CN110128546A - 一种用于rna示踪的融合蛋白及其应用 - Google Patents

一种用于rna示踪的融合蛋白及其应用 Download PDF

Info

Publication number
CN110128546A
CN110128546A CN201910351227.3A CN201910351227A CN110128546A CN 110128546 A CN110128546 A CN 110128546A CN 201910351227 A CN201910351227 A CN 201910351227A CN 110128546 A CN110128546 A CN 110128546A
Authority
CN
China
Prior art keywords
leu
lys
glu
gly
asn
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910351227.3A
Other languages
English (en)
Other versions
CN110128546B (zh
Inventor
韩春雨
高峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hebei University of Science and Technology
Original Assignee
Hebei University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hebei University of Science and Technology filed Critical Hebei University of Science and Technology
Priority to CN201910351227.3A priority Critical patent/CN110128546B/zh
Publication of CN110128546A publication Critical patent/CN110128546A/zh
Priority to PCT/CN2020/087527 priority patent/WO2020221255A1/zh
Application granted granted Critical
Publication of CN110128546B publication Critical patent/CN110128546B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明涉及一种用于RNA核酸示踪的融合蛋白，将荧光蛋白分为N段和C段后，插入RNA结合蛋白，构建得到如下结构的融合蛋白：荧光蛋白N‑RNA结合蛋白‑荧光蛋白C。本发明同时涉及该融合蛋白的应用。本发明的融合蛋白可作为RNA示踪工具，具有灵敏度高和背景噪音信号低等优点。

