于作物蔬菜和水果生物防治的新手段,尤其是培育抗真菌转基因的应用成为当前各国生物防治研究领域中的热点。抗真菌蛋白可以分为:包括几丁质酶和几丁质酶样蛋白,几丁质结合蛋白,亲环蛋白样蛋白,防御素和防御素样蛋白,脱氧核糖核酸酶,胚胎丰富蛋白,葡聚糖酶,凝集素,脂质转移蛋白,过氧化物酶,蛋白酶抑制剂,核糖核酸酶,核糖体失活蛋白,和类似丘脑蛋白等抗真菌蛋白。编码这些抗真菌蛋白的基因许多已经被利用分子生物学等相关技术,获得转化植株,从而使之获得更好的抗真菌侵袭性。采用转基因方法来提高宿主对真菌的抗性,可以减少有害农药对环境的损坏,并且比传统的抗菌方法具有更高的效率。
几丁质是由N-乙酰-D-氨基葡萄糖(N-acetyl-D-glucosamine,GlcNAc)通过β-1,4-糖苷键连接而成的一种多糖,在地球上含量仅次于纤维素。几丁质广泛存在于病原真菌细胞壁、线虫卵壳和节肢动物中。几丁质酶可以水解几丁质产生几丁寡糖和N-乙酰氨基葡萄糖,且广泛存在于自然界的生物中,具有重要的生理功能,在植物病虫害的生物防治中发挥着重要作用。其可以直接参与真菌细胞壁的溶解、孢子萌发、细胞自溶、孢子形成和真菌寄生等诸多过程。目前关于几丁质酶的研究,已经从酶蛋白的分离、纯化发展到了分子水平。现已从多种微生物及植物中克隆到了几丁质酶编码基因,并将这些基因进行异源转化和表达。利用几丁质酶破坏真菌细胞壁,能够有效防御真菌对植物及人类的侵染,在农业与医学方面具有重大意义。然而,截至目前,来源于假结核耶尔森氏菌抗真菌蛋白及其编码基因还未见有报道,本发明专利旨在获得一种几丁质酶型抗真菌蛋白,将有助于几丁质酶在将来实际应用中的优化与改造。
发明内容
本发明的目的在于提供一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法,是一种具有几丁质酶活性的蛋白。
其具体技术方案为:
一种假结核耶尔森氏菌抗真菌蛋白,所述蛋白的氨基酸序列如SEQ ID NO:1所示;或在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或多个氨基酸序列获得具有同等活性的蛋白。
一种假结核耶尔森氏菌抗真菌蛋白,述蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
本发明所述的假结核耶尔森氏菌抗真菌蛋白用于制备抗真菌药物的应用。
本发明所述的假结核耶尔森氏菌抗真菌蛋白用于制备抗霉菌药物的应用。
本发明所述的假结核耶尔森氏菌抗真菌蛋白用于制备防腐剂的应用。
本发明所述的假结核耶尔森氏菌抗真菌蛋白用于制备水果保鲜剂的应用。
本发明所述的假结核耶尔森氏菌抗真菌蛋白用于制备蔬菜保鲜剂的应用。
一种表达载体,所述的载体为大肠杆菌BL21(DE3)(pET28a-YPK_D305)。
本发明所述的假结核耶尔森氏菌抗真菌蛋白的分离纯化方法,包括:
1)活化:将已构建好的蛋白表达菌株大肠杆菌BL21(DE3)(pET28a-YPK_D305),挑单菌落,接种于含有50μg/mL卡那霉素的液体LB培养基中,于220rpm,37℃摇床中培养至平台期;
2)转接扩大培养:按1%的接种比例将菌转接于含有50μg/mL卡那霉素的LB液体培养基中,37℃,180rpm摇动培养;检测OD600至0.6时,将摇床温度调至26℃,并按终浓度0.5mM加入诱导剂IPTG,开始诱导;过夜培养,16~18小时后,诱导完毕;
3)收菌:用低温离心机在4℃下收集菌体,8000rpm,10分钟,重复此步骤,直至收集到所有菌液中的菌体,然后用Binding buffer洗涤菌体两次;
4)破碎菌体:加入适量Binding buffer,使菌体悬浮,破碎菌体后再次悬浮菌体,4℃条件下,用超声波细胞破碎仪破碎,功率为200W,工作4s,间歇5s,循环数为99,直至将菌液超清,10000rpm,4℃下离心30分钟,收集上清;
