CN109678939B

CN109678939B - 一种FnCpf1突变体

Info

Publication number: CN109678939B
Application number: CN201910008859.XA
Authority: CN
Inventors: 罗云孜; 王丽苹; 王浩君
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2018-04-27
Filing date: 2019-01-04
Publication date: 2022-03-04
Anticipated expiration: 2039-01-04
Also published as: CN109593763B; CN109593763A; CN109678939A

Abstract

本发明提供了一种FnCpf1其突变体，它是在野生型FnCpf1基础上，经过氨基酸位点突变得到，所述野生型FnCpf1的氨基酸序列如SEQ ID NO：1所示，所述突变的位点包含Asn607、Lys180、Lys660、Asp616中的至少一个突变。本发明FnCpf1突变体的是识别序列更多，应用范围更广。

Description

一种FnCpf1突变体

技术领域

本发明属于分子生物学领域，具体涉及一种FnCpf1突变体。

背景技术

二型限制限制性内切酶(RE)的发现促进了DNA重组技术的广泛应用。随后,合成生物学的飞速发展触发了对灵活多用的体外DNA组装/编辑策略的需求。迄今,体外DNA组装/编辑方法可分为两类:酶切连接和同源序列引导的同源重组。目前,最常用的酶切连接主要包括传统的基于限制性内切酶的酶切连接和Golden Gate组装。Golden Gate是基于IIS的限制性内切酶的一步克隆方法，并已优化用于多片段DNA的按序组装(Werner,Engler etal.2012)。但是，对于既定的DNA序列，IIS内切酶识别位点分布的不确定性和其存在数量的有限性，其编辑会受到限制。对于基于同源重组的体外编辑方法而言，例如Gibson组装，片段数的增加或者片段中高GC序列的出现，编辑效率均会受到影响而下降。另外，由于其序列依赖性，我们无法实现对于高相似序列的重复编辑，例如将同一个启动子重复引入至目标基因的不同位置。这些编辑方法中存在的限制也强调了对于开发新方法的需求性和重要性。

快速增长的技术-规律成簇的间隔短回文重复(CRISPR)系统近几年发展迅速，其中最广泛应用的CRISPR系统是来自于化脓链球菌的CRISPR/Cas9。该系统在研究者的设计和优化下，被用于辅助体外的分子克隆。但是，这些方法都是利用同源重组的方法将cas9剪切产生的平末端与目标载体相连，所以对于序列高相似性的DNA片段的编辑往往会受到一定的局限。

2015年，张锋等发现并鉴定了一种新的第二类V型的系统CRISPR-Cpf1，随后研究者发现并证明了其在多种有机体中的应用潜能和价值。相比于cas9的体外编辑，Cpf1具有以下优势：1)Cpf1剪切双链DNA产生粘性末端；2)Cpf1的引导不需要反向作用的RNA,所以引导crRNA的长度更短(～42bp)；3)Cpf1可以自己加工crRNA簇形成单个成熟的crRNA,使其易于开发用于多基因编辑。因此，基于Cpf1的体外编辑方法可以为cas9的应用提供另一种选择性。

在众多已经鉴定的Cpf1蛋白中，来源于Francisella novicida的FnCpf1具有一个明显的优势-FnCpf1识别的PAM较短(3bp),并且当间歇序列的长度为23bp时，特异性的剪切PAM远端的第18个碱基和互补链的第23碱基，产生5bp的粘性末端。由于Cpf1的剪切由crRNA(识别的间歇区通常为23bp)介导,相同的DNA序列出现的概率大大减少，因此很好的规避了识别位点非理想带来的限制。综上，FnCpf1介导的体外DNA编辑方法可以解决很多现有技术上的缺陷，但是FnCpf1识别范围限定于TTN，使用范围仍然有限。

发明内容

为了解决前述问题，本发明提供了一种识别范围更广的Cpf1突变体。

本发明FnCpf1突变体，它是在野生型FnCpf1基础上，经过氨基酸位点突变得到，所述野生型FnCpf1的氨基酸序列如SEQ ID NO：1所示，所述突变的位点包含Asn607、Lys180、Lys660、Asp616中的至少一个突变。