Description

一种用于RNA示踪的融合蛋白及其应用
技术领域
本发明属于核酸示踪技术领域，具体涉及一种用于RNA示踪的融合蛋白及其应用。
背景技术
细胞内三类大分子DNA、RNA、蛋白质，它们的功能是人们一直所感兴趣的。位置决定功能，将这些大分子定位明确就显得尤为重要。
信使RNA（mRNA）和非编码RNA（ncRNA）的复杂时空动态几乎影响细胞功能的所有方面。RNA与大量RNA结合蛋白结合，动态调节RNA定位和功能。总之，RNA直接调节大量细胞过程，需要强有力的方法来荧光标记和跟踪活细胞中的RNA。
1998年Roy M.Long等第一次将GFP蛋白与MS2-MCP蛋白融合运用于RNA定位。这种方法的一个限制是需要许多拷贝的MS2 RNA标签来增强荧光对比度，并且RNA标签与MS2-FP复合物结合的大尺寸可能扰乱RNA定位。尽管如此，该系统仍是活细胞RNA成像的黄金标准，因为它已被成功用于在单分子动力学水平上示踪mRNA。这种方法的另一个限制是MS2-FP系统有很高的背景噪音，直接影响到了定位效果。
Cas蛋白家族是近几年的研究热门，失活Cas的核酸酶的活性而获得了dead Cas（dCas），dCas只会在sgRNA的引导下特异性的结合到目的位点，而不会产生切割。如果在dCas上融合荧光蛋白（FP），就可以特异性地定位目的RNA。这种巧妙的设计方法主要还是为了减少自发荧光，但并没有从根本上解决问题，造成灵敏度低，直接影响到了定位效果。
发明内容
本发明的目的是提供一种具有灵敏度高、背景噪音信号低的新型RNA示踪工具及其应用。
本发明采用如下技术方案：
一种用于RNA核酸示踪的融合蛋白，将荧光蛋白分为N段和C段后，插入RNA结合蛋白，构建得到如下结构的融合蛋白：荧光蛋白N-RNA结合蛋白-荧光蛋白C。
优选的，所述融合蛋白还包括连接蛋白，构建得到如下结构的融合蛋白：荧光蛋白N-连接蛋白A-RNA结合蛋白-连接蛋白B-荧光蛋白C。
优选的，所述荧光蛋白为Venus荧光蛋白。
优选的，所述荧光蛋白N包含如SEQ ID No.10所示的氨基酸序列。
优选的，所述荧光蛋白C包含如SEQ ID No.11所示的氨基酸序列。
优选的，所述连接蛋白A包含如SEQ ID No.12所示的氨基酸序列。
优选的，所述连接蛋白B包含如SEQ ID No.13所示的氨基酸序列。
进一步的，所述RNA结合蛋白包括但不限于dcasE、dcas6Fv、dcse3、dDbCasE、dEaCasE、dMsCasE、dSpCasE、dXtCasE或dLwcas13a中的一种。
进一步的，所述融合蛋白包含如SEQ ID No.1~ SEQ ID No.9所示的氨基酸序列中的一种。
优选的，所述融合蛋白包含与SEQ ID No.1~ SEQ ID No.9中的任意一种具有70％~100％相似性的氨基酸序列。
一种上述的融合蛋白在RNA示踪中的应用。
一种利用上述的融合蛋白的RNA示踪方法。
本发明的有益效果在于：本发明的融合蛋白可以对RNA进行示踪定位，帮助人们发现RNA动力学和功能之间错综复杂的联系，其灵敏性高、背景噪音信号低、实时性强，用于活细胞中RNA可视化的工具，以利于阐明mRNA和ncRNA等生命周期的动力学机制的研究。
附图说明
图1为融合蛋白在RNA示踪过程中的原理图。
图2为VN-dLwcas13a-VC在RNA示踪过程中的原理图。
图3为VN-dcasE-VC的GK质粒表达载体构建示意图。
图3中，VN154表示Venus N端第1~154的氨基酸序列，VC154表示Venus N端第155之后的氨基酸序列。
图4用于示踪的靶位点表达质粒RM-16*CBS-C1构建图。
图5为实施例3的荧光显微镜的镜检照片。
具体实施方式
下面将结合具体实施例和附图对本发明进行进一步详细的说明，但不仅限于此。
实施例1
一种用于RNA核酸示踪的融合蛋白，将荧光蛋白分为C段和N段后，插入RNA结合蛋白，构建得到如下结构的融合蛋白：荧光蛋白N-RNA结合蛋白-荧光蛋白C。
优选的，所述融合蛋白还包括连接蛋白，构建得到如下结构的融合蛋白：荧光蛋白N-连接蛋白A-RNA结合蛋白-连接蛋白B-荧光蛋白C。
所述荧光蛋白包括但不限于Venus荧光蛋白。
作为优选，将Venus荧光蛋白进行分段，即得到荧光蛋白N（以下简称为VN）和荧光蛋白C（以下简称为VC）。将VN、RNA结合蛋白、VC构建为融合蛋白VN-RNA结合蛋白-VC。
更优化的，所述荧光蛋白N包含如SEQ ID No.10所示的氨基酸序列。所述荧光蛋白C包含如SEQ ID No.11所示的氨基酸序列。
构建融合蛋白，需要使用连接蛋白将蛋白片段进行连接，形成VN-连接蛋白A-RNA结合蛋白-连接蛋白B-VC。所述连接蛋白A包含如SEQ ID No.12所示的氨基酸序列。所述连接蛋白B包含如SEQ ID No.13所示的氨基酸序列。需要说明的是，与SEQ ID No.12或SEQ IDNo.13具有70％~100％相似性的氨基酸序列也属于本申请的保护范围。
所述RNA结合蛋白包括但不限于dcasE、dcas6Fv、dcse3、dDbCasE、dEaCasE、dMsCasE、dSpCasE、dXtCasE或dLwcas13a中的一种。
更具体的优化后，得到如下9种融合蛋白。其结构均为VN-连接蛋白A-RNA结合蛋白-连接蛋白B-VC，由于其连接蛋白的位置和结构相同，为了简化叙述，故在下述表示过程中省略该部分。
1、VN-dcasE-VC
包含如SEQ ID No.1所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：GAGUUCCCCGCGCCAGCGGGGAUAAACCG。其中，RNA结合蛋白dcasE来源于E.coli K12。
2、VN-dcas6Fv-VC
包含如SEQ ID No.2所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：GUUCACCGCCGCACAGGCGGCUUAGAAA。其中，RNA结合蛋白dcas6Fv来源于Shewanella putrefaciens CN-32。
3、VN-dcse3-VC
包含如SEQ ID No.3所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：GUAGUCCCCACGCGUGTGGGGAUGGACC。其中，RNA结合蛋白dcse3来源于T. thermophilus HB8。
4、VN-dDbCasE-VC
包含如SEQ ID No.4所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：AUGUUCCCCACGCCUGUGGGGAUGAACCG。其中，RNA结合蛋白dDbCasE来源于Deltaproteobacteria bacterium。
5、VN-dEaCasE-VC
包含如SEQ ID No.5所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：GUGUUCCCCGCGUAUGCGGGGAUAAACCG。其中，RNA结合蛋白dEaCasE来源于Erwinia amylovora。
6、VN- dMsCasE -VC
包含如SEQ ID No.6所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：GCGUUCCCCACAGCCGUGGGGAUUAACCG。其中，RNA结合蛋白dMsCasE来源于Marinomonas sp。
7、VN- dSpCasE -VC
包含如SEQ ID No.7所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：CUGUUCCCCGUAAGCACGGGGAUAAACCG。其中，RNA结合蛋白dSpCasE来源于Shewanella putrefaciens。
8、VN-dXtCasE-VC
包含如SEQ ID No.8所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的RNA序列为：CUGUUCCCCGUGAGUACGGGGAUAAACCG。其中，RNA结合蛋白dXtCasE来源于Xenorhabdus thuongxuanensis。
如图1所示，上述八种融合蛋白的RNA示踪原理为：RNA结合蛋白能够特异结合RNA，将RNA结合蛋白插入到荧光蛋白中，形成全新的“荧光蛋白N-RNA结合蛋白-荧光蛋白C”。由于荧光蛋白被拆分为两部分，且在没有RNA结合蛋白识别的底物时，融合蛋白并不稳定，因此在荧光显微镜下不发光，“荧光蛋白N-RNA结合蛋白-荧光蛋白C”没有可检测的荧光信号。当存在靶向RNA底物时，RNA结合蛋白与靶向RNA识别后结合，使得“荧光蛋白N-RNA结合蛋白-荧光蛋白C”趋向稳定，此时，荧光蛋白N与荧光蛋白C重新形成稳定的荧光蛋白，会有明显的荧光信号。
9、VN-dLwcas13a -VC
包含如SEQ ID No.9所示的氨基酸序列或与其具有70％~100％相似性的氨基酸序列，该融合蛋白识别的crRNA，crRNA与靶向RNA互补结合，实现靶向RNA的示踪。其中，RNA结合蛋白dLwcas13a来源于Leptotrichia wadei。
如图2所示，上述VN-dLwcas13a -VC融合蛋白的RNA示踪原理为：与其他8个融合蛋白的原理相似，区别在于RNA结合蛋白识别靶向RNA不再是直接识别，而是通过crRNA作为媒介来实现。
Lwcas13a是一类CRISPR-Cas蛋白，能够特异结合crRNA，形成Lwcas13a-crRNA复合体，然后将与crRNA互补的靶向RNA并进行切割。Dead Lwcas13a（dLwcas13a）是Lwcas13a酶活中心失活突变体，能够结合与crRNA互补的靶向RNA，但不能切割。将dLwcas13a插入进荧光蛋白中，形成新的“荧光蛋白N-dLwcas13a-荧光蛋白C”，在没有靶向RNA时，荧光蛋白N与荧光蛋白C不能稳定的形成完整的荧光蛋白，没有荧光信号，在有靶向的RNA底物时，有荧光信号。
实施例3
一种利用上述的融合蛋白的RNA示踪方法。将本发明的融合蛋白插入质粒载体中，再利用该质粒载体转染或导入细胞，由于融合蛋白本身不发荧光，而与靶RNA结合后发出荧光，荧光显微镜下可捕捉到靶RNA。
下面以VN-dcasE-VC为例，说明该RNA示踪方法。
1、细胞培养。
2、细胞转染（按照R-4000操作说明进行）
1）分24孔板，密度达到50%左右转染，用于转染的VN-dCasE-VC蛋白表达载体的构建图如图3所示，用于示踪的靶位点表达质粒RM-16*CBS-C1的构建图如图4所示。
2）提前30分钟将完全培养基更换为5%血清的DMEM培养基；
3）取两支1.5ml EP管，分别加入25μl Opti-MEM；
4）一支EP管中加入50ng VN-dCasE-VC蛋白表达质粒+800ng RM-16*CBS-C1表达质粒，另一支EP管中加入0.5μl R-4000。室温静置5min钟，将两管溶液混合，室温再静置20min；
5）将孵育后的混合物轻轻滴加到24孔板，轻轻旋转混匀，继续培养细胞；
6）20h后倒置荧光显微镜镜检；镜检结果如图5所示。