5)上柱:将上一步中的上清分批全部加入到事先活化好的Ni-NTA亲和纯化柱中,直至全部液体通过柱子流下;
6)洗涤:用Washing buffer将柱子洗两遍;
7)洗脱:用Elution buffer洗脱,且每次洗脱液收集在1.5 mL事先预冷的EP管中,放置于冰上;
8)透析:将收集到的洗脱液注入透析袋中,于透析液中4℃下过夜透析;
9)分装:将蛋白溶液分装,-80℃保存。
与现有技术相比,本发明的有益效果:
本发明首次报道来源于假结核耶尔森氏菌的具有几丁质酶活性的抗真菌型效应蛋白YPK_D305,该蛋白具有降解真菌细胞壁的几丁质酶活性,与以报道的目前广泛应用的抗真菌蛋白基因均不具有同源性,是一种抗真菌的蛋白和基因。进行抗真菌实验,可对白色念珠菌、酿酒酵母菌、假禾谷镰孢菌、禾谷镰孢菌及辣椒疫霉菌等病原真菌产生明显的抑制效果。本发明所涉及的YPK_D305抗真菌编码基因及蛋白具有更高、更广谱性的抗真菌活性。
附图说明
附图是用来提供对本公开的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本公开,但并不构成对本公开的限制。在附图中:
图1是YPK_D305基因PCR扩增结果;
图2是YPK_D305重组质粒小诱导结果;
图3是YPK_D305重组蛋白纯化结果;
图4是YPK_D305的几丁质酶活性检测结果;
图5是YPK_D305对白色念珠菌抗菌结果;
图6是YPK_D305对酿酒酵母菌抗菌结果;
图7是YPK_D305蛋白对假禾谷镰孢菌、禾谷镰孢菌和辣椒疫霉菌的抑菌效果;
图8是YPK_D305基因缺失突变体和互补菌株对假禾谷镰孢菌、辣椒疫霉、脉孢霉菌和苹果轮纹菌的抑菌效果;
图9是本发明构建的蛋白表达菌株大肠杆菌BL21(DE3)(pET28a-YPK_D305)。
具体实施方式
下面结合具体实施方案对本发明的技术方案作进一步详细地说明。
本发明提供了一种假结核耶尔森氏菌抗真菌蛋白,蛋白的氨基酸序列如SEQ IDNO:1所示,或在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或多个氨基酸序列获得具有同等活性的蛋白。
本发明的蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
本发明的假结核耶尔森氏菌抗真菌蛋白用于制备抗真菌药物的应用。
本发明的假结核耶尔森氏菌抗真菌蛋白用于制备抗霉菌药物的应用。
本发明的假结核耶尔森氏菌抗真菌蛋白用于制备防腐剂的应用。
本发明的假结核耶尔森氏菌抗真菌蛋白用于制备水果保鲜剂的应用。
本发明的假结核耶尔森氏菌抗真菌蛋白用于制备蔬菜保鲜剂的应用。
一种表达载体,为大肠杆菌BL21(DE3)(pET28a-YPK_D305)。
一种假结核耶尔森氏菌抗真菌蛋白的诱导和纯化方法,包括以下步骤:
1)活化:将已构建好的蛋白表达菌株大肠杆菌BL21(DE3)(pET28a-YPK_D305),挑单菌落,接种于含有50μg/mL卡那霉素 5mL液体LB培养基中,于220rpm,37℃摇床中培养至平台期;
2)转接扩大培养:按1%的比例将菌转接于含有50μg/mL卡那霉素的500mL LB液体培养基的1L的三角瓶中,37℃,180rpm摇动培养;检测OD600,至0.6时,将摇床温度调至26℃,并按终浓度0.5mM加入诱导剂IPTG,开始诱导;过夜培养,约16-18小时后,诱导完毕;
3)收菌:用低温离心机在4℃下收集菌体,8000rpm,10分钟,重复此步骤,直至收集到所有菌液中的菌体,然后用Binding buffer (25mMTris-HCl,150mMNaCl,30mMimidazole)洗涤菌体两次;
