CN114751990B - 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用 - Google Patents

酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用 Download PDF

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CN114751990B
CN114751990B CN202210338602.2A CN202210338602A CN114751990B CN 114751990 B CN114751990 B CN 114751990B CN 202210338602 A CN202210338602 A CN 202210338602A CN 114751990 B CN114751990 B CN 114751990B
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俞汉青
李宇昇
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Abstract

本发明涉及生物技术领域,尤其涉及酰胺高丝氨酸内酯受体‑增强绿色荧光融合蛋白及其制备方法与应用。本发明通过直接合成及PCR方法,使用柔性linker连接信号分子受体蛋白与增强的绿色荧光蛋白,此融合蛋白可以直接与信号分子的抑制物相作用,构象改变后则其后的增强绿色荧光蛋白可以正确折叠并产生信号;反之受体蛋白则与增强绿色荧光蛋白相互干扰,不产生荧光信号。通过这一方法,可以通过明显的亮暗反应判断待筛选物的抑制能力,并且本方法直接利用融合蛋白对抑制物的结合作用,不仅可以免除酰胺高丝氨酸内酯纯品的加入,而且避免了非特异性结合造成的假阳性。

Description

酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方 法与应用
技术领域
本发明涉及生物技术领域,尤其涉及酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用。
背景技术
信号分子是一种由细菌自身合成、分泌并累积于生长环境中的一类小分子,其浓度与种群密度关联,因此细菌可以通过受体蛋白感测信号分子浓度,进而根据种群密度对基因的表达进行调节。前述过程被称为群感效应,而信号分子在群感效应过程中处于关键地位。酰胺高丝氨酸内酯是一类广泛存在于革兰氏阴性菌中的信号分子,多种致病菌或条件致病菌具有通过酰胺高丝氨酸介导的群感效应获得竞争优势,或强化自身对宿主的侵染能力。
以酰胺高丝氨酸内酯介导的群感效应为例,信号分子抑制物是一种能够替代酰胺高丝氨酸内酯与细菌受体蛋白结合的物质,但与其结合后,受体蛋白却失去原有功能,群感效应由此被破坏。由于破坏群感效应是一种不易引起细菌抗药性的方法,因此以信号分子为目标的微生物防控技术具有良好的应用前景。近年来,通过信号分子抑制物,尤其是酰胺高丝氨酸内酯的抑制物,控制致病菌感染成为一个研究热点,与此同时,筛选信号分子抑制物的尝试也引起广泛关注。
传统的筛选酰胺高丝氨酸内酯抑制物筛选方法基于竞争反应,即在加入信号分子抑制物待筛选物的同时,仍需提供酰胺高丝氨酸内酯纯品,通过二者对细菌受体蛋白的竞争性结合,造成检测菌产生信号的减弱,完成筛选。传统方法的缺点在于需要酰胺高丝氨酸内酯纯品,大大提高了检测成本;此外通过观测信号减弱,显著性不强,且决定于酰胺高丝氨酸内酯与待筛选物的比例;最后,如果是具有全细胞毒性的物质,甚至可能引起假阳性的现象。
发明内容
有鉴于此,本发明要解决的技术问题在于提供酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白(ainR-eGFP)及其制备方法与应用,利用该ainR-eGFP重组蛋白进行信号分子抑制物的筛选,相比于传统方法具有成本低、操作简便、不需要信号分子标准品、避免具有全细胞毒性的物质产生的假阳性、信号响应明显的优势。
本发明提供的融合蛋白,包括酰胺高丝氨酸内酯受体、linker和增强绿色荧光蛋白;其中,所述酰胺高丝氨酸内酯受体来自费氏弧菌。
本发明所述的融合蛋白包含ainR、eGFP和连接两个蛋白的linker序列。一些实施例中,本发明提供的融合蛋白的结构N端至C端依次为ainR-linker-eGFP。
前期试验中尝试采用其它菌种来源的,具有类似功能的蛋白序列,但所得融合蛋白均不能在体外对信号分子抑制物进行良好的检测。并且,融合蛋白的连接顺序对功能影响显著,ainR-linker-eGFP能够实现良好的检测效果,而eGFP-linker-ainR则不具有类似功能。本发明构建的融合蛋白中,ainR来自费氏弧菌,其氨基酸序列如SEQ ID NO:1所示,编码该氨基酸的核酸序列如SEQ ID NO:5所示。
一些实施例中,所述增强绿色荧光蛋白的氨基酸序列如SEQ ID NO:2所示。编码SEQ ID NO:2所示氨基酸序列的核酸序列如SEQ ID NO:6所示。
Linker序列的添加可以保证融合蛋白中的两种蛋白形成正确的空间结构,更好的发挥生物学活性。通常,linker的选择与融合蛋白中两种成分的接头序列密切相关,本发明在实验中,对linker序列的选择进行了研究。结果表明,长度大于15个氨基酸残基的柔性或刚性linker序列,例如(GGGGS)3和(EAAAK)3,均可达到较好效果。但直接连接的方式或采用其它较短linker序列,例如(GGGGS)或(EAAAK),则无法达到预期效果。本发明中,所述linker为刚性linker或柔性linker,其至少由15个氨基酸残基组成。一些实施例中,所述linker为(GGGGS)3,编码该linker序列的核酸为ggcggcggcggcagcggcggcggcggcagcggcggcggcggcagc(SEQ ID NO:12)。
本发明还提供了编码所述融合蛋白的核酸。
本发明所述的融合蛋白包括包含ainR、eGFP和连接两个蛋白的linker序列,一些实施例中,编码ainR的核酸序列如SEQ ID NO:5所示,编码eGFP的核酸序列如SEQ ID NO:6所示。
一些实施例中,所述融合蛋白的氨基酸序列如SEQ ID NO:3所示。编码SEQ ID NO:3所示氨基酸序列的融合蛋白的核酸的序列如SEQ ID NO:4所示。
本发明还提供了含有该融合蛋白的转录单元。
所述转录单元是由启动子开始,以终止子结束的DNA序列。在启动子和终止子之间,包括编码本发明所述融合蛋白的核酸。所述转录单元中还可以包括终止子和/或启动子的增强子。在本发明的一些具体实施例中,所述转录单元的启动子为T7启动子,终止子为T7终止子。
本发明还提供了含有所述核酸的表达载体。
本发明所述的表达载体是指具有能够在适当的宿主中表达靶基因的DNA分子。本发明对所述的质粒载体的骨架没有限制,只要其可以在宿主细胞中表达都在本发明的保护范围内。一些实施例中,所述表达载体的骨架载体采用的是pET系列载体。具体的,采用pET28a作为骨架载体。