图5为采用vert A1倒置荧光显微镜，20倍镜头进行拍照。VN-dCasE-VC系统在293T细胞能够进行RFP monomer RNA的定位（f），并且在没有结合位点时没有泄露（c）。a，b，c 分别是红色荧光通道、光镜和绿色荧光通道的照片，是没有dCasE结合位点的对照组。d，e，f分别是红色荧光通道，光镜，绿色荧光通道的照片，是有dCasE结合位点的实验组。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，但并不限于此，本领域的技术人员很容易根据上述实施例领会本发明的精神，并作出不同的引申和变化，但只要不脱离本发明的精神，都在本发明的保护范围之内。
SEQUENCE LISTING
<110> 河北科技大学
<120> 一种用于RNA示踪的融合蛋白及其应用
<130> 2019
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 474
<212> PRT
<213> 人工序列
<400> 1
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Tyr Leu Ser Lys Val Ile Ile Ala Arg Ala Trp Ser Arg
180 185 190
Asp Leu Tyr Gln Leu Ala Gln Gly Leu Trp His Leu Phe Pro Asn Arg
195 200 205
Pro Asp Ala Ala Arg Asp Phe Leu Phe His Val Glu Lys Arg Asn Thr
210 215 220
Pro Glu Gly Cys His Val Leu Leu Gln Ser Ala Gln Met Pro Val Ser
225 230 235 240
Thr Ala Val Ala Thr Val Ile Lys Thr Lys Gln Val Glu Phe Gln Leu
245 250 255
Gln Val Gly Val Pro Leu Tyr Phe Arg Leu Arg Ala Asn Pro Ile Lys
260 265 270
Thr Ile Leu Asp Asn Gln Lys Arg Leu Asp Ser Lys Gly Asn Ile Lys
275 280 285
Arg Cys Arg Val Pro Leu Ile Lys Glu Ala Glu Gln Ile Ala Trp Leu
290 295 300
Gln Arg Lys Leu Gly Asn Ala Ala Arg Val Glu Asp Val His Pro Ile
305 310 315 320
Ser Glu Arg Pro Gln Tyr Phe Ser Gly Asp Gly Lys Ser Gly Lys Ile
325 330 335
Gln Thr Val Cys Phe Glu Gly Val Leu Thr Ile Asn Asp Ala Pro Ala
340 345 350
Leu Ile Asp Leu Val Gln Gln Gly Ile Gly Pro Ala Lys Ser Met Gly
355 360 365
Cys Gly Leu Leu Ser Leu Ala Pro Leu Leu Gln Ser Thr Thr Cys Thr
370 375 380
Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
385 390 395 400
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp
405 410 415
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
420 425 430
Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn
435 440 445
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
450 455 460
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470
<210> 2
<211> 458
<212> PRT
<213> 人工序列
<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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Asn Ser Tyr Ile Asp Ile Arg Leu Lys Pro Asp Ala Glu
180 185 190
Met Arg Glu Ala Glu Leu Ser Ser Lys Val Phe Thr Lys Phe Ala Lys
195 200 205
Ala Leu Val Thr Leu Asn Ser His Lys Ile Gly Ile Ser Phe Pro Gln
210 215 220
Met Lys Leu Ser Leu Gly Gln Leu Phe Arg Ile His Gly Asp Ala Ser
225 230 235 240
Leu Leu His Asp Leu Gln Gly Leu Asp Trp Leu Gly Pro Leu Ala Gly
245 250 255
Tyr Cys Gln Val Thr Ala Val Ser Ala Val Pro Asp His Val Gln Tyr
260 265 270
Arg Ile Val Ser Val Lys Arg Ser Asn Leu Ser Lys Ala Lys Leu Lys
275 280 285
Arg Leu Ile Ala Arg Gly Ser Ile Asp Lys Asp Gly Glu Lys Arg Tyr
290 295 300
Lys Val Lys Met Leu Gly Gln Gly Phe Asp Asn Pro Tyr Leu Asp Leu
305 310 315 320
Phe Ser Ser Ser Thr Gly Gln Val Tyr Arg Lys Phe Phe Glu Phe Ser
325 330 335
Asp Ile Gln Ala His Pro Leu Asp Gly Glu Phe Asp Ser Tyr Gly Leu
340 345 350
Ser Lys Thr Ala Thr Val Pro Trp Phe Leu Gln Ser Thr Thr Cys Thr
355 360 365
Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
370 375 380
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp
385 390 395 400
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
405 410 415
Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn
420 425 430
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
435 440 445
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
450 455
<210> 3
<211> 486
<212> PRT
<213> 人工序列
<400> 3
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Trp Leu Thr Lys Leu Val Leu Asn Pro Ala Ser Lys Ala
180 185 190
Ala Arg Arg Asp Leu Ala Asn Pro Tyr Glu Met Ala Arg Thr Leu Ser
195 200 205
Lys Ala Val Ser Arg Ala Leu Glu Gly Gly Glu Glu Arg Leu Leu Trp
210 215 220
Arg Leu Glu Pro Thr Arg Gly Leu Glu Pro Pro Val Val Leu Val Gln
225 230 235 240
Thr Leu Thr Glu Pro Asp Trp Ser Val Leu Glu Gly Gly Tyr Ala Gln
245 250 255
Val Phe Pro Pro Lys Pro Phe His Pro Ala Phe Arg Pro Gly Gln Arg
260 265 270
Leu Arg Phe Arg Leu Arg Ala Asn Pro Ala Lys Arg Leu Ala Ala Thr
275 280 285
Gly Lys Arg Val Ala Leu Lys Thr Pro Ala Glu Lys Val Ala Trp Leu
290 295 300
Glu Arg Arg Leu Glu Glu Gly Gly Phe Arg Leu Leu Glu Gly Glu Arg
305 310 315 320
Gly Pro Trp Val Gln Ile Leu Gln Asp Thr Phe Leu Glu Val Arg Arg
325 330 335
Lys Lys Asp Gly Glu Glu Ala Gly Lys Leu Leu Gln Val Gln Ala Val
340 345 350
Leu Phe Glu Gly Arg Leu Glu Val Val Asp Pro Glu Arg Ala Leu Ala
355 360 365
Thr Leu Lys Arg Gly Val Gly Pro Gly Lys Ala Leu Gly Leu Gly Leu
370 375 380
Leu Ser Val Ala Pro Leu Gln Ser Thr Thr Cys Thr Ala Gly Ala Glu
385 390 395 400
Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
405 410 415
His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln
420 425 430
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
435 440 445
Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
450 455 460
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
465 470 475 480
Met Asp Glu Leu Tyr Lys
485
<210> 4
<211> 476
<212> PRT
<213> 人工序列
<400> 4