4)破碎菌体:加入适量Binding buffer,充分震荡,将菌体悬浮起来,经低温高压细胞破碎仪压榨破碎菌体后,置于超声波破碎仪中进行超声用Binding buffer充分悬浮菌体,在4℃条件下,用超声波细胞破碎仪破碎,功率为200W,工作4s ,间歇5s,循环数为99,直至将菌液超清,10000rpm,4℃下离心30分钟,将上清转移到干净的三角瓶中;
5)上柱:将上一步中的上清分批全部加入到事先活化好的Ni-NTA亲和纯化柱中,直至全部液体通过柱子流下;
6)洗涤:用Washing buffer(25mMTris-HCl,150mMNaCl,100mM imidazole)将柱子洗两遍;
7)洗脱:用Elution buffer(25mMTris-HCl,150mMNaCl,250mM imidazole)洗脱,且每次洗脱液收集在1.5 mL事先预冷的EP管中,放置于冰上;
8)透析:将煮好的透析袋用去离子水清洗干净,将收集到的蛋白洗脱液注入透析袋中,两端用夹子夹好,放入透析液(10mMTris-HCl,100mMNaCl)中(透析液中加入DTT,以防止蛋白被氧化),4℃下过夜透析;
9)分装:将蛋白溶液分装,-80℃保存;跑SDS-PAGE胶来检测蛋白的纯化情况及进行抗真菌实验。
鉴于真菌细胞壁的主要成分是几丁质,因此通过生物信息学预测和分泌组学数据分析,在假结核耶尔森氏菌全基因组中筛选出YPK_D305编码的基因,并经表达载体构建、蛋白的诱导与纯化得到本发明的假结核耶尔森氏菌抗真菌蛋白,同时,通过YPK_D305基因缺失突变体和互补菌株的构建证明,本发明给出的假结核耶尔森氏菌抗真菌蛋白确实具有抗真菌的作用,特别是对于白色念珠菌、酿酒酵母菌以及霉菌等具有代表性的真菌,均具有良好的抑制活性。
YPK_D305诱导表达载体的构建:
获得基因YPK_D305,利用特异引物从野生型假结核耶尔森氏菌基因组DNA中扩增YPK_D305引物序列如下:
正向引物YPK_D305-F:GGAATTCCATATGATGGAGGTTAGCGACAAACTGCAGTT;
反向引物YPK_D305-R:
CCGCTCGAGTTTACCGTTAGCAGGTTCAG;
(1)YPK_D305基因的扩增
表1YPK_D305基因PCR扩增体系
反应程序为:
预变性95℃,5 min;(变性95℃,30 s;退火50℃,60 s;延伸72℃,1 min/kb)×10个循环;(变性95℃,30 s;退火52℃,60 s;延伸72℃,1 min/kb)×20个循环;最后72℃延伸10 min;16℃保温。结束后将PCR产物进行电泳检测。
(2)YPK_D305产物双酶切反应体系(150μL)
表2YPK_D305PCR产物双酶切反应体系(150μL)
离心混匀后,放入37℃水浴中,酶切过夜(14-16hours) ;加入15μL LoadingBuffer(10×),100℃加热3min ,终止酶活;取少量酶切产物进行琼脂糖凝胶电泳检测;
(3)YPK_D305酶切片段的回收(参考天根质粒回收试剂盒说明书)
1)柱平衡:将CB2放入预先准备好的收集管中,用移液枪吸取500μL BL,加入到吸附柱CB2中,静置5min,12,000rpm,离心1min,弃去管中废液;
2)将YPK_D305PCR酶切产物加等倍提交的PC溶液混匀后,全部转移至吸附柱CB2中,静置5min,12,000rpm离心1min,弃去管中废液;
3)加入600μl PW至吸附柱中,12,000rpm,离心1min,弃去管中废液;
4)重复步骤3;
5)12,000rpm,离心3min,以甩干柱子里残余的液体,将吸附柱于通风处,晾干至无乙醇气味;