本本发明还提供了转化或转染所述表达载体的重组宿主。所述转染(transfection)是真核细胞在一定条件下主动或被动导入外源DNA片段而获得新的表型的过程。所述转化(transformation)是某一基因型的细胞从周围介质中吸收来自另一基因型的DNA而使它的基因型和表现型发生相应变化的现象。本发明所述重组宿主的构建方法采用电转化法,所述重组宿主可为微生物、植物细胞或动物细胞。优选的,所述重组宿主为微生物,更优选的重组宿主为原核生物。一些实施例中,所述重组宿主的宿主为大肠杆菌。一些具体实施例中,所述大肠杆菌为BL21(DE3)。
本发明所述融合蛋白的制备方法,包括培养所述重组宿主,诱导所述融合蛋白的表达。
具体的培养、诱导方法包括:37℃以LB培养基培养重组大肠杆菌至OD600为0.3,加入诱导剂IPTG;培养至OD600值为1.0获得含有融合蛋白的培养物。
本发明所述融合蛋白存在于菌体中,其可直接作为检测试剂使用。
本发明还提供了一种酰胺高丝氨酸内酯抑制物的检测试剂,其包括如下I)~IV中的任一项:
I)、所述融合蛋白;
II)、所述制备方法制得的融合蛋白;
III)、所述重组宿主;
IV)、所述重组宿主的培养产物。
本发明还提供了一种酰胺高丝氨酸内酯抑制物的检测方法,其包括:使如下I)~IV)中的任一项与待测物接触,根据荧光情况判断待测物是否具有抑制作用:
I)、所述融合蛋白;
II)、所述制备方法制得的融合蛋白;
III)、所述重组宿主;
IV)、所述重组宿主的培养产物。
所述荧光为488nm激发光源处的荧光。
一些实施例中,所述检测方法为,将待测物与重组菌株的发酵液接触,继续培养6h,然后于488nm处检测荧光,若观测到菌体发出明显的绿色荧光,则代表所测物质具有潜在的信号分子抑制效果。
本发明通过直接合成及PCR方法,使用柔性linker连接信号分子受体蛋白与增强的绿色荧光蛋白,此融合蛋白可以直接与信号分子的抑制物相作用,构象改变后则其后的增强绿色荧光蛋白可以正确折叠并产生信号;反之受体蛋白则与增强绿色荧光蛋白相互干扰,不产生荧光信号。通过这一方法,可以通过明显的亮暗反应判断待筛选物的抑制能力,并且本方法直接利用融合蛋白对抑制物的结合作用,不仅可以免除酰胺高丝氨酸内酯纯品的加入,而且避免了非特异性结合造成的假阳性。
附图说明
图1示表达融合蛋白的重组大肠杆菌对3,4-二氯-2(5H)-呋喃酮的荧光响应;
图2示表达融合蛋白的重组大肠杆菌对不同浓度呋喃酮的荧光响应随时间的变化;
图3示对代表其它几种代表性酰胺高丝氨酸内酯信号分子抑制物的响应,包括;呋喃酮、4-羟基-2(5H)-呋喃酮和3,4-二溴-2(5H)-呋喃酮;
图4示本方法6h时对不同呋喃酮及衍生物的荧光响应值;
图5示实施例2的检测效果;
图6示对比例1的检测结果;
图7示对比例2的检测结果。
具体实施方式
本发明提供了提供酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。本发明所涉及的信号分子受体蛋白、linker、荧光蛋白的基因序列来自Genbank,pET28a,大肠杆菌BL21(DE3)为市售。
表1.基因及对应氨基酸、核苷酸序列
下面结合实施例,进一步阐述本发明:
实施例1:酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白的构建
1、融合片段的获得
1.1化学合成表1所示的受体蛋白ainR核苷酸序列;
化学合成表1所示的增强绿色荧光蛋白eGFP核苷酸序列;
选择受体蛋白与荧光蛋白之间连接序列,本发明采用柔性序列(GGGGS)3,其核苷酸序列见表1;
1.2目标片段扩增
根据选择的连接序列与表达载体,设计扩增受体蛋白的正向与反向引物序列R-F与R-R,具体序列见表1,以合成的受体蛋白序列为模板,以R-F和R-R为引物,PCR扩增步骤及参数为:94℃预变性5min;之后94℃变性30s;55℃退火30s;随后72℃延伸2min;32个循环后72℃保温10min。扩增获得片段A;产物进行核酸电泳验证。
根据选择的连接序列与表达载体,设计扩增增强绿色荧光蛋白的正向与反向引物序列F-F与F-R,具体序列见表1,以合成的增强绿色荧光蛋白序列为模板,以F-F和F-R为引物,扩增目标基因。PCR扩增步骤及参数为:94℃预变性5min;之后94℃变性30s;55℃退火30s;随后72℃延伸2min;32个循环后72℃保温10min。扩增获得片段B,产物进行核酸电泳验证。
1.5前述两个步骤中得到的PCR产物经过市售的PCR产物纯化试剂盒进行纯化。
2、重组质粒的构建
2.1于37℃,200rpm的条件下以恒温摇床过夜培养含pET28a质粒的大肠杆菌;
2.2利用质粒提取试剂盒对过夜培养的菌液进行质粒提取;
2.3提取的pET28a质粒使用Nco I与Xho I限制性内切酶进行双酶切;
2.4酶切产物利用市售PCR产物纯化试剂盒纯化;
2.5将纯化后的受体蛋白序列、增强荧光蛋白序列以及酶切后的pET28a质粒混合(10ng、10ng及100ng,反应体系为100μl),经多片段重组酶连接,获得表达融合蛋白的重组质粒;其中,编码融合蛋白cviR-linker-eGFP的核酸序列如SEQ ID NO:4所示。
3、表达融合蛋白的重组大肠杆菌构建:
3.1将重组质粒转化入100μl大肠杆菌BL21(DE3),并涂布于含有卡那霉素的LB琼脂平板上,于37℃恒温培养箱中过夜培养;
3.2从平板上挑取单克隆至含有5ml添加卡那霉素的LB液体培养基的透气管中,于37℃,200rpm条件下恒温摇床过夜培养;
3.3将过夜培养的菌液转移至超净台内,吸取10μl使用引物R-F与F-R进行PCR扩增用于序列验证;
3.4另吸取500μl菌液加入含有500μl浓度为50%的甘油管中,于零下80℃进行菌种保存。
实施例2利用重组大肠杆菌表达融合蛋白检测酰胺高丝氨酸内酯的抑制物:
接种重组大肠杆菌于5ml含有卡那霉素的LB液体培养基中,并于37℃,200rpm培养至OD600=0.3,加入诱导剂IPTG后,继续培养至OD600=1.0;
吸取重组大肠杆菌50μl,加入40μl新鲜LB,并加入待确定信号分子抑制物溶液10μl(此应用中采用呋喃酮及其三种衍生物,分别为3,4-二氯-2(5H)-呋喃酮、4-羟基-2(5H)-呋喃酮、3,4-二溴-2(5H)-呋喃酮,四者浓度均为20mg/L),于96孔板或1.5ml离心管中继续培养6h;
荧光检测:
吸取少量菌液涂布于载玻片,盖上盖玻片;
使用荧光显微镜的488nm激发光为光源,于100×油镜下观察。若观测到菌体发出明显的绿色荧光,则代表所测物质具有潜在的信号分子抑制效果;
对于荧光显微镜照片,可将其转换为灰度图片并统计灰度值,并可以灰度值的变化对信号分子抑制物的强度进行量化。如对已有广泛研究的呋喃酮及其衍生类物质,可根据荧光强弱判别其抑制能力相对强弱,其抑制强度强弱与本方法判别结果一致。
实施例3
采用刚性linker时,可以取得类似效果。构建融合蛋白时采用的方法和步骤与前述一致,但引物序列需要进行相应的调整:
R-R:ttttgccgccgcttctttagccgctgcttcttttgccgcagcttcctatagccatttatctacaac
F-F:gaagctgcggcaaaagaagcagcggctaaagaagcggcggcaaaaatggtgagcaagggcgcc
gagc
刚性linker序列为:
检测结果如图5所示,无抑制物(左)时菌体不产生荧光,而加入抑制物呋喃酮(右)则出现明显的荧光响应。