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Phe Leu Ser Lys Ile Phe Ile Pro Trp Gln Lys Ala Arg Asn Pro
180 185 190
Tyr Asp Ile Ala Arg Glu Leu Trp His Leu Phe Pro Asn Arg Pro Asp
195 200 205
Glu Ser Arg Gly Phe Leu Phe Arg Val Glu Lys Gln Gln Lys Gly Leu
210 215 220
Gly Ala Glu Ile Leu Met Gln Ser Asn Ser Glu Val Ser Ala Val Asn
225 230 235 240
Gly Thr Val Arg Ile Ile Gly Ser Arg Lys Tyr Val Phe Ser Leu Gln
245 250 255
Lys Gly Gln Lys Ile Arg Phe Arg Leu Arg Ala Asn Pro Ile Lys Thr
260 265 270
Ile Lys Asp Glu Asp Gly Arg Leu Asn Gln Arg Asp Glu Ile Lys Lys
275 280 285
Cys Arg Val Pro Leu Ile Arg Glu Glu Asp Gln Lys Ala Trp Ile Glu
290 295 300
Arg Lys Leu Glu Lys Ile Cys Ser Leu Asp Ala Leu Val Phe Gln Gln
305 310 315 320
Glu Met Pro Leu Tyr Phe Arg Lys Ser Asn Ser Lys Arg Ala Gly Lys
325 330 335
Ile Gln Thr Val Leu Phe Asp Gly Val Leu Asn Ile Asn Asp Pro Asp
340 345 350
Ala Leu Ile Glu Met Ile Lys Asn Gly Ile Gly Pro Ala Lys Ala Phe
355 360 365
Gly Cys Gly Leu Leu Ser Met Ala Arg Phe Leu Leu Gln Ser Thr Thr
370 375 380
Cys Thr Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys
385 390 395 400
Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu
405 410 415
Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu
420 425 430
Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp
435 440 445
Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala
450 455 460
Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470 475
<210> 5
<211> 476
<212> PRT
<213> 人工序列
<400> 5
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Ile Tyr Leu Ser Gln Ile Ala Val Pro Trp Ser Trp Ala
180 185 190
Lys Asp Pro Tyr Gln Leu Ala Arg Ala Leu Trp Gln Leu Phe Pro Asp
195 200 205
Arg Pro Ser Asp Arg Arg Asp Phe Leu Phe Arg Val Glu Thr Arg His
210 215 220
Ala Arg Ala Gly Gln Trp Val Leu Leu Gln Ser Leu Gln Ala Pro Gln
225 230 235 240
Asn Cys Ala Ala Ala Gln Val Leu Ala Ser Lys Val Thr Gln Phe Ala
245 250 255
Leu Ser Pro Gly Gln Arg Leu His Phe Arg Leu Arg Ala Asn Pro Val
260 265 270
Lys Asn Ile Lys Asp Asn Arg Gly Arg Val Asn Ser Arg Gly Glu Val
275 280 285
Lys Ser Cys Arg Val Pro Leu Ile Asp Asp Asn Gln Leu Met Gln Trp
290 295 300
Leu Val Arg Lys Leu Gln Asp Ala Ala Val Leu His Ser Ala Ser Val
305 310 315 320
Ser Lys Glu Pro Ala Leu Cys Phe Asn Lys Gln Ala Val Ala Gly Lys
325 330 335
Ile Gln Pro Val Cys Phe Glu Gly Ile Leu Gln Val Thr Ser Glu Thr
340 345 350
His Phe Tyr Gln Cys Leu Val Asn Gly Ile Gly Pro Ala Lys Ser Met
355 360 365
Gly Cys Gly Met Leu Ser Ile Ala Arg Ala Leu Leu Gln Ser Thr Thr
370 375 380
Cys Thr Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys
385 390 395 400
Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu
405 410 415
Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu
420 425 430
Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp
435 440 445
Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala
450 455 460
Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470 475
<210> 6
<211> 478
<212> PRT
<213> 人工序列
<400> 6
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Tyr Leu Ser Lys Val Met Val His Trp Lys Trp Ile Glu
180 185 190
Asn Pro Tyr Lys Leu Ala Lys Ala Leu Trp Gln Leu Phe Pro Tyr Arg
195 200 205
Asp Gly Gln Lys Arg Asp Phe Gln Phe Arg Val Glu Gln Tyr Gln Lys
210 215 220
Gly Ile Gly Ala Leu Val Leu Leu Gln Ser Glu Gln Glu Pro Ile Asn
225 230 235 240
Ala Asp Val Ala Met Ile Met Ala Ser Lys Ser Phe Lys Pro Asn Ile
245 250 255
Glu Thr Gly Ser Thr Ile Arg Phe Lys Leu Arg Ala Asn Pro Val Lys
260 265 270
Thr Ile Lys Asp Lys His Gly Arg Leu Thr Lys Lys Gly Glu Pro Lys
275 280 285
Lys Cys Arg Val Pro Leu Leu Arg Glu Glu Asp Gln Ile Asn Trp Leu
290 295 300
Gln Arg Lys Leu Glu Gly Lys Ala Thr Leu Gln Ser Ile Gln Val Gln
305 310 315 320
Asp Glu Lys Pro Leu Tyr Phe Tyr Lys Glu Ser Glu Asp Arg Arg Gly
325 330 335
Gln Ile Lys Pro Val Cys Phe Glu Gly Val Leu Ser Ile Asp Asn Ala
340 345 350
Glu Ala Phe Ser Gln Met Leu Gln Thr Gly Ile Gly Ala Ala Lys Gly
355 360 365
Met Gly Cys Gly Met Leu Ser Leu Ala Pro Val Gly Leu Leu Gln Ser
370 375 380
Thr Thr Cys Thr Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly
385 390 395 400
Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val
405 410 415
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
420 425 430
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser
435 440 445
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
450 455 460
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470 475
<210> 7
<211> 474
<212> PRT
<213> 人工序列
<400> 7
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Phe Leu Ser Lys Val Trp Ile Gly Trp Leu Trp Ala Lys
180 185 190
Asp Pro Tyr Gln Leu Ala Arg Ala Leu Trp Gln Leu Phe Pro Asp Arg
195 200 205
Pro Thr Asp Glu Arg Asp Phe Leu Phe Arg Val Glu Glu Gln Gln Arg
210 215 220
Gly Lys Gly Ala Met Ala Leu Leu Leu Ser Ala Glu Gln Pro Gln Ser
225 230 235 240
Ala Ser Val Ala Glu Val Ile Ala Ser Lys Pro Met Pro Gln Asn Ile