6)把吸附柱放于1.5 mL EP上,向回收柱内中间位置,悬空加入灭菌ddH2O 80μL(已于55℃预热),12,000rpm,离心2min,为提高回收效率,可重复一次;
7)于-20℃贮存。
(4)表达质粒pET28a提取(参考天根质粒提取试剂盒方法)
1)在吸附柱CP3中,用移液器加500μL的BL,12000rpm ,离心1min,弃去废液;
2)从含pET28a表达载体的大肠杆菌TG1平板上各挑取3个单克隆接种到5mL含Km的液体LB培养基中,37℃、220r摇床,过夜培养;
3)离心收集菌体:将过夜培养的含pET28a表达空载菌液TG1 3Ml ,12000rpm ,离心 2min收集到1.5 mL EP管中,加250μL溶液P1,彻底悬浮菌体;加250μL溶液P2,上下翻转4-6次 (要轻柔),使菌体充分裂解,注意避免裂解过度;加350μL溶液P3,立即上下翻转4-6次(要轻柔) ,至出现白色絮状沉淀,冰浴5min,12000rpm ,离心20min;转移上清至吸附柱中,12000rpm ,离心1min,重复一次,弃去废液;加500μL PD,12000rpm,离心2min,弃去废液;加入600μLPW于吸附柱中,2000rpm,离心1min,重复一次;将吸附柱重新放回收集管中,12000rpm,离心2min;将吸附柱于通风处,晾干至无乙醇气味;把吸附柱置于灭菌处理新的1.5 mL EP管,向回收柱中间位置悬空加入灭菌ddH2O 100μL(已于55℃预热),12 ,000rpm,离心2min,重复一次;-20℃贮存。
(5)表达质粒pET28a双酶切
表3pET28a质粒双酶切反应体系(200μL)
离心混匀后,放入37℃培养箱中,过夜酶切;加入20μL Binding Buffer(10×)混匀,终止酶活,将产物进行琼脂糖电泳检测分析;对目标片段进行回收。
(6)重组表达载体的构建
1)按如下反应体系加入各反应物
表4 载体片段连接体系(15 μL)
置于16℃连接仪,连接过夜。
2)热激转化(全过程于冰上进行)取出TG1感受态细胞;于超净台中将连接产物完全加入感受态细胞,切勿吹打;冰浴半小时,42℃热激90秒,立即放至冰上,冰浴3-5分钟;加入800μL LB培养基,(37℃预热无抗),100rpm,37℃摇床,复苏40-55分钟;4000rpm,离心3min,弃上清。重悬沉淀,涂板,37℃,过夜培养。
3)菌落PCR阳性克隆的筛选和鉴定
表5重组质粒转化TG1的菌落PCR初步筛选(20 μL)
预变性95℃,5 min;(变性95℃,30 s;退火50℃,60 s;延伸72℃,1 min/kb)×10个循环;(变性95℃,30 s;退火52℃,60 s;延伸72℃,1 min/kb)×20个循环;最后72℃延伸10 min;16℃保温。结束后将PCR产物进行电泳检测;筛选出正确的阳性克隆;挑取阳性克隆至5 mL LB培养基中,37℃,120 rpm摇床,过夜培养。进行小量双酶切验证;并对酶切验证的阳性克隆进行测序,筛选出测序结果正确的阳性克隆质粒,保存备用。
(7)获得表达克隆
取出预先制备好的-80℃保存的L21(DE3)感受态细胞,在超净台中加入1μL阳性克隆质粒,轻轻混匀后;冰浴半小时,42℃热激90 s,立即放至冰上,冰浴3-5 min;加入800μLLB培养基,(37℃预热无抗),100rpm,37℃摇床,复苏60-90 min;取50-100μL菌液涂板,37℃,过夜培养,获得重组表达克隆的单菌落。结果见附图1。
构建好的蛋白表达菌株大肠杆菌BL21(DE3)(pET28a-YPK_D305)见图9。
YPK_D305蛋白的诱导与纯化:
1)活化:将已构建好的蛋白表达菌株大肠杆菌BL21(DE3)(pET28a-YPK_D305),挑单菌落,接种于含有50μg/mL卡那霉素的 5mL液体LB培养基中,于220rpm,37℃摇床中培养至平台期;