对比例1
当采用来自紫色素杆菌(Chromobacterium violaceum)的信号分子受体蛋白cviR进行融合蛋白的构建则无法获得检测能力。cviR序列为:
构建方法及步骤与前述一致,但扩增其序列的引物需要进行相应调整:
R-F:gaaggagatataccatgggcatggtgatctcgaaacccatc
R-R:ggatccgccgccacccgacccaccaccgcccgagccaccgccaccttcgttcgctacggtcga
其余引物均相同。
检测结果如图6所示,无抑制物(左)或加入抑制物呋喃酮(右)均出现较强荧光,无法有效检出信号分子抑制物。
对比例2
当采用反向构建,即eGFP-linker-aniR的次序合成融合蛋白时,则不具有检测效果,无论抑制物是否存在活性,均无荧光反应。构建时引物需调整为:
R-F:ggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcatgttaactactttg
R-R:ctatagccatttatctacaacttgctgcaattgtgcttta
F-F:atggtgagcaagggcgccgagctgttcaccggcatcgtgc
F-R:gctgccgccgccgccgctgccgccgccgccgctgccgccgccgcccttgtacagctcatc检测结果如图7所示,无抑制物(左)或加入抑制物呋喃酮(右)均无荧光,无法有效检出信号分子抑制物。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学技术大学
<120> 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用
<130> MP2007876
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 820
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Leu Thr Thr Leu Ser Lys Val Tyr Leu Leu Leu Thr Thr Ser Ala
1 5 10 15
Ile Ile Leu Leu Trp Val Gly Tyr Phe Val Arg Ser Leu Tyr Lys Glu
20 25 30
Arg Thr Lys Val Asn Pro Tyr Ile Tyr Ser Ser Tyr Ile Phe Tyr Ala
35 40 45
Leu Phe Ile Ile Leu Trp Ile Leu Ser Asn Ala Tyr Phe Gln Ser Pro
50 55 60
Leu Leu Thr Tyr Phe Asp Glu Ser Ala Ala Ile Phe Met Ala Leu Phe
65 70 75 80
Ala Asn Met Thr Ser Tyr Leu Ala Phe Ala Phe Ala Phe Leu Phe Ser
85 90 95
Cys Arg Leu Ala Ser Lys His Pro Asp Lys Arg Leu Ser Lys Trp Gln
100 105 110
Phe Gly Leu Thr Ser Ile Thr Thr Phe Ala Ala Leu Ile Val Asn Val
115 120 125
Ile Pro Asn Leu Thr Val Ile Gly Val Thr Ile Gln Ala Pro Ser Val
130 135 140
Phe Thr Ile Glu Phe Gly Pro Phe Ala Pro Leu Phe Phe Leu Asn Ala
145 150 155 160
Phe Leu Phe Val Ile Leu Thr Ser Ile Asn Phe Phe Lys Leu Arg Lys
165 170 175
Ser Asn Ile Lys Leu Asn Lys Glu Lys Ser Ile Tyr Leu Met Val Gly
180 185 190
Ile Phe Ile Tyr Met Ile Ser Thr Ile Ala Ser Gln Ile Ile Ile Pro
195 200 205
Val Ile Trp Ala Asp Phe Ser Tyr Thr Trp Val Pro Pro Ala Leu Ser
210 215 220
Val Thr Glu Ala Leu Leu Ile Gly Tyr Thr Leu Leu Tyr His Arg Leu
225 230 235 240
Tyr Ser Phe Lys Tyr Leu Leu Phe Trp Ser Leu Ser Tyr Ser Ile Asn
245 250 255
Leu Ile Leu Tyr Leu Ile Pro Ile Ile Ile Ile Tyr Asp Leu Thr Thr
260 265 270
Pro Ser Asp Leu Leu Tyr Ile Cys Ile Ile Glu Ile Ile Phe Thr Gly
275 280 285
Leu Phe Trp Asp Lys Thr Leu Lys Lys Thr Lys Lys Ile Ala Ser Ile
290 295 300
Ile Ile Tyr Lys Asp Lys Gln Thr Pro Val Glu Lys Ile Tyr Lys Ile
305 310 315 320
Ala Glu Glu Phe Lys Tyr Ser Ser Ser Asn Ala Ile Ile Lys Leu Ala
325 330 335
Ser Ile Leu Asn Thr Pro Lys Glu Glu Leu Leu Leu Ile Gly Lys Asn
340 345 350
Thr Asn Tyr Asn Ile Phe Ile Pro His Leu Asn Gln Ser His Ser Ala
355 360 365
Leu Val Lys Asp Glu Leu Asp Tyr Gln Ile His Tyr Ser Pro Lys Thr
370 375 380
Ala Asn Ala Glu Leu His Gln Val Gln Glu Lys Met Ser Glu Ser Lys
385 390 395 400
Thr Ala Leu Ile Leu Pro Ile Phe Gly Glu Asn Lys Leu Ile Ser His
405 410 415
Phe Leu Ile Ser Ala Asn Lys His Asp Asn Thr Thr Phe Ser Asn Glu
420 425 430
Glu Ile Ser Ala Ile Gln Trp Val Leu Thr Lys Val Gln Gly Tyr Ile
435 440 445
Glu Ser Glu Arg Lys Val Arg Gln Ser Gln Ala Leu Ala Asn Ser Ile
450 455 460
Ala His Glu Met Arg Asn Pro Leu Ser Gln Leu Gln Tyr His Phe Glu