245 250 255
Ala Ala Gly Ala Arg Leu Arg Phe Arg Leu Arg Ala Asn Pro Val Lys
260 265 270
Ala Ile Lys Asp Ser Lys Arg Leu Asp Lys Asn Gly Glu Ala Lys Ser
275 280 285
Ile Arg Val Pro Leu Ile His Glu Glu Glu Gln Leu Lys Trp Leu Leu
290 295 300
Arg Lys Phe Ala Gly Val Ala Glu Leu Glu Thr Ala Arg Val Trp Gln
305 310 315 320
Glu Pro Ala Leu Phe Phe Arg Lys Gln Asp Met Gly Gly Lys Ile Gln
325 330 335
Pro Val Cys Phe Glu Gly Val Leu Thr Val Leu Asp Asn Gln Ala Leu
340 345 350
Phe Glu Leu Leu Arg Gln Gly Ile Gly Pro Ala Lys Ala Met Gly Cys
355 360 365
Gly Leu Leu Ser Leu Ala Ala Ala Leu Leu Gln Ser Thr Thr Cys Thr
370 375 380
Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
385 390 395 400
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp
405 410 415
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
420 425 430
Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn
435 440 445
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
450 455 460
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470
<210> 8
<211> 475
<212> PRT
<213> 人工序列
<400> 8
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Val Phe Leu Ser Lys Val Trp Ile Asp Trp Arg Trp Ala Lys
180 185 190
Asp Pro Tyr Gln Leu Ala Arg Ala Leu Trp Gln Leu Phe Pro His Arg
195 200 205
Pro Asn Asp Ala Arg Asp Phe Leu Phe Arg Ile Glu Ala Gln Ser Phe
210 215 220
Gly Arg Gly Ala Glu Ala Leu Leu Gln Ser Val Gln Ala Pro Ser Ser
225 230 235 240
Ala Gln Ala Ala Gln Val Ile Val Ser Lys Pro Ile Gln Trp Ser Ile
245 250 255
Pro Asp Gly Ala Lys Leu Arg Phe Lys Leu Arg Ala Asn Pro Ile Lys
260 265 270
Thr Ile Lys Asp Gly Glu Gln Arg Arg Asp Arg Asn Gly Lys Ile Lys
275 280 285
Ser Cys Arg Val Pro Leu Ile His Glu Glu Glu Gln Leu Gln Trp Leu
290 295 300
Ser Arg Lys Leu Ala Gly Ala Ala Leu Leu Ser Thr Ala Trp Val Thr
305 310 315 320
Pro Glu Ser Ser Ile Tyr Phe Tyr Lys Asp Asp Ile Arg Gly Lys Ile
325 330 335
Gln Pro Val Cys Phe Glu Gly Gln Ile Ile Val Gln Glu Asn Glu Asp
340 345 350
Phe Ile Ala Leu Leu Asn Gln Gly Ile Gly Pro Ala Lys Ala Met Gly
355 360 365
Cys Gly Leu Leu Ser Leu Ser Leu Ser Leu Leu Gln Ser Thr Thr Cys
370 375 380
Thr Ala Gly Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala
385 390 395 400
Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala
405 410 415
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
420 425 430
Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro
435 440 445
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
450 455 460
Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
465 470 475
<210> 9
<211> 1426
<212> PRT
<213> 人工序列
<400> 9
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr Leu Glu Arg Pro Ala Cys
145 150 155 160
Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn His Lys Leu Ala
165 170 175
Thr Met Lys Val Thr Lys Val Asp Gly Ile Ser His Lys Lys Tyr Ile
180 185 190
Glu Glu Gly Lys Leu Val Lys Ser Thr Ser Glu Glu Asn Arg Thr Ser
195 200 205
Glu Arg Leu Ser Glu Leu Leu Ser Ile Arg Leu Asp Ile Tyr Ile Lys
210 215 220
Asn Pro Asp Asn Ala Ser Glu Glu Glu Asn Arg Ile Arg Arg Glu Asn
225 230 235 240
Leu Lys Lys Phe Phe Ser Asn Lys Val Leu His Leu Lys Asp Ser Val
245 250 255
Leu Tyr Leu Lys Asn Arg Lys Glu Lys Asn Ala Val Gln Asp Lys Asn
260 265 270
Tyr Ser Glu Glu Asp Ile Ser Glu Tyr Asp Leu Lys Asn Lys Asn Ser
275 280 285
Phe Ser Val Leu Lys Lys Ile Leu Leu Asn Glu Asp Val Asn Ser Glu
290 295 300
Glu Leu Glu Ile Phe Arg Lys Asp Val Glu Ala Lys Leu Asn Lys Ile
305 310 315 320
Asn Ser Leu Lys Tyr Ser Phe Glu Glu Asn Lys Ala Asn Tyr Gln Lys
325 330 335
Ile Asn Glu Asn Asn Val Glu Lys Val Gly Gly Lys Ser Lys Arg Asn
340 345 350
Ile Ile Tyr Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asn Asp Tyr Ile
355 360 365
Asn Asn Val Gln Glu Ala Phe Asp Lys Leu Tyr Lys Lys Glu Asp Ile
370 375 380
Glu Lys Leu Phe Phe Leu Ile Glu Asn Ser Lys Lys His Glu Lys Tyr
385 390 395 400
Lys Ile Arg Glu Tyr Tyr His Lys Ile Ile Gly Arg Lys Asn Asp Lys
405 410 415
Glu Asn Phe Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn
420 425 430
Ile Lys Glu Leu Ile Glu Lys Ile Pro Asp Met Ser Glu Leu Lys Lys
435 440 445
Ser Gln Val Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp
450 455 460
Lys Asn Ile Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser
465 470 475 480
Gln Leu Leu Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn
485 490 495
Asp Lys Ile Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile
500 505 510
Glu Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly
515 520 525
Lys Tyr Asn Tyr Tyr Leu Gln Val Gly Glu Ile Ala Thr Ser Asp Phe
530 535 540
Ile Ala Arg Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly
545 550 555 560
Val Ser Ser Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu
565 570 575
Asn Glu Asn Asp Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn
580 585 590
Asn Lys Gly Glu Glu Lys Tyr Val Ser Gly Glu Val Asp Lys Ile Tyr
595 600 605
