CN116162169A - 一种基于tlr4和md2的fret蛋白质传感器、应用、重组菌株重组质粒和构建方法 - Google Patents
一种基于tlr4和md2的fret蛋白质传感器、应用、重组菌株重组质粒和构建方法 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，涉及重组蛋白MD2‑TLR4‑mTurquoise2和重组蛋白MD2‑TLR4‑mNeonGreen构成的FRET蛋白质传感器及其应用，以及相关的重组菌株、重组质粒和构建方法。本发明使用人源Toll4样受体4(TLR4)和髓样分化蛋白2(MD2)融合基因分别与荧光共振能量转移(FRET)能量供体mTurquoise2和能量受体mNeonGreen基因融合，构建FRET的信号模块MD2‑TLR4‑mTurquoise2和MD2‑TLR4‑mNeonGreen。将融合基因克隆至酵母表达载体pPIC9k，构建表达载体pPIC9k‑MD2‑TLR4‑mTurquoise2和pPIC9k‑MD2‑TLR4‑mNeonGreen，转化至酵母菌GS115，表达重组蛋白MD2‑TLR4‑mTurquoise2和MD2‑TLR4‑mNeonGreen，镍柱纯化获得重组蛋白，并将其应用于环境样品中细菌内毒素Lipopolysaccharide(LPS)的FRET检测。
Description
技术领域
本发明属于基因工程技术领域，涉及重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen构成的FRET蛋白质传感器及其应用，以及相关的重组菌株、重组质粒和构建方法。
背景技术
内毒素是革兰氏阴性菌细胞壁的产物，是常见的致热源，属于强免疫刺激因子，致使机体产生一系列炎症反应，对人体危害巨大。内毒素主要检测方法为鲎试验法(国家药典规定方法)，检测限为0.05EU/mL，原理为海洋节肢动物“鲎”的血液变形细胞，能被细菌内毒素激活而凝固，通过对凝胶的分析以达到对内毒素的定性定量检测。但是鲎试验法存在假阳型结果，所需鲎试剂需要捕杀国家保护动物鲎，与环境保护相悖。所以需要设计更合理的检测环境中细菌内毒素的方法。
荧光共振能量转移(FRET)：是一种光物理现象，通过偶极-偶极相互作用，将供体荧光团的共振能量以非辐射方式转移到受体上。当供体和受体之间距离范围为1nm-10nm时，激发态供体的偶极-偶极相互作用刺激受体进入激发态，同时供体弛豫到非荧光基态，产生能量转移。
人源Toll4样受体4(TLR4)与髓样分化蛋白2(MD2)是细胞识别细菌内毒素的重要组成，与内毒素脂多糖LPS结合时，它们在细胞表面形成复合物，其中LPS的6条脂质链中有5条与MD2的疏水囊结合，而MD2表面的剩余脂质链与TLR4结合，LPS磷酸基团也与TLR4带正电荷的残基相互作用，LPS结合特异性引发MD2-TLR4形成同型二聚体复合物。
发明内容
本发明使用人源Toll4样受体4(TLR4)和髓样分化蛋白2(MD2)融合基因分别与荧光共振能量转移(FRET)能量供体mTurquoise2和能量受体mNeonGreen基因融合，构建FRET的信号模块MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen。将融合基因克隆至酵母表达载体pPIC9k，构建表达载体pPIC9k-MD2-TLR4-mTurquoise2和pPIC9k-MD2-TLR4-mNeonGreen，转化至酵母菌GS115，表达重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen，镍柱纯化获得重组蛋白，并将其应用于环境水样细菌内毒素Lipopolysaccharide(LPS)的FRET检测。
具体采用的技术方案为：一种由重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen构成的FRET蛋白质传感器，重组蛋白MD2-TLR4-mTurquoise2氨基酸序列如SEQ ID NO:1所示，重组蛋白MD2-TLR4-mNeonGreen氨基酸序列如SEQ ID NO:2所示。
所述的FRET蛋白质传感器的用途为检测环境水样中的细菌内毒素。
一种用于表达所述重组蛋白MD2-TLR4-mTurquoise2的重组酵母菌GS115/pPIC9k-MD2-TLR4-mTurquoise2和一种用于表达所述重组蛋白MD2-TLR4-mNeonGreen的重组酵母菌GS115/pPIC9k-MD2-TLR4-mNeonGreen。
而且，构建用于表达重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen的重组菌株的方法为：用SalⅠ单酶切构建的重组载体pPIC9k-md2-toll4-mTurquoise2和pPIC9k-md2-toll4-mNeonGreen，电转化进入毕氏酵母GS115，使用抗遗传霉素G418筛选高拷贝转化子，对高拷贝转化子基因组DNA进行PCR鉴定。
而且，诱导重组菌株表达重组蛋白的方法为：通过重组毕氏酵母菌表达重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen，发酵液采用镍柱层析，SDS-PAGE验证重组蛋白大小，并考察重组蛋白光谱特征。
而且，重组载体pPIC9k-md2-toll4-mTurquoise2和pPIC9k-md2-toll4-mNeonGreen的构建方法为：
步骤1.使用人源Toll4样受体4和髓样分化蛋白2通过重叠PCR技术得到融合基因md2-toll4，再分别与荧光共振能量转移能量供体mTurquoise2和能量受体mNeonGreen分别通过重叠PCR方法融合，获得融合基因md2-toll4-mTurquoise2和md2-toll4-mNeonGreen；
步骤2.对步骤1所得md2-toll4-mTurquoise2和md2-toll4-mNeonGreen进行PCR扩增；
步骤3.用EcoRⅠ和NotⅠ双酶切步骤2中的PCR产物md2-toll4-mTurquoise2、md2-toll4-mNeonGreen和毕氏酵母表达载体pPIC9k，通过清洁回收对应条带，将回收产物md2-toll4-mTurquoise2和pPIC9k以及md2-toll4-mNeonGreen和pPIC9k，按照按等摩尔比混合用T4 DNA连接酶，4℃连接过夜，并热击转化至大肠杆菌TOP10，挑取单克隆，PCR扩增鉴定、用EcoRⅠ和NotⅠ双酶切鉴定及测序验证，得到重组载体pPIC9k-md2-toll4-mTurquoise2和重组载体pPIC9k-md2-toll4-mNeonGreen。
本发明的有益效果在于：1、提供了一组能迅速检测环境样品(特别是环境水样)中的细菌内毒素的重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen；2、提供了用于表达重组蛋白MD2-TLR4-mTurquoise2的重组酵母菌GS115/pPIC9k-MD2-TLR4-mTurquoise2和用于表达重组蛋白MD2-TLR4-mNeonGreen的重组酵母菌GS115/pPIC9k-MD2-TLR4-mNeonGreen；3、提供了重组酵母菌的构建方法；4、提供了诱导重组酵母菌表达对应重组蛋白的方法；5、提供了重组载体pPIC9k-md2-toll4-mTurquoise2和重组载体pPIC9k-md2-toll4-mNeonGreen的构建方法。
附图说明
图1：A)目的片段的PCR扩增结果，1，2：md2(426bp)；3，4：toll4(1831bp)；5，6：toll4-mTurquoise2(2548bp)；7，8：toll4-mNeonGreen(2539bp)；9，10：md2-toll4-mTurquoise2(2974bp)；11，12：md2-toll4-mNeonGreen(2965bp)；B)目的片段双酶切鉴定结果，M：DL15000 bp Marker；1：pPIC9k质粒双酶切产物(9500bp)；2：md2-toll4-mTurquoise2双酶切产物(3000bp)；3：md2-toll4-mNeonGreen双酶切产物(3000bp)。
图2：A)PCR鉴定阳性质粒；1，2：pPIC9k-md2-toll4-mTurquoise2 PCR鉴定(3000bp)；3，4：pPIC9k-md2-toll4-mNeonGreen PCR鉴定(3000bp)；B)双酶切鉴定阳性质粒；1，2：pPIC9k-md2-toll4-mTurquoise2双酶切鉴定(3000bp)；3，4：pPIC9k-toll4-mNeonGreen双酶切鉴定(3000bp)。
图3：PCR鉴定重组酵母菌株；1，2：AOX1引物扩增GS115/md2-toll4-mTurquoise2(2200bp和3500bp)；3：阳性对照AOX1引物扩增pPIC9k-md2-toll4-mTurquoise2(3500bp)；4，5：AOX1引物鉴定GS115/md2-toll4-mNeonGreen(2200bp和3500bp)；6：阳性对照AOX1引物扩增md2-toll4-mNeonGreen(3500bp)；7，8：特异性引物扩增GS115/md2-toll4-mTurquoise2(3000bp)；9：阳性对照特异性引物扩增pPIC9k-md2-toll4-mTurquoise2(3000bp)；10，11：特异性引物扩增GS115/md2-toll4-mNeonGreen(3000bp)；12：阳性对照特异性引物扩增pPIC9k-md2-toll4-mNeonGreen(3000bp)。
图4：重组酵母表达产物SDS-PAGE分析，M：蛋白质Marker；1：MD2-TLR4-mTurquoise2(107.6kDa)；2：MD2-TLR4-mNeonGreen(107.8kDa)。
图5：A)重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen紫外-可见吸收光谱；B)重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen的荧光发射光谱。
图6：A)重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen不同浓度比下FRET响应归一化图；B)重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen浓度比例对FRET信号的影响。
图7：A)不同浓度LPS下基于MD2-TLR4的FRET生物传感器荧光光谱归一化图；a-l：LPS浓度0ng/mL，0.1ng/mL，1ng/mL，10ng/mL，20ng/mL，30ng/mL，40ng/mL，50ng/mL，80ng/mL，100ng/mL，200ng/mL，300ng/mL；B)基于MD2-TLR4的FRET生物传感器对不同浓度LPS响应。
图8：A)基于MD2-TLR4的FRET传感器对不同菌体的选择性；B)基于MD2-TLR4的FRET传感器对不同干扰成分的FRET响应。
图9：基于MD2-TLR4的FRET传感器对环境水样品的FRET响应。
具体实施方式
下面结合附图和实施例对本发明进行详细具体说明，本发明的内容不局限于以下实施例。
本发明的原理为：
将TLR4(toll4)、MD2(md2)基因分别和FRET供体绿色荧光蛋白(mNeonGreen)、受体青色荧光蛋白(mTurquoise22)基因融合，构建重组载体pPIC9k-md2-toll4-mTurquoise22和pPIC9k-md2-toll4-mNeonGreen，电转化进毕氏酵母GS115，进行重组蛋白表达和纯化及对LPS的荧光检测。
重组质粒构建如下：通过NCBI获得toll4、md2、mNeonGreen、mTurquoise22基因序列，由擎科生物公司合成，通过对应特异性引物扩增出对应片段，使用重叠PCR，将toll4、md2融合，得到md2-toll4，再将md2-toll4分别与mTurquoise22和mNeonGreen融合得到md2-toll4-mTurquoise2和md2-toll4-mNeonGreen。使用EcoRⅠ和NotⅠ双酶切上述融合基因和pPIC9k质粒，使用T4 DNA连接酶连接，转化进入大肠杆菌TOP10，通过特异性引物菌落PCR鉴定和双酶切鉴定后，测序验证。
重组菌构建如下：将上述重组载体pPIC9k-md2-toll4-mTurquoise2和pPIC9k-md2-toll4-mNeonGreen使用Sal I单酶切线性化后电转化酵母GS115，通过遗传霉素筛选，对阳性重组子提基因组DNA进行PCR验证。
将阳性重组子进行诱导表达，通过盐析和镍柱纯化得到目的蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen，对内毒素样品进行检测。
以下是本发明的具体实施例：
1.md2-toll4-mTurquoise2和md2-toll4-mNeonGreen的PCR扩增
扩增体系为基因模板、正反向引物、无菌水、KOD-plus体系。
98℃，预变性5min；98℃，变性30s；对应Tm值，退火30s；72℃，延伸1min/1000bp；从变形到延伸35个循环；68℃，5min。Tm：mTurquoise2(71℃)，mNeonGreen(71℃)，toll4(71℃)，md2(61℃)，toll4-mTurquoise2(71℃)，toll4-mNeonGreen(71℃)，md2-toll4-mTurquoise2(71℃)，md2-toll4-mNeonGreen(71℃)。
PCR产物用1％琼脂糖凝胶电泳分析，扩增目的条带使用DNA凝胶回收试剂盒回收。(图1)
2.重组载体的构建
使用EcoRⅠ和NotⅠ双酶切md2-toll4-mTurquoise2、md2-toll4-mNeonGreen和pPIC9k质粒，使用T4 DNA连接酶连接md2-toll4-mTurquoise2和pPIC9k、md2-toll4-mNeonGreen和pPIC9k，得pPIC9k-MD2-TLR4-mTurquoise2和pPIC9k-MD2-TLR4-mNeonGreen，转化进入大肠杆菌TOP10，通过特异性引物验证和双酶切验证载体构建，送测序验证。(图2)
3.重组酵母菌的构建
pPIC9k-MD2-TLR4-mTurquoise2和pPIC9k-MD2-TLR4-mNeonGreen使用SalⅠ酶切线性化，通过电转化进入毕氏酵母GS115，使用G418筛选高拷贝转化子，通用引物5’AOX验证阳性重组子。PCR反应程序为：98℃，预变性5min；98℃，变性30s；55℃，退火30s；72℃，延伸2min；35个循环；68℃，5min。PCR产物用1％琼脂糖凝胶电泳分析。
(图3)
4.重组蛋白的表达和纯化
GS115/md2-toll4-mTurquoise2和GS115/md2-toll4-mNeonGreen阳性菌接种于BMGY培养基，30℃，250rpm培养18-20h，至0D600＝2-6转接于BMMY培养基表达96h，每24h加入甲醇诱导表达。取培养基上清镍柱层析获得供体蛋白MD2-TLR4-mTurquoise2和受体蛋白MD2-TLR4-mNeonGreen，SDS-PAGE验证目的蛋白大小，使用荧光光谱仪和紫外分光光度计分析供体蛋白和受体蛋白的光谱特征。(图4和图5)
5.LPS的FRET检测
固定LPS浓度为0.5μg/mL定值，MD2-TLR4-mTurquoise2终浓度为0.02M，为固定值，逐渐增加能量受体MD2-TLR4-mNeonGreen的浓度比例。供体受体浓度比例为1：1，1：5，1：10，1：15，1：20，1：25，检测组加入0.5μg/mL LPS，其余用PBS补齐至总体系200μL。(图6)
在能量供体和受体最优浓度比例下，对不同浓度的LPS进行检测，检测浓度为0.1ng/mL，1ng/mL，10ng/mL，20ng/mL，30ng/mL，40ng/mL，50ng/mL，80ng/mL，100ng/mL，200ng/mL，300ng/mL。(图7)
反应体系置于冰上孵育结合30min，使用日立F-2500荧光分光光度计检测FRET信号。激发光波长：434nm；检测波长测定范围：460-600nm；扫描速度：300nm/min；激发和发射光缝宽度：20nm；PMT：700V。
6.干扰实验
在优化的FRET体系能量供体和受体浓度比例下，实验组不加入LPS，以检测体系中的干扰物质的FRET响应，对照组成分为对应干扰物质所在体系加入LPS。重复实验三次。(图8)
7.环境水样品的LPS检测
选取南湖水和自来水作为检测对象，反应体系为FRET供体受体浓度最优比例1：20，重复实验三次结构如图9所示。本应用还能适用于食品、土壤等情况，而对于固态的环境样品，先加水稀释后再进行检测即可。
Claims (6)
1.一种由重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen构成的FRET蛋白质传感器，重组蛋白MD2-TLR4-mTurquoise2氨基酸序列如SEQ ID NO:1所示，重组蛋白MD2-TLR4-mNeonGreen氨基酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的FRET蛋白质传感器的用途为检测环境样品中的细菌内毒素。
3.一种用于表达权利要求1所述重组蛋白MD2-TLR4-mTurquoise2的重组酵母菌GS115/pPIC9k-MD2-TLR4-mTurquoise2和一种用于表达权利要求1所述重组蛋白MD2-TLR4-mNeonGreen的重组酵母菌GS115/pPIC9k-MD2-TLR4-mNeonGreen。
4.根据权利要求1所述的FRET蛋白质传感器，其特征在于构建用于表达重组蛋白MD2-TLR4-mTurquoise2和重组蛋白MD2-TLR4-mNeonGreen的重组菌株的方法为：用SalⅠ单酶切构建的重组载体pPIC9k-md2-toll4-mTurquoise2和pPIC9k-md2-toll4-mNeonGreen，电转化进入毕氏酵母GS115，使用抗遗传霉素G418筛选高拷贝转化子，对高拷贝转化子基因组DNA进行PCR鉴定。
5.根据权利要求4所述的FRET蛋白质传感器，其特征在于诱导重组菌株表达重组蛋白的方法为：通过重组毕氏酵母菌表达重组蛋白MD2-TLR4-mTurquoise2和MD2-TLR4-mNeonGreen，发酵液采用镍柱层析，SDS-PAGE验证重组蛋白大小，并考察重组蛋白光谱特征。
6.根据权利要求4所述的FRET蛋白质传感器，其特征在于，重组载体pPIC9k-md2-toll4-mTurquoise2和pPIC9k-md2-toll4-mNeonGreen的构建方法为：
步骤1.使用人源Toll4样受体4和髓样分化蛋白2通过重叠PCR技术得到融合基因md2-toll4，再分别与荧光共振能量转移能量供体mTurquoise2和能量受体mNeonGreen分别通过重叠PCR方法融合，获得融合基因md2-toll4-mTurquoise2和md2-toll4-mNeonGreen；
步骤2.对步骤1所得md2-toll4-mTurquoise2和md2-toll4-mNeonGreen进行PCR扩增；
步骤3.用EcoRⅠ和NotⅠ双酶切步骤2中的PCR产物md2-toll4-mTurquoise2、md2-toll4-mNeonGreen和毕氏酵母表达载体pPIC9k，通过清洁回收对应条带，将回收产物md2-toll4-mTurquoise2和pPIC9k以及md2-toll4-mNeonGreen和pPIC9k，按照按等摩尔比混合用T4DNA连接酶，4℃连接过夜，并热击转化至大肠杆菌TOP10，挑取单克隆，PCR扩增鉴定、用EcoRⅠ和NotⅠ双酶切鉴定及测序验证，得到重组载体pPIC9k-md2-toll4-mTurquoise2和重组载体pPIC9k-md2-toll4-mNeonGreen。
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