其中，它包含Asn607Arg、Lys180Ser、Lys660Arg、Asp616Asn的至少一个突变。

Asn607Arg，是指野生型FnCpf1第607位的氨基酸Asn突变成为了Arg，其余突变的解释方式相同。

其中，它还包含Lys671Arg突变、Lys613Val和/或Asn617Arg突变。

其中，它的氨基酸序列为SEQ ID NO：2或3所示。

本发明还提供了上述Cpf1突变体的核苷酸序列。

本发明还提供了一种重组载体，它包含编码上述Cpf1突变体的核苷酸序列；所述的重组载体是原核载体。

本发明还提供了一种重组菌，它包含上述的重组载体；优选地，所述细菌为大肠杆菌BL21(DE3)菌株。

本发明还提供了制备上述Cpf1突变体的方法。

本发明还提供了上述Cpf1突变体、核苷酸序列、重组载体，或重组菌在基因编辑中的用途。

其中，所述基因编辑包含基因敲除、基因突变、靶向基因激活/抑制、DNA连接、DNA多片段组装、DNA片段插入、DNA片段替换、碱基替换。

SEQ ID NO：1：野生型FnCpf1的氨基酸序列1-1300：MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN*

SEQ ID NO：2：EP15的氨基酸序列

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFSGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLARGWDKNKEPNNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYRLLPGANKMLPRVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN*

SEQ ID NO：3：EP16的氨基酸序列

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFSGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLARGWDKNVEPNRTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYRLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN*

EP15的核苷酸序列(SEQ ID NO：4)为：

ATGAGCATCTATCAGGAGTTCGTGAATAAGTACAGCCTGTCCAAGACCCTGCGGTTTGAGCTGATCCCCCAGGGCAAGACACTGGAGAACATCAAGGCCAGGGGCCTGATCCTGGACGATGAGAAGCGCGCCAAGGACTATAAGAAGGCCAAGCAGATCATCGATAAGTACCACCAGTTCTTTATCGAGGAGATCCTGAGCAGCGTGTGCATCTCTGAGGATCTGCTGCAGAATTACAGCGACGTGTATTTCAAGCTGAAGAAGTCTGACGATGACAACCTGCAGAAGGACTTCAAGAGCGCCAAGGACACCATCAAGAAGCAGATCAGCGAGTATATCAAGGACTCCGAGAAGTTTAAGAATCTGTTCAACCAGAATCTGATCGATGCCAAGAAGGGCCAGGAGTCCGACCTGATCCTGTGGCTGAAGCAGTCTAAGGACAATGGCATCGAGCTGTTCAAGGCCAACTCTGATATCACCGATATCGACGAGGCCCTGGAGATCATCAAGAGCTTTAAGGGCTGGACCACATACTTTAGCGGCTTCCACGAGAACAGGAAGAACGTGTACAGCAGCAACGACATCCCTACAAGCATCATCTACCGCATCGTGGATGACAATCTGCCAAAGTTCCTGGAGAACAAGGCCAAGTATGAGTCCCTGAAGGACAAGGCCCCCGAGGCCATCAATTACGAGCAGATCAAGAAGGATCTGGCCGAGGAGCTGACCTTCGATATCGACTATAAGACATCCGAGGTGAACCAGCGGGTGTTTTCTCTGGACGAGGTGTTTGAGATCGCCAATTTCAACAATTACCTGAACCAGTCCGGCATCACCAAGTTCAATACAATCATCGGCGGCAAGTTTGTGAACGGCGAGAATACCAAGAGAAAGGGCATCAACGAGTACATCAATCTGTATAGCCAGCAGATCAACGACAAGACCCTGAAGAAGTACAAGATGAGCGTGCTGTTCAAGCAGATCCTGTCCGATACAGAGTCTAAGAGCTTTGTGATCGATAAGCTGGAGGATGACTCTGACGTGGTGACCACAATGCAGAGCTTTTATGAGCAGATCGCCGCCTTCAAGACCGTGGAGGAGAAGTCTATCAAGGAGACACTGAGCCTGCTGTTCGATGACCTGAAGGCCCAGAAGCTGGACCTGTCTAAGATCTACTTCAAGAACGATAAGTCCCTGACCGACCTGTCTCAGCAGGTGTTTGATGACTATAGCGTGATCGGCACCGCCGTGCTGGAGTACATCACACAGCAGATCGCCCCAAAGAACCTGGATAATCCCTCTAAGAAGGAGCAGGAGCTGATCGCCAAGAAGACCGAGAAGGCCAAGTATCTGAGCCTGGAGACAATCAAGCTGGCCCTGGAGGAGTTCAATAAGCACCGGGATATCGACAAGCAGTGCAGATTTGAGGAGATCCTGGCCAACTTCGCCGCCATCCCCATGATCTTTGATGAGATCGCCCAGAACAAGGACAATCTGGCCCAGATCTCCATCAAGTACCAGAACCAGGGCAAGAAGGACCTGCTGCAGGCCTCTGCCGAGGATGACGTGAAGGCCATCAAGGATCTGCTGGACCAGACCAACAATCTGCTGCACAAGCTGAAGATCTTCCACATCTCCCAGTCTGAGGATAAGGCCAATATCCTGGATAAGGACGAGCACTTTTATCTGGTGTTCGAGGAGTGTTACTTCGAGCTGGCCAACATCGTGCCCCTGTACAACAAGATCAGAAATTATATCACACAGAAGCCTTACTCCGACGAGAAGTTTAAGCTGAACTTCGAGAACAGCACCCTGGCCAGAGGCTGGGATAAGAATAAGGAGCCTAACAACACAGCCATCCTGTTCATCAAGGATGACAAGTACTATCTGGGCGTGATGAATAAGAAGAACAATAAGATCTTCGATGACAAGGCCATCAAGGAGAACAAGGGCGAGGGCTACAAGAAGATCGTGTATAGGCTGCTGCCCGGCGCCAATAAGATGCTGCCTAGGGTGTTCTTTTCCGCCAAGTCTATCAAGTTCTACAACCCATCCGAGGACATCCTGCGGATCAGAAATCACTCCACCCACACAAAGAACGGCTCTCCCCAGAAGGGCTATGAGAAGTTTGAGTTCAATATCGAGGATTGCCGGAAGTTTATCGACTTCTACAAGCAGAGCATCTCCAAGCACCCTGAGTGGAAGGATTTTGGCTTCAGGTTTAGCGACACCCAGCGGTACAACTCCATCGACGAGTTCTACAGAGAGGTGGAGAATCAGGGCTATAAGCTGACATTTGAGAACATCTCTGAGAGCTACATCGACAGCGTGGTGAATCAGGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTCAGCGCCTATTCCAAGGGCCGGCCAAACCTGCACACCCTGTACTGGAAGGCCCTGTTCGATGAGAGAAATCTGCAGGACGTGGTGTATAAGCTGAACGGCGAGGCCGAGCTGTTTTACAGGAAGCAGTCCATCCCTAAGAAGATCACACACCCAGCCAAGGAGGCCATCGCCAACAAGAATAAGGACAATCCTAAGAAGGAGAGCGTGTTCGAGTACGATCTGATCAAGGACAAGCGGTTCACCGAGGATAAGTTCTTTTTCCACTGTCCAATCACAATCAACTTCAAGTCCTCTGGCGCCAACAAGTTTAATGACGAGATCAATCTGCTGCTGAAGGAGAAGGCCAACGATGTGCACATCCTGAGCATCGACCGGGGCGAGAGACACCTGGCCTACTATACCCTGGTGGATGGCAAGGGCAATATCATCAAGCAGGATACCTTCAACATCATCGGCAATGACAGGATGAAGACAAACTACCACGATAAGCTGGCCGCCATCGAGAAGGATAGGGACTCCGCCCGCAAGGACTGGAAGAAGATCAACAATATCAAGGAGATGAAGGAGGGCTATCTGTCTCAGGTGGTGCACGAGATCGCCAAGCTGGTCATCGAGTACAATGCCATCGTGGTGTTCGAGGATCTGAACTTCGGCTTTAAGAGGGGCCGCTTTAAGGTGGAGAAGCAGGTGTATCAGAAGCTGGAGAAGATGCTGATCGAGAAGCTGAATTACCTGGTGTTTAAGGATAACGAGTTCGACAAGACCGGAGGCGTGCTGAGGGCATACCAGCTGACCGCCCCCTTTGAGACATTCAAGAAGATGGGCAAGCAGACAGGCATCATCTACTATGTGCCAGCCGGCTTCACCTCCAAGATCTGCCCCGTGACAGGCTTTGTGAACCAGCTGTACCCTAAGTATGAGTCCGTGTCTAAGAGCCAGGAGTTTTTCAGCAAGTTCGATAAGATCTGTTATAATCTGGACAAGGGCTACTTCGAGTTTTCCTTCGATTATAAGAACTTTGGCGACAAGGCCGCCAAGGGCAAGTGGACCATCGCCTCTTTCGGCAGCCGGCTGATCAACTTTAGAAATTCCGATAAGAACCACAATTGGGACACCCGGGAGGTGTACCCAACAAAGGAGCTGGAGAAGCTGCTGAAGGACTACAGCATCGAGTATGGCCACGGCGAGTGCATCAAGGCCGCCATCTGTGGCGAGAGCGATAAGAAGTTTTTCGCCAAGCTGACCTCCGTGCTGAATACAATCCTGCAGATGCGGAACAGCAAGACCGGCACAGAGCTGGACTACCTGATCTCCCCCGTGGCCGATGTGAACGGCAACTTCTTCGACAGCAGACAGGCCCCCAAGAATATGCCTCAGGATGCCGACGCCAACGGCGCCTATCACATCGGCCTGAAGGGCCTGATGCTGCTGGGCAGGATCAAGAACAATCAGGAGGGCAAGAAGCTGAACCTGGTCATCAAGAACGAGGAGTACTTTGAGTTCGTGCAGAACCGCAACAATTGA

EP16的核苷酸序列(SEQ ID NO：5)为：

ATGAGCATCTATCAGGAGTTCGTGAATAAGTACAGCCTGTCCAAGACCCTGCGGTTTGAGCTGATCCCCCAGGGCAAGACACTGGAGAACATCAAGGCCAGGGGCCTGATCCTGGACGATGAGAAGCGCGCCAAGGACTATAAGAAGGCCAAGCAGATCATCGATAAGTACCACCAGTTCTTTATCGAGGAGATCCTGAGCAGCGTGTGCATCTCTGAGGATCTGCTGCAGAATTACAGCGACGTGTATTTCAAGCTGAAGAAGTCTGACGATGACAACCTGCAGAAGGACTTCAAGAGCGCCAAGGACACCATCAAGAAGCAGATCAGCGAGTATATCAAGGACTCCGAGAAGTTTAAGAATCTGTTCAACCAGAATCTGATCGATGCCAAGAAGGGCCAGGAGTCCGACCTGATCCTGTGGCTGAAGCAGTCTAAGGACAATGGCATCGAGCTGTTCAAGGCCAACTCTGATATCACCGATATCGACGAGGCCCTGGAGATCATCAAGAGCTTTAAGGGCTGGACCACATACTTTAGCGGCTTCCACGAGAACAGGAAGAACGTGTACAGCAGCAACGACATCCCTACAAGCATCATCTACCGCATCGTGGATGACAATCTGCCAAAGTTCCTGGAGAACAAGGCCAAGTATGAGTCCCTGAAGGACAAGGCCCCCGAGGCCATCAATTACGAGCAGATCAAGAAGGATCTGGCCGAGGAGCTGACCTTCGATATCGACTATAAGACATCCGAGGTGAACCAGCGGGTGTTTTCTCTGGACGAGGTGTTTGAGATCGCCAATTTCAACAATTACCTGAACCAGTCCGGCATCACCAAGTTCAATACAATCATCGGCGGCAAGTTTGTGAACGGCGAGAATACCAAGAGAAAGGGCATCAACGAGTACATCAATCTGTATAGCCAGCAGATCAACGACAAGACCCTGAAGAAGTACAAGATGAGCGTGCTGTTCAAGCAGATCCTGTCCGATACAGAGTCTAAGAGCTTTGTGATCGATAAGCTGGAGGATGACTCTGACGTGGTGACCACAATGCAGAGCTTTTATGAGCAGATCGCCGCCTTCAAGACCGTGGAGGAGAAGTCTATCAAGGAGACACTGAGCCTGCTGTTCGATGACCTGAAGGCCCAGAAGCTGGACCTGTCTAAGATCTACTTCAAGAACGATAAGTCCCTGACCGACCTGTCTCAGCAGGTGTTTGATGACTATAGCGTGATCGGCACCGCCGTGCTGGAGTACATCACACAGCAGATCGCCCCAAAGAACCTGGATAATCCCTCTAAGAAGGAGCAGGAGCTGATCGCCAAGAAGACCGAGAAGGCCAAGTATCTGAGCCTGGAGACAATCAAGCTGGCCCTGGAGGAGTTCAATAAGCACCGGGATATCGACAAGCAGTGCAGATTTGAGGAGATCCTGGCCAACTTCGCCGCCATCCCCATGATCTTTGATGAGATCGCCCAGAACAAGGACAATCTGGCCCAGATCTCCATCAAGTACCAGAACCAGGGCAAGAAGGACCTGCTGCAGGCCTCTGCCGAGGATGACGTGAAGGCCATCAAGGATCTGCTGGACCAGACCAACAATCTGCTGCACAAGCTGAAGATCTTCCACATCTCCCAGTCTGAGGATAAGGCCAATATCCTGGATAAGGACGAGCACTTTTATCTGGTGTTCGAGGAGTGTTACTTCGAGCTGGCCAACATCGTGCCCCTGTACAACAAGATCAGAAATTATATCACACAGAAGCCTTACTCCGACGAGAAGTTTAAGCTGAACTTCGAGAACAGCACCCTGGCCAGAGGCTGGGATAAGAATGTGGAGCCTAACAGAACAGCCATCCTGTTCATCAAGGATGACAAGTACTATCTGGGCGTGATGAATAAGAAGAACAATAAGATCTTCGATGACAAGGCCATCAAGGAGAACAAGGGCGAGGGCTACAAGAAGATCGTGTATAGGCTGCTGCCCGGCGCCAATAAGATGCTGCCTAAGGTGTTCTTTTCCGCCAAGTCTATCAAGTTCTACAACCCATCCGAGGACATCCTGCGGATCAGAAATCACTCCACCCACACAAAGAACGGCTCTCCCCAGAAGGGCTATGAGAAGTTTGAGTTCAATATCGAGGATTGCCGGAAGTTTATCGACTTCTACAAGCAGAGCATCTCCAAGCACCCTGAGTGGAAGGATTTTGGCTTCAGGTTTAGCGACACCCAGCGGTACAACTCCATCGACGAGTTCTACAGAGAGGTGGAGAATCAGGGCTATAAGCTGACATTTGAGAACATCTCTGAGAGCTACATCGACAGCGTGGTGAATCAGGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTCAGCGCCTATTCCAAGGGCCGGCCAAACCTGCACACCCTGTACTGGAAGGCCCTGTTCGATGAGAGAAATCTGCAGGACGTGGTGTATAAGCTGAACGGCGAGGCCGAGCTGTTTTACAGGAAGCAGTCCATCCCTAAGAAGATCACACACCCAGCCAAGGAGGCCATCGCCAACAAGAATAAGGACAATCCTAAGAAGGAGAGCGTGTTCGAGTACGATCTGATCAAGGACAAGCGGTTCACCGAGGATAAGTTCTTTTTCCACTGTCCAATCACAATCAACTTCAAGTCCTCTGGCGCCAACAAGTTTAATGACGAGATCAATCTGCTGCTGAAGGAGAAGGCCAACGATGTGCACATCCTGAGCATCGACCGGGGCGAGAGACACCTGGCCTACTATACCCTGGTGGATGGCAAGGGCAATATCATCAAGCAGGATACCTTCAACATCATCGGCAATGACAGGATGAAGACAAACTACCACGATAAGCTGGCCGCCATCGAGAAGGATAGGGACTCCGCCCGCAAGGACTGGAAGAAGATCAACAATATCAAGGAGATGAAGGAGGGCTATCTGTCTCAGGTGGTGCACGAGATCGCCAAGCTGGTCATCGAGTACAATGCCATCGTGGTGTTCGAGGATCTGAACTTCGGCTTTAAGAGGGGCCGCTTTAAGGTGGAGAAGCAGGTGTATCAGAAGCTGGAGAAGATGCTGATCGAGAAGCTGAATTACCTGGTGTTTAAGGATAACGAGTTCGACAAGACCGGAGGCGTGCTGAGGGCATACCAGCTGACCGCCCCCTTTGAGACATTCAAGAAGATGGGCAAGCAGACAGGCATCATCTACTATGTGCCAGCCGGCTTCACCTCCAAGATCTGCCCCGTGACAGGCTTTGTGAACCAGCTGTACCCTAAGTATGAGTCCGTGTCTAAGAGCCAGGAGTTTTTCAGCAAGTTCGATAAGATCTGTTATAATCTGGACAAGGGCTACTTCGAGTTTTCCTTCGATTATAAGAACTTTGGCGACAAGGCCGCCAAGGGCAAGTGGACCATCGCCTCTTTCGGCAGCCGGCTGATCAACTTTAGAAATTCCGATAAGAACCACAATTGGGACACCCGGGAGGTGTACCCAACAAAGGAGCTGGAGAAGCTGCTGAAGGACTACAGCATCGAGTATGGCCACGGCGAGTGCATCAAGGCCGCCATCTGTGGCGAGAGCGATAAGAAGTTTTTCGCCAAGCTGACCTCCGTGCTGAATACAATCCTGCAGATGCGGAACAGCAAGACCGGCACAGAGCTGGACTACCTGATCTCCCCCGTGGCCGATGTGAACGGCAACTTCTTCGACAGCAGACAGGCCCCCAAGAATATGCCTCAGGATGCCGACGCCAACGGCGCCTATCACATCGGCCTGAAGGGCCTGATGCTGCTGGGCAGGATCAAGAACAATCAGGAGGGCAAGAAGCTGAACCTGGTCATCAAGAACGAGGAGTACTTTGAGTTCGTGCAGAACCGCAACAATTGA

本发明改进后的FnCpf1突变体对序列的识别范围广，是野生型FnCpf1识别范围的2～2.5倍，可以应用于CRISPR-Cpf1系统，编辑DNA片段，可以克服传统野生型FnCpf1识别范围太窄，应用范围太窄的问题，应用前景优良。

显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。

以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。

附图说明

图1野生型FnCpf1及突变体蛋白EP15和EP16的SDS-PAGE电泳图谱，Cpf1蛋白大小151KDa

图2野生FnCpf1的PAM检测结果图，模板大小：1234bp,产物大小：707bp和527bp；

图3FnCpf1突变体EP15的PAM检测结果图，模板大小：1234bp,产物大小：707bp和527bp；

图4FnCpf1突变体EP16的PAM检测结果图，模板大小：1234bp,产物大小：707bp和527bp。

具体实施方式

下面以实施例作进一步说明，但本发明不局限于这些实施例。

实施例1FnCpf1突变体的制备以及效果验证一、酶的制备(克隆：表达和纯化野生型和突变型FnCpf1蛋白)

1、制备突变质粒

1)FnCpf1(WP003040289)表达质粒来源于上海中科院。随后FnCpf1突变体的表达质粒以此为基础，进行定点突变PCR，在：野生型FnCpf 1(氨基酸序列如SEQ ID NO：1所示)的基础上，拟突变制备突变体EP15和EP16，