Asn Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr
610 615 620
Ser Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe
625 630 635 640
Ala Asn Ile Asp Glu Ala Ile Ser Ser Ile Ala His Gly Ile Val His
645 650 655
Phe Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile
660 665 670
Ala Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu
675 680 685
Lys Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val
690 695 700
Phe Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr
705 710 715 720
Lys Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr
725 730 735
Lys Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe
740 745 750
Trp Ser Val Pro Lys Asp Lys Glu Glu Lys Asp Ala Gln Ile Tyr Leu
755 760 765
Leu Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn
770 775 780
Ser Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys
785 790 795 800
Gln Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn
805 810 815
Ile Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg
820 825 830
Glu Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp
835 840 845
Phe Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys
850 855 860
Asn Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp
865 870 875 880
Ile Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp
885 890 895
Lys Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro
900 905 910
His Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu Gly Lys Ile Leu
915 920 925
Lys Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu
930 935 940
Asn His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln
945 950 955 960
Ser Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn
965 970 975
Leu Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu
980 985 990
Ala Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys
995 1000 1005
Asp Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe
1010 1015 1020
Asp Gly Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys
1025 1030 1035
Lys Tyr Gly Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala
1040 1045 1050
Lys Tyr Lys Ile Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys
1055 1060 1065
Lys Asn Glu Ile Glu Lys Asn Tyr Thr Met Gln Gln Asn Leu His
1070 1075 1080
Arg Lys Tyr Ala Arg Pro Lys Lys Asp Glu Lys Phe Asn Asp Glu
1085 1090 1095
Asp Tyr Lys Glu Tyr Glu Lys Ala Ile Gly Asn Ile Gln Lys Tyr
1100 1105 1110
Thr His Leu Lys Asn Lys Val Glu Phe Asn Glu Leu Asn Leu Leu
1115 1120 1125
Gln Gly Leu Leu Leu Lys Ile Leu His Arg Leu Val Gly Tyr Thr
1130 1135 1140
Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg Leu Lys Gly Glu Phe
1145 1150 1155
Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn Phe Asp Asn Ser
1160 1165 1170
Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu Lys Tyr Ile
1175 1180 1185
Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu Lys Arg Ser
1190 1195 1200
Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln Glu Lys Lys
1205 1210 1215
Asp Leu Tyr Ile Ala Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro
1220 1225 1230
His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn Leu Arg Lys
1235 1240 1245
Leu Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile Met Lys Ser
1250 1255 1260
Ile Val Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala Thr Phe Lys
1265 1270 1275
Ile Gly Ala Asp Lys Lys Ile Glu Ile Gln Thr Leu Glu Ser Glu
1280 1285 1290
Lys Ile Val His Leu Lys Asn Leu Lys Lys Lys Lys Leu Met Thr
1295 1300 1305
Asp Arg Asn Ser Glu Glu Leu Cys Glu Leu Val Lys Val Met Phe
1310 1315 1320
Glu Tyr Lys Ala Leu Glu Leu Gln Ser Thr Thr Cys Thr Ala Gly
1325 1330 1335
Ala Glu Phe Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
1340 1345 1350
Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp
1355 1360 1365
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
1370 1375 1380
Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp
1385 1390 1395
Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
1400 1405 1410
Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
1415 1420 1425
<210> 10
<211> 154
<212> PRT
<213> 人工序列
<400> 10
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 Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala 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 Thr
145 150
<210> 11
<211> 85
<212> PRT
<213> 人工序列
<400> 11
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
1 5 10 15
Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn
20 25 30
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
35 40 45
Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
50 55 60
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
65 70 75 80
Asp Glu Leu Tyr Lys
85
<210> 12
<211> 23
<212> PRT
<213> 人工序列
<400> 12
Leu Glu Arg Pro Ala Cys Lys Ile Pro Asn Asp Leu Lys Gln Lys Val
1 5 10 15
Met Asn His Lys Leu Ala Thr
20
<210> 13
<211> 12
<212> PRT
<213> 人工序列
<400> 13
Leu Gln Ser Thr Thr Cys Thr Ala Gly Ala Glu Phe
1 5 10