465 470 475 480
Lys Ile Lys His His Tyr Gln Lys Asn Thr Glu His Glu Lys Gln Glu
485 490 495
Gln Leu Ile Lys Asn Glu Leu Asn Gln Gly Cys Leu Ala Ile Gln Lys
500 505 510
Gly Ala Gln Leu Ile Asp Ile Ile Leu Ser Glu Ala Lys Asn Thr Ala
515 520 525
Ile Ser Asp Asp Leu Phe His His His Ser Ile Ser Leu Leu Thr Gln
530 535 540
Gln Ile Ile Asp Glu Tyr Val Phe Asp Ser Glu Glu Met Lys Gln Lys
545 550 555 560
Ile Thr Leu Asp Leu Glu Asp Asp Phe Ile Val Asn Ile Asn Asp Thr
565 570 575
Leu Tyr Gly Phe Ile Leu Phe Asn Leu Leu Arg Asn Ala Thr Tyr Tyr
580 585 590
Phe Asp Glu Tyr Asn Ser Ser Ile Ser Ile Arg Leu Val Lys Gly Phe
595 600 605
Ala Thr Asn Lys Leu Ile Phe Arg Asp Thr Gly Pro Gly Ile Asp Ser
610 615 620
His Ile Leu Pro Asn Ile Phe Asp Asp Phe Phe Thr His Asn Lys Glu
625 630 635 640
Gly Gly Ser Gly Leu Gly Leu Ser Tyr Cys Leu Arg Val Met His Ala
645 650 655
Phe Glu Gly Asn Ile Ala Cys Tyr Ser Thr Lys Gly Glu Phe Thr Glu
660 665 670
Phe Val Leu Ser Phe Pro His Ile Glu Gly Asp Ile Asn Ala Leu Asn
675 680 685
Ser His Lys Ser Asn Thr Pro Pro Leu Ile Asn Lys Lys Asp Asn Ser
690 695 700
Leu Lys Thr Val Leu Ile Val Asp Asp Lys Lys Val Gln Arg Met Leu
705 710 715 720
Ile His Thr Phe Ile Asn Lys Asp Asn Leu Thr Leu Leu Gln Ala Glu
725 730 735
Asn Gly Glu Glu Ala Val Glu Ile Ala Thr Asn Asn Lys Leu Asp Leu
740 745 750
Ile Phe Met Asp Ser Arg Met Pro Val Met Asn Gly Ile Asp Ala Ala
755 760 765
Lys Lys Ile Lys Ile Ile Tyr Pro Asn Leu Pro Ile Ile Ala Leu Thr
770 775 780
Gly Glu Ser Ser His Glu Glu Ile Ser Ala Ile Thr Gln Val Met Asp
785 790 795 800
Gly Tyr Leu Thr Lys Pro Val Ser Lys Ala Gln Leu Gln Gln Val Val
805 810 815
Asp Lys Trp Leu
820
<210> 2
<211> 239
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 3
<211> 1074
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Leu Thr Thr Leu Ser Lys Val Tyr Leu Leu Leu Thr Thr Ser Ala
1 5 10 15
Ile Ile Leu Leu Trp Val Gly Tyr Phe Val Arg Ser Leu Tyr Lys Glu
20 25 30
Arg Thr Lys Val Asn Pro Tyr Ile Tyr Ser Ser Tyr Ile Phe Tyr Ala
35 40 45
Leu Phe Ile Ile Leu Trp Ile Leu Ser Asn Ala Tyr Phe Gln Ser Pro
50 55 60
Leu Leu Thr Tyr Phe Asp Glu Ser Ala Ala Ile Phe Met Ala Leu Phe
65 70 75 80
Ala Asn Met Thr Ser Tyr Leu Ala Phe Ala Phe Ala Phe Leu Phe Ser
85 90 95
Cys Arg Leu Ala Ser Lys His Pro Asp Lys Arg Leu Ser Lys Trp Gln
100 105 110
Phe Gly Leu Thr Ser Ile Thr Thr Phe Ala Ala Leu Ile Val Asn Val
115 120 125
Ile Pro Asn Leu Thr Val Ile Gly Val Thr Ile Gln Ala Pro Ser Val
130 135 140
Phe Thr Ile Glu Phe Gly Pro Phe Ala Pro Leu Phe Phe Leu Asn Ala
145 150 155 160
Phe Leu Phe Val Ile Leu Thr Ser Ile Asn Phe Phe Lys Leu Arg Lys
165 170 175
Ser Asn Ile Lys Leu Asn Lys Glu Lys Ser Ile Tyr Leu Met Val Gly
180 185 190
Ile Phe Ile Tyr Met Ile Ser Thr Ile Ala Ser Gln Ile Ile Ile Pro
195 200 205
Val Ile Trp Ala Asp Phe Ser Tyr Thr Trp Val Pro Pro Ala Leu Ser
210 215 220
Val Thr Glu Ala Leu Leu Ile Gly Tyr Thr Leu Leu Tyr His Arg Leu
225 230 235 240
Tyr Ser Phe Lys Tyr Leu Leu Phe Trp Ser Leu Ser Tyr Ser Ile Asn
245 250 255
Leu Ile Leu Tyr Leu Ile Pro Ile Ile Ile Ile Tyr Asp Leu Thr Thr
260 265 270
Pro Ser Asp Leu Leu Tyr Ile Cys Ile Ile Glu Ile Ile Phe Thr Gly