2)转接扩大培养:按1%的比例将菌转接于含有50μg/mL卡那霉素的500mL LB液体培养基的1L的三角瓶中,37℃,180rpm摇动培养;检测OD600,至0.6时,将摇床温度调至26℃,并按终浓度0.5mM加入诱导剂IPTG,开始诱导;过夜培养,约16-18小时后,诱导完毕,其中蛋白小诱导的结果见附图2;
3)收菌:用低温离心机在4℃下收集菌体,8000rpm,10分钟,重复此步骤,直至收集到所有菌液中的菌体,然后用Binding buffer (25mMTris-HCl,150mMNaCl,30mMimidazole)洗涤菌体两次;
4)破碎菌体:加入适量Binding buffer,充分震荡,将菌体悬浮起来,经低温高压细胞破碎仪压榨破碎菌体后,置于超声波破碎仪中进行超声用Binding buffer充分悬浮菌体,在4℃条件下,用超声波细胞破碎仪破碎,功率为200W,工作4s ,间歇5s,循环数为99,直至将菌液超清,10000rpm,4℃下离心30分钟,将上清转移到干净的三角瓶中;
5)上柱:将上一步中的上清分批全部加入到事先活化好的Ni-NTA亲和纯化柱中,直至全部液体通过柱子流下;
6)洗涤:用Washing buffer(25mMTris-HCl,150mMNaCl,100mM imidazole)将柱子洗两遍;
7)洗脱:用Elution buffer(25mMTris-HCl,150mMNaCl,250mM imidazole)洗脱,且每次洗脱液收集在1.5 mL事先预冷的EP管中,放置于冰上。
8)透析:将煮好的透析袋用去离子水清洗干净,将收集到的蛋白洗脱液注入透析袋中,两端用夹子夹好,放入透析液(10mMTris-HCl,100mMNaCl)中(透析液中加入DTT,以防止蛋白被氧化),4℃下过夜透析;
9)分装:将蛋白溶液分装,-80℃保存;跑SDS-PAGE胶来检测蛋白的纯化情况及进行抗真菌实验。结果见附图3。
YPK_D305基因缺失突变体和互补菌株的构建:
(1)设计相关引物,见表6:
表6相关引物
(2)以假结核耶尔森氏菌的基因组DNA为PCR模板,使用YPK_D305-UF-BglII /YPK_ D305-UR扩增YPK_D305的上游片段,使用YPK_D305-DF /YPK_D305-DR-XhoI扩增YPK_D305的下游片段,按照表7的重叠PCR反应体系和PCR反应程序进行重叠PCR,得到重叠片段。使用BglII/XbaI对重叠片段进行双酶切,使用BglII/XbaI对pDM4进行双酶切,后续经过回收---连接---转化至大肠杆菌S17-1λ pir---菌落PCR验证---酶切验证---测序等步骤得到转有pDM4-ΔYPK_D305质粒的S17-1λ pir菌株,其中pDM4载体抗性为氯霉素(Cm)抗性。
表7重叠PCR反应体系
(3)假结核耶尔森氏菌YPK_D305基因缺失突变体ΔYPK_D305的构建,具体步骤如下:
①将带有pDM4-ΔYPK_D305质粒的大肠杆菌S17-1λ pir(供体菌)和假结核耶尔森氏菌野生型菌株(受体菌)分别培养在5 mL的含有Cm的LB和含有萘啶酮酸(Ni)的YLB液体培养基中,在37℃和26℃,220 rpm条件下培养至稳定期;
②取700 μL供体菌和700 μL受体菌到1.5 mL离心管中,轻柔颠倒混匀,4000 rpm离心3 min,弃上清,将菌液滴在无抗性的YLB平板(提前置于37℃培养箱干燥24 h)上,26℃倒置培养8 h;
③将平板上的菌苔刮至1.5 mL离心管(已加入1 mL无抗性的YLB液体培养基)中,涡旋混匀,取10 μL的菌液涂布在的含有Ni和Cm的双抗性YLB平板中,置于26℃培养箱中培养24 h。接取单菌落于含有Ni的YLB液体培养基中,26℃摇床过夜培养至稳定期,取0.5~2 μL菌液涂布在含有20%蔗糖和Ni的YLB平板上;