Claims (8)

1.一种用于RNA核酸示踪的融合蛋白，其特征在于，将荧光蛋白分为N段和C段后，插入RNA结合蛋白，构建得到如下结构的融合蛋白：荧光蛋白N-RNA结合蛋白-荧光蛋白C。
2.根据权利要求1所述的融合蛋白，其特征在于，所述荧光蛋白能够在相应波长的光照下显现特定颜色。
3.根据权利要求1所述的融合蛋白，其特征在于，包含如SEQ ID No.1~ SEQ ID No.9所示的氨基酸序列中的一种。
4.根据权利要求2所述的融合蛋白，其特征在于，包含与SEQ ID No.1~ SEQ ID No.9中的任意一种具有70％~100％相似性的氨基酸序列。
5.根据权利要求1所述的融合蛋白，其特征在于，所述荧光蛋白N包含如SEQ ID No.10所示的氨基酸序列。
6.根据权利要求1所述的融合蛋白，其特征在于，所述荧光蛋白C包含如SEQ ID No.11所示的氨基酸序列。
7.一种如权利要求1~6任一项所述的融合蛋白在RNA示踪中的应用。
8.一种RNA示踪方法，其特征在于，利用权利要求1~4任一项所述的融合蛋白。
CN201910351227.3A 2019-04-28 2019-04-28 一种用于rna示踪的融合蛋白及其应用 Active CN110128546B (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201910351227.3A CN110128546B (zh) 2019-04-28 2019-04-28 一种用于rna示踪的融合蛋白及其应用
PCT/CN2020/087527 WO2020221255A1 (zh) 2019-04-28 2020-04-28 一种rna结合融合蛋白及其用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910351227.3A CN110128546B (zh) 2019-04-28 2019-04-28 一种用于rna示踪的融合蛋白及其应用