275 280 285
Leu Phe Trp Asp Lys Thr Leu Lys Lys Thr Lys Lys Ile Ala Ser Ile
290 295 300
Ile Ile Tyr Lys Asp Lys Gln Thr Pro Val Glu Lys Ile Tyr Lys Ile
305 310 315 320
Ala Glu Glu Phe Lys Tyr Ser Ser Ser Asn Ala Ile Ile Lys Leu Ala
325 330 335
Ser Ile Leu Asn Thr Pro Lys Glu Glu Leu Leu Leu Ile Gly Lys Asn
340 345 350
Thr Asn Tyr Asn Ile Phe Ile Pro His Leu Asn Gln Ser His Ser Ala
355 360 365
Leu Val Lys Asp Glu Leu Asp Tyr Gln Ile His Tyr Ser Pro Lys Thr
370 375 380
Ala Asn Ala Glu Leu His Gln Val Gln Glu Lys Met Ser Glu Ser Lys
385 390 395 400
Thr Ala Leu Ile Leu Pro Ile Phe Gly Glu Asn Lys Leu Ile Ser His
405 410 415
Phe Leu Ile Ser Ala Asn Lys His Asp Asn Thr Thr Phe Ser Asn Glu
420 425 430
Glu Ile Ser Ala Ile Gln Trp Val Leu Thr Lys Val Gln Gly Tyr Ile
435 440 445
Glu Ser Glu Arg Lys Val Arg Gln Ser Gln Ala Leu Ala Asn Ser Ile
450 455 460
Ala His Glu Met Arg Asn Pro Leu Ser Gln Leu Gln Tyr His Phe Glu
465 470 475 480
Lys Ile Lys His His Tyr Gln Lys Asn Thr Glu His Glu Lys Gln Glu
485 490 495
Gln Leu Ile Lys Asn Glu Leu Asn Gln Gly Cys Leu Ala Ile Gln Lys
500 505 510
Gly Ala Gln Leu Ile Asp Ile Ile Leu Ser Glu Ala Lys Asn Thr Ala
515 520 525
Ile Ser Asp Asp Leu Phe His His His Ser Ile Ser Leu Leu Thr Gln
530 535 540
Gln Ile Ile Asp Glu Tyr Val Phe Asp Ser Glu Glu Met Lys Gln Lys
545 550 555 560
Ile Thr Leu Asp Leu Glu Asp Asp Phe Ile Val Asn Ile Asn Asp Thr
565 570 575
Leu Tyr Gly Phe Ile Leu Phe Asn Leu Leu Arg Asn Ala Thr Tyr Tyr
580 585 590
Phe Asp Glu Tyr Asn Ser Ser Ile Ser Ile Arg Leu Val Lys Gly Phe
595 600 605
Ala Thr Asn Lys Leu Ile Phe Arg Asp Thr Gly Pro Gly Ile Asp Ser
610 615 620
His Ile Leu Pro Asn Ile Phe Asp Asp Phe Phe Thr His Asn Lys Glu
625 630 635 640
Gly Gly Ser Gly Leu Gly Leu Ser Tyr Cys Leu Arg Val Met His Ala
645 650 655
Phe Glu Gly Asn Ile Ala Cys Tyr Ser Thr Lys Gly Glu Phe Thr Glu
660 665 670
Phe Val Leu Ser Phe Pro His Ile Glu Gly Asp Ile Asn Ala Leu Asn
675 680 685
Ser His Lys Ser Asn Thr Pro Pro Leu Ile Asn Lys Lys Asp Asn Ser
690 695 700
Leu Lys Thr Val Leu Ile Val Asp Asp Lys Lys Val Gln Arg Met Leu
705 710 715 720
Ile His Thr Phe Ile Asn Lys Asp Asn Leu Thr Leu Leu Gln Ala Glu
725 730 735
Asn Gly Glu Glu Ala Val Glu Ile Ala Thr Asn Asn Lys Leu Asp Leu
740 745 750
Ile Phe Met Asp Ser Arg Met Pro Val Met Asn Gly Ile Asp Ala Ala
755 760 765
Lys Lys Ile Lys Ile Ile Tyr Pro Asn Leu Pro Ile Ile Ala Leu Thr
770 775 780
Gly Glu Ser Ser His Glu Glu Ile Ser Ala Ile Thr Gln Val Met Asp
785 790 795 800
Gly Tyr Leu Thr Lys Pro Val Ser Lys Ala Gln Leu Gln Gln Val Val
805 810 815
Asp Lys Trp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
820 825 830
Gly Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
835 840 845
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
850 855 860
Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
865 870 875 880
Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
885 890 895
Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
900 905 910
His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
915 920 925
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
930 935 940
Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
945 950 955 960
Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys
965 970 975
Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys
980 985 990
Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu
995 1000 1005
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
1010 1015 1020
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
1025 1030 1035 1040
Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
1045 1050 1055
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
1060 1065 1070
Tyr Lys
<210> 4
<211> 3225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgttaacta ctttgcctaa agtttattta ctactgacaa cctcagcaat catcttgctt 60
tgggtagggt attttgtccg ctctctatat aaagagcgga caaaggtaaa cccttacatt 120
tattcatcat acattctcta tgcattattt attatcttat ggatactgag taatgcttac 180
tttcagtctc cattactcac atactttgat gaatcaaccg ctatttttat ggcgctattt 240
gctaatatga cctcatactt agcttttgct tttgcttttc tattttcgtg tcgccttgct 300
tcaaaacacc cagataaacg actatctaaa tggcagtttc gtttaacgag cattacaacg 360
cttgtcgcat taattgttaa tgttatccct aacttaacgg ttataggagt aacaatccaa 420
gctcctagcg tattcaccat tgaatttggt ccctttgctc ctttgttttt cttaaacgca 480
tttctattcg taattctgac gagcattaat ttctttaagc ttagaaaaag taatataaaa 540
ctcaataaag aaaaatccat ttatttaatg attgggatat ttatttacat gatctcaacc 600
attgcatctc aaataattat accagtcata tgggctgatt tctcatacac atgggttcct 660
cctgcgttat ccgttactga agctttatta attggatata cattacttta ccatagatta 720
tatagcttta aatatttgct attttggtca ttatcatatt ctataaatct aattttgtat 780
ctaacgccta ttattataat ttacgattta acaaccccat ctgatttatt atacatttgt 840
attgttgcaa taatatttac agggctactt tgggataaaa cgcttaaaaa gacaaaaaaa 900
atcgcttcca ttattatata taaagacaaa cagacacctg ttgagaaaat atataaaatt 960
gctgaagagt tcaaatactc aagtagtaat gcaattatta aattagcatc aattttaaac 1020
acgccaaaag aagaattatt attgattggt aaaaacacta attataatat ttttattcca 1080
catttaaacc aaagccactc tgctttggtg aaagatgaac ttgattacca aattcattac 1140
tcccctaaaa ctgctaacgc tgaacttcat caagtacaag agaaaatgag tgaaagtaag 1200
acggcattga tcttacctat ttttggtgaa aacaagctaa tatcgcattt tttaatttcg 1260
gcaaataaac acgataatac gacattttca aatgaagaga tttcagctat tcaatgggta 1320
ctaactaaag ttcaaggcta cattgagagt gaacgaaaag ttcgtcaatc acaggccctt 1380
gctaactcta tcgcacacga aatgcgcaac cctctatcac aacttcagta tcattttgaa 1440
aaaatcaaac atcactatca aaaaaatact gaacatgaaa aacaagaaga tctaataaaa 1500
aatgaattaa accaagggtg tttagcgatt caaaaaggtg cgcaattaat tgatattatt 1560
ttgagtgaag ccaaaaatac cgcgatcagt gatgacctat ttcatcacca ttctatttca 1620
ttattaactc aacaaattat tgatgaatat gtatttgatt cagaagaaat gaagcaaaaa 1680
atcactctag atcttgaaga tgatttcgtc gtaaatatca atgatactct gtatggattc 1740
atactgttta atttattgag aaacgcaact tattactttg atgaatataa cagctccata 1800
tcaattcgtt tagttaaagg gtttgctacc aataaattaa tatttagaga tacaggacct 1860
ggaattgatt cacatatcct ccctaatatt tttgacgact tttttactca taataaagaa 1920
ggaggcagtg gattaggttt atcttattgt ttgcgtgtta tgcatgcttt tgaaggaaac 1980
atcgcatgtt attccaccaa aggagagttc actgaatttg tacttagttt ccctcatata 2040
gaaggagaca ttaacgcatt aaactcccac aaaccaaata ctccaccgtt aattaataag 2100
aaagataatt cgcttaaaac agtattaatt gttgatgata aaaaagtaca acgtatgtta 2160
attcatactt ttattaacaa agataattta acacttttac aagcagaaaa tggtgaagaa 2220
gccgtagaaa tagccacaaa taacaagctt gatcttatct ttatggattc tcgtatgcca 2280
gtaatgaatg gaattgatgc agctaaaaaa attaagataa tctatcctaa tttaccgatt 2340
atcgcgttaa ctggcgaatc cagtcatgaa gaaatcagcg ctataacaca agttatggat 2400
ggctatctaa caaagccagt gtctaaagca caattgcagc aagttgtaga taaatggcta 2460
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcatggt gagcaagggc 2520
gccgagctgt tcaccggcat cgtgcccatc ctgatcgagc tgaatggcga tgtgaatggc 2580
cacaagttca gcgtgagcgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 2640
aagttcatct gcaccaccgg caagctgcct gtgccctggc ccaccctggt gaccaccctg 2700
agctacggcg tgcagtgctt ctcacgctac cccgatcaca tgaagcagca cgacttcttc 2760
aagagcgcca tgcctgaggg ctacatccag gagcgcacca tcttcttcga ggatgacggc 2820
aactacaagt cgcgcgccga ggtgaagttc gagggcgata ccctggtgaa tcgcatcgag 2880
ctgaccggca ccgatttcaa ggaggatggc aacatcctgg gcaataagat ggagtacaac 2940
tacaacgccc acaatgtgta catcatgacc gacaaggcca agaatggcat caaggtgaac 3000
ttcaagatcc gccacaacat cgaggatggc agcgtgcagc tggccgacca ctaccagcag 3060
aataccccca tcggcgatgg ccctgtgctg ctgcccgata accactacct gtccacccag 3120
agcgccctgt ccaaggaccc caacgagaag cgcgatcaca tgatctactt cggcttcgtg 3180
accgccgccg ccatcaccca cggcatggat gagctgtaca agtga 3225
<210> 5
<211> 2463
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgttaacta ctttgcctaa agtttattta ctactgacaa cctcagcaat catcttgctt 60
tgggtagggt attttgtccg ctctctatat aaagagcgga caaaggtaaa cccttacatt 120
tattcatcat acattctcta tgcattattt attatcttat ggatactgag taatgcttac 180
tttcagtctc cattactcac atactttgat gaatcaaccg ctatttttat ggcgctattt 240
gctaatatga cctcatactt agcttttgct tttgcttttc tattttcgtg tcgccttgct 300
tcaaaacacc cagataaacg actatctaaa tggcagtttc gtttaacgag cattacaacg 360
cttgtcgcat taattgttaa tgttatccct aacttaacgg ttataggagt aacaatccaa 420
gctcctagcg tattcaccat tgaatttggt ccctttgctc ctttgttttt cttaaacgca 480
tttctattcg taattctgac gagcattaat ttctttaagc ttagaaaaag taatataaaa 540
ctcaataaag aaaaatccat ttatttaatg attgggatat ttatttacat gatctcaacc 600
attgcatctc aaataattat accagtcata tgggctgatt tctcatacac atgggttcct 660
cctgcgttat ccgttactga agctttatta attggatata cattacttta ccatagatta 720
tatagcttta aatatttgct attttggtca ttatcatatt ctataaatct aattttgtat 780
ctaacgccta ttattataat ttacgattta acaaccccat ctgatttatt atacatttgt 840
attgttgcaa taatatttac agggctactt tgggataaaa cgcttaaaaa gacaaaaaaa 900
atcgcttcca ttattatata taaagacaaa cagacacctg ttgagaaaat atataaaatt 960
gctgaagagt tcaaatactc aagtagtaat gcaattatta aattagcatc aattttaaac 1020
acgccaaaag aagaattatt attgattggt aaaaacacta attataatat ttttattcca 1080
catttaaacc aaagccactc tgctttggtg aaagatgaac ttgattacca aattcattac 1140
tcccctaaaa ctgctaacgc tgaacttcat caagtacaag agaaaatgag tgaaagtaag 1200
acggcattga tcttacctat ttttggtgaa aacaagctaa tatcgcattt tttaatttcg 1260
gcaaataaac acgataatac gacattttca aatgaagaga tttcagctat tcaatgggta 1320
ctaactaaag ttcaaggcta cattgagagt gaacgaaaag ttcgtcaatc acaggccctt 1380
gctaactcta tcgcacacga aatgcgcaac cctctatcac aacttcagta tcattttgaa 1440
aaaatcaaac atcactatca aaaaaatact gaacatgaaa aacaagaaga tctaataaaa 1500
aatgaattaa accaagggtg tttagcgatt caaaaaggtg cgcaattaat tgatattatt 1560
ttgagtgaag ccaaaaatac cgcgatcagt gatgacctat ttcatcacca ttctatttca 1620
ttattaactc aacaaattat tgatgaatat gtatttgatt cagaagaaat gaagcaaaaa 1680
atcactctag atcttgaaga tgatttcgtc gtaaatatca atgatactct gtatggattc 1740
atactgttta atttattgag aaacgcaact tattactttg atgaatataa cagctccata 1800
tcaattcgtt tagttaaagg gtttgctacc aataaattaa tatttagaga tacaggacct 1860
ggaattgatt cacatatcct ccctaatatt tttgacgact tttttactca taataaagaa 1920
ggaggcagtg gattaggttt atcttattgt ttgcgtgtta tgcatgcttt tgaaggaaac 1980
atcgcatgtt attccaccaa aggagagttc actgaatttg tacttagttt ccctcatata 2040
gaaggagaca ttaacgcatt aaactcccac aaaccaaata ctccaccgtt aattaataag 2100
aaagataatt cgcttaaaac agtattaatt gttgatgata aaaaagtaca acgtatgtta 2160
attcatactt ttattaacaa agataattta acacttttac aagcagaaaa tggtgaagaa 2220
gccgtagaaa tagccacaaa taacaagctt gatcttatct ttatggattc tcgtatgcca 2280
gtaatgaatg gaattgatgc agctaaaaaa attaagataa tctatcctaa tttaccgatt 2340
atcgcgttaa ctggcgaatc cagtcatgaa gaaatcagcg ctataacaca agttatggat 2400
ggctatctaa caaagccagt gtctaaagca caattgcagc aagttgtaga taaatggcta 2460
tag 2463
<210> 6
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggtgagca agggcgccga gctgttcacc ggcatcgtgc ccatcctgat cgagctgaat 60
ggcgatgtga atggccacaa gttcagcgtg agcggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc ctggcccacc 180
ctggtgacca ccctgagcta cggcgtgcag tgcttctcac gctaccccga tcacatgaag 240
cagcacgact tcttcaagag cgccatgcct gagggctaca tccaggagcg caccatcttc 300
ttcgaggatg acggcaacta caagtcgcgc gccgaggtga agttcgaggg cgataccctg 360
gtgaatcgca tcgagctgac cggcaccgat ttcaaggagg atggcaacat cctgggcaat 420
aagatggagt acaactacaa cgcccacaat gtgtacatca tgaccgacaa ggccaagaat 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg atggcagcgt gcagctggcc 540
gaccactacc agcagaatac ccccatcggc gatggccctg tgctgctgcc cgataaccac 600
tacctgtcca cccagagcgc cctgtccaag gaccccaacg agaagcgcga tcacatgatc 660
tacttcggct tcgtgaccgc cgccgccatc acccacggca tggatgagct gtacaagtga 720
<210> 7
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgttaacta ctttgcctaa agtttattta ctactgacaa 40
<210> 8
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctgccgccg ccgccgctgc cgccgccgcc gctgccgccg ccgcctagcc atttatctac 60
<210> 9
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcatggt gagcaagggc 60
<210> 10
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcacttgtac agctcatcca tgccgtgggt gatggcggcg gcggt 45
<210> 11
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 12
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45

Claims (8)

1.融合蛋白,其特征在于,其氨基酸序列如SEQ ID NO:3所示。
2.编码权利要求1所述融合蛋白的核酸。
3.根据权利要求2所述的核酸,其特征在于,所述核酸的序列如SEQ ID NO:4所示。
4.含有权利要求2或3所述核酸的表达载体。
5.转化或转染权利要求4所述表达载体的重组宿主。
6.权利要求1所述融合蛋白的制备方法,其特征在于,培养权利要求5所述重组宿主,诱导所述融合蛋白的表达。
7.一种酰胺高丝氨酸内酯抑制物的检测试剂,其特征在于,包括如下I)~II)中的任一项:
I)、权利要求1所述融合蛋白;
II)、权利要求5所述重组宿主。
8.一种酰胺高丝氨酸内酯抑制物的检测方法,其特征在于,使如下I)~II)中的任一项与待测物接触,根据荧光情况判断待测物是否具有抑制作用:
I)、权利要求1所述融合蛋白;
II)、权利要求5所述重组宿主。
CN202210338602.2A 2022-04-01 2022-04-01 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用 Active CN114751990B (zh)

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