④获得的单菌落经PCR验证、抗性确定和测序验证正确后,即为假结核耶尔森氏菌YPK_D305基因缺失突变体ΔYPK_D305。
(4)ΔYPK_D305(YPK_D305)互补菌株的构建,具体步骤如下:
①以假结核耶尔森氏菌基因组DNA为PCR模板,使用表4中的特异性引物YPK_D305-F- BglII /YPK_D305-R-XbaI扩增YPK_D305片段,使用BglII/XbaI分别对片段和pKT100载体进行双酶切,后续经过回收---连接---转化至大肠杆菌TG1---菌落PCR验证---酶切验证---测序等步骤得到转有pKT100-YPK_D305质粒的TG1菌株,其中pKT100载体抗性为卡那霉素(Km)抗性。
②制备假结核耶尔森氏菌野生型WT和突变体ΔYPK_D305菌株的电转感受态,具体步骤如下:菌株液体培养---4℃,4500 rpm离心3 min收集菌体---无菌ddH2O重悬菌体---4℃,4500 rpm离心3 min收集菌体---重复无菌ddH2O重悬菌体和离心步骤---预冷的10%的甘油重悬菌体---4500 rpm离心3 min收集菌体---10%甘油重悬菌体---分装后置于-80℃保存。
③冰上将感受态融化,加入质粒pKT100或pKT100-YPK_D305,混匀后加入无菌的预冷电转杯,1.8 KV条件下电转后转移至1 mL新鲜的YLB培养基中,26℃摇床复苏2 h,4500rpm离心3 min,将弃除上清的菌悬液涂布至含有Ni和Km的双抗平板,得到含有质粒的相关菌株,包括WT(pKT100)、ΔYPK_D305(pKT100)突变体和ΔYPK_D305(pKT100-YPK_D305),其中附图中pKT100使用Vector表示,即表示为假结核耶尔森氏菌野生型WT(Vector)、ΔYPK_ D305(Vector)突变体和ΔYPK_D305(YPK_D305)互补菌株。
即该试验所获得菌株在结果图中的标注方式是WT(Vector)、ΔYPK_D305(Vector) 和ΔYPK_D305 (YPK_D305)。
YPK_D305纯化蛋白的几丁质酶活实验(参考索莱宝几丁质酶活性检测试剂盒方法):
1、可见分光光度计预热30min。波长调至540nm,蒸馏水调零。
2、将标准溶液用蒸馏水稀释为4、3、2.5、2、1μmol/mL 的标准溶液备用。
3、在EP管中分别加入:
4、几丁质酶活的计算
1)标准曲线的绘制:以△A标准为y轴,标准溶液浓度为x轴,绘制标准曲线,得到标准方程y=kx+b。将△A测定带入标准方程中,得到x(μmol/mL)。
2)按照蛋白质浓度计算:酶活性定义:37℃下,每mg蛋白每小时分解几丁质产生1μmol N-乙酰氨基葡萄糖的酶量为一个酶活性单位。几丁质酶活性(U/mg prot)=x×V样÷(V样×Cpr)÷T=x÷Cpr。
其中,V样:反应体系中样本体积,0.5mL;V样总:加入提取液体积,1.5mL;W:样本质量,g;Cpr:样本蛋白浓度,mg/mL;T:反应时间,1h;细胞数量:以104为单位,万个。
通过检测假结核耶尔森氏菌效应蛋白YPK_D305的几丁质酶活,确定该效应蛋白可以降解胶体几丁质,且随着时间的延长,降解产物N-乙酰氨基葡萄糖的含量增加,几丁质酶活增强,同时加热失活的YPK_D305蛋白不具有几丁质酶活性,结果见附图4。
YPK_D305抑制真菌实验:
选取白色念珠菌(Candida albicans)和酿酒酵母菌(Saccharomyces cerevisiae)作为靶标真菌,分别于野生型、YPK_D305突变株及其互补菌株进行杀真菌实验。以白色念珠菌为例,在无抗性的M9固体培养基平板上放置一张0.22 μM的滤膜,将白色念珠菌和假结核耶尔森氏菌菌液均归一化为OD600为1.0,以体积比4:1(攻击菌:靶标菌细胞比=1000:1)混合,然后将30μL混合菌体点样至YPD琼脂平板上,30℃培养一段时间,用连续稀释法和在YPD培养基上的活菌计数来计算存活的目标真菌细胞。
通过对假结核耶尔森氏菌野生型WT(Vector)、突变体△YPK_D305(Vector)和互补菌株△YPK_D305 (YPK_D305)进行真菌抑制实验,发现和野生型菌株共培养的真菌数目显著低于△YPK_D305突变体,而和△YPK_D305 (YPK_D305)互补菌株共培养的真菌数目恢复至野生型水平,表明YPK_D305具有抑制真菌的功能,结果见附图5,6。
YPK_D305抑制霉菌实验:
1)制备适于霉菌生长的土豆培养基,配方如下:马铃薯200g、葡萄糖200g、琼脂15~20g和蒸馏水1L;将200g的马铃薯去皮切碎,于沸水中煮烂,用八层纱布过滤得土豆汁,后加入200g葡萄糖,加入琼脂20g,加水定容1L,121℃,25min灭菌,制成PDA培养基平板;
2)从冻存管中活化霉菌,置于26 ℃培养箱中活化;
3)使用打孔器取相同大小的菌核,分别使用YPK_D305纯化蛋白和ΔYPK_D305及其回补菌株处理霉菌菌核。于26 ℃条件下,共孵育48 h;
4)取出菌核,用ddH2O冲洗,置于新鲜PDA平板上,在26 ℃培养箱培养。
5)作为对照,分别用透析液、热失活蛋白、GST、野生型和空培养基对菌核进行同样的杀菌培养处理,48 h后观察抑菌效果。
图7和8显示的结果表示:通过将不同霉菌分别与YPK_D305纯化蛋白和ΔYPK_D305及其回补菌株共孵育后,发现其对部分霉菌具有菌核生长抑制作用,无论是ΔYPK_D305或是热灭活的YPK_D305蛋白以及各种对照,其处理的次生菌核均无任何生长限制。
其中,附图7为蛋白孵育结果,YPK_D305纯化蛋白分别对假禾谷镰孢菌(Fusarium pseudograminearum)、禾谷镰孢菌(Fusarium graminearum)和辣椒疫霉(Phytophthora capsici)有显著抑制作用,附图8为菌液孵育结果,分别对辣椒疫霉(Phytophthora capsici)、假禾谷镰孢菌(Fusarium pseudograminearum)、麦孢霉菌(Neuros Pora)和苹果轮纹(Botryosphaeria dothidea)有显著抑制作用。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,在本公开的技术构思范围内,可以对本公开的技术方案进行多种简单变型,这些简单变型均属于本公开的保护范围。
核苷酸序列表电子文件
<110>西北农林科技大学
<120>一种假结核耶尔森氏菌抗真菌蛋白、应用及分离纯化方法
<160>2
<210> 1
<211>255
<212> PRT
<213>YPK_D305氨基酸序列
<400> 1
MGRTAQLLLVSCLLALFMPPAMAEVSDKLQLSHKVYAHDYQAFWLWSGVN 50
PQPALQQANQVYLHQGEVVIRQRAAWFQKMGLPSSRLTLPAMWVTVRITT 100
LDVPDDILAILIDLPRRWAAAGNQVIGLQIDFDAGTYRLDDYAGFLRRVR 150
TKLDPNFALGVTGLLDWAKTGSIQQLNALPIDELVIQTYQGRSTVNQYSR 200
YLPALLQLRLPFKIGLVQHGEWDPQWEQYLAASPFYRGEVVFLLNHLRSE 250
PANGK 255
<210>2
<211>765
<212>DNA
<213>YPK_D305基因序列
<400>2
ATGGGCAGAACGGCTCAACTATTACTGGTAAGCTGCCTATTAGCGCTATT 50
TATGCCGCCCGCGATGGCAGAGGTTAGCGACAAACTGCAGTTGAGCCATA 100
AGGTGTATGCCCATGATTATCAGGCGTTTTGGTTGTGGTCAGGTGTGAAC 150
CCACAGCCCGCTTTACAGCAGGCGAATCAGGTTTATCTGCATCAGGGGGA 200
AGTGGTTATTCGCCAACGAGCTGCCTGGTTTCAGAAAATGGGGCTACCCA 250
GCTCTAGGCTGACACTGCCTGCGATGTGGGTCACGGTGCGCATCACCACG 300
TTGGATGTACCAGATGATATTTTGGCGATTCTGATTGATTTACCCCGGCG 350
GTGGGCCGCCGCTGGAAATCAGGTCATCGGGCTACAGATTGATTTTGATG 400
CAGGCACCTATCGTCTTGATGATTACGCTGGGTTCCTGCGCCGGGTGCGC 450
ACTAAGCTGGATCCAAATTTTGCACTTGGTGTAACCGGCTTATTGGACTG 500
GGCAAAAACCGGTAGCATCCAGCAGCTCAATGCGTTGCCGATTGATGAAC 550
TGGTGATACAAACCTATCAGGGGCGATCAACCGTTAATCAGTACTCACGT 600
TACTTACCCGCCTTGCTGCAATTACGTCTTCCCTTCAAGATTGGTTTGGT 650
TCAACACGGGGAGTGGGATCCGCAATGGGAACAGTATCTGGCTGCTTCCC 700
CTTTTTATCGGGGAGAAGTCGTCTTTTTGCTTAATCATCTTCGCTCTGAA 750
CCTGCTAACGGTAAA 765
Claims (5)
1.假结核耶尔森氏菌抗真菌蛋白用于制备抗真菌药物的应用;所述的假结核耶尔森氏菌抗真菌蛋白的氨基酸序列如SEQ ID NO:1所示;
或者,所述的假结核耶尔森氏菌抗真菌蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
2.假结核耶尔森氏菌抗真菌蛋白用于制备抗霉菌药物的应用,所述的假结核耶尔森氏菌抗真菌蛋白的氨基酸序列如SEQ ID NO:1所示;
或者,所述的假结核耶尔森氏菌抗真菌蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
3.假结核耶尔森氏菌抗真菌蛋白用于制备防腐剂的应用,所述的假结核耶尔森氏菌抗真菌蛋白的氨基酸序列如SEQ ID NO:1所示;
或者,所述的假结核耶尔森氏菌抗真菌蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
4.假结核耶尔森氏菌抗真菌蛋白用于制备水果保鲜剂的应用;所述的假结核耶尔森氏菌抗真菌蛋白的氨基酸序列如SEQ ID NO:1所示;
或者,所述的假结核耶尔森氏菌抗真菌蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
5.假结核耶尔森氏菌抗真菌蛋白用于制备蔬菜保鲜剂的应用,所述的假结核耶尔森氏菌抗真菌蛋白的氨基酸序列如SEQ ID NO:1所示;
或者,所述的假结核耶尔森氏菌抗真菌蛋白由如SEQ ID NO:2所示的核苷酸序列编码得到。
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