Publications (2)

Publication Number Publication Date
CN110128546A true CN110128546A (zh) 2019-08-16
CN110128546B CN110128546B (zh) 2022-05-17

Family

ID=67575546

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910351227.3A Active CN110128546B (zh) 2019-04-28 2019-04-28 一种用于rna示踪的融合蛋白及其应用

Country Status (2)

Country Link
CN (1) CN110128546B (zh)
WO (1) WO2020221255A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020221255A1 (zh) * 2019-04-28 2020-11-05 河北科技大学 一种rna结合融合蛋白及其用途
CN113621647A (zh) * 2021-08-20 2021-11-09 湖南大学 用于内源性rna成像的遗传编码传感器及其制备方法和应用
WO2023284380A1 (zh) * 2021-07-15 2023-01-19 呈诺再生医学科技(北京)有限公司 细胞作为基因治疗的载体在临床中的应用
WO2023029532A1 (en) * 2021-08-30 2023-03-09 Huigene Therapeutics Co., Ltd. Engineered cas6 protein and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006037622A1 (en) * 2004-10-05 2006-04-13 DKFZ Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Screening process for the detection and characterization of protein-protein-interactions in vivo by fluorescence cross correlation spectroscopy
CN105624178A (zh) * 2014-10-31 2016-06-01 中国科学院武汉病毒研究所 一种基于荧光蛋白iRFP的双分子荧光片段互补系统及应用
US20170058271A1 (en) * 2015-08-28 2017-03-02 The General Hospital Corporation Engineered CRISPR-Cas9 Nucleases
CN106868031A (zh) * 2017-02-24 2017-06-20 北京大学 一种基于分级组装的多个sgRNA串联并行表达的克隆方法及应用
CN109385450A (zh) * 2017-08-02 2019-02-26 中国科学院武汉病毒研究所 一种基于荧光蛋白Venus的三片段荧光互补系统及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110128546B (zh) * 2019-04-28 2022-05-17 河北科技大学 一种用于rna示踪的融合蛋白及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006037622A1 (en) * 2004-10-05 2006-04-13 DKFZ Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Screening process for the detection and characterization of protein-protein-interactions in vivo by fluorescence cross correlation spectroscopy
CN105624178A (zh) * 2014-10-31 2016-06-01 中国科学院武汉病毒研究所 一种基于荧光蛋白iRFP的双分子荧光片段互补系统及应用
US20170058271A1 (en) * 2015-08-28 2017-03-02 The General Hospital Corporation Engineered CRISPR-Cas9 Nucleases
CN106868031A (zh) * 2017-02-24 2017-06-20 北京大学 一种基于分级组装的多个sgRNA串联并行表达的克隆方法及应用
CN109385450A (zh) * 2017-08-02 2019-02-26 中国科学院武汉病毒研究所 一种基于荧光蛋白Venus的三片段荧光互补系统及应用

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
CHUNYU HAN等: "Background free tracking of single RNA in living cells using catalytically inactive CasE", 《BIORXIV》 *
DR.SPENCER C. KNIGHT等: "Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies", 《ANGEW CHEM INT ED ENGL》 *
FENG GAO等: "A Cas6-based RNA tracking platform functioning in a fluorescence-activation mode", 《NUCLEIC ACIDS RESEARCH》 *
OLIVER RACKHAM等: "Visualization of RNA-protein interactions in living cells:FMRP and IMP1 interact on mRNAs", 《THE EMBO JOURNAL》 *
SIYU CHEN等: "CRISPR-Cas system for RNA detection and imagine", 《CHEMICAL RESEARCH IN CHINESE UNIVERSITY》 *
THOMAS J. MAGLIERY等: "Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism", 《JACS》 *
任文琳等: "CRISPR/Cas9介导的基因组编辑技术及其在RNA编辑中的潜在应用", 《生物技术进展》 *
孙兆明等: "生长抑制因子1基因核定位序列-绿荧光蛋白融合表达载体的构建及表达", 《中华医学遗传学杂志》 *
谢胜松等: "CRISPR/Cas9系统中sgRNA设计与脱靶效应评估", 《遗传》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020221255A1 (zh) * 2019-04-28 2020-11-05 河北科技大学 一种rna结合融合蛋白及其用途
WO2023284380A1 (zh) * 2021-07-15 2023-01-19 呈诺再生医学科技(北京)有限公司 细胞作为基因治疗的载体在临床中的应用
CN113621647A (zh) * 2021-08-20 2021-11-09 湖南大学 用于内源性rna成像的遗传编码传感器及其制备方法和应用
CN113621647B (zh) * 2021-08-20 2023-10-13 湖南大学 用于内源性rna成像的遗传编码传感器及其制备方法和应用
WO2023029532A1 (en) * 2021-08-30 2023-03-09 Huigene Therapeutics Co., Ltd. Engineered cas6 protein and uses thereof

Also Published As

Publication number Publication date
WO2020221255A1 (zh) 2020-11-05
CN110128546B (zh) 2022-05-17

Similar Documents

Publication Publication Date Title
CN110128546A (zh) 一种用于rna示踪的融合蛋白及其应用
Kreis et al. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins
Corvera et al. A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.
Case et al. The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain
CN103228669B (zh) 基于荧光共振能量转移的原理的单分子型fret生物传感器接头
US11325952B2 (en) Light-gated signaling modulation
CN106916795A (zh) 一种可调节的荧光素酶分段融合蛋白、其制备方法及应用
AU7634400A (en) Isolation of precursor cells and their use for tissue repair
CN104991072A (zh) 一种昆虫体外蛋白质相互作用检测系统的制备方法及应用
CN110468153A (zh) 一种远红光调控的基于CRISPR/Cas9系统的基因组转录装置、构建方法及应用
AU2012227790B2 (en) Probe for analyzing biological tissue and method for utilizing same
Bogorodskiy et al. Accessing mitochondrial protein import in living cells by protein microinjection
Marter et al. Optimized mRuby3 is a Suitable Fluorescent Protein for in vivo Co-localization Studies with GFP in the Diatom Phaeodactylum tricornutum
Grady et al. A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation
CN109897093A (zh) 多种改进型橙/红色荧光蛋白
US6656696B2 (en) Compositions and methods for monitoring the phosphorylation of natural binding partners
CN110229220A (zh) 一种基于纤维小体的毕赤酵母表面共展示系统及其构建方法和应用
CN108834418A (zh) 荧光蛋白质
JP6032862B2 (ja) サイクリックｇｍｐ解析方法
CN109777833A (zh) T细胞特异接头蛋白slp-76类泛素化的方法及相应的体外高效鉴定
Shi et al. Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein–Protein Interactions and Cellular Protein Complex Localizations
CN102181465B (zh) 一种荧光筛选克隆载体及其制备与应用
US20110008778A1 (en) Method of Generating p° Cells
US20240093206A1 (en) System of stable gene expression in cell lines and methods of making and using the same
CN111748035B (zh) 一组新型的fn3类抗体突变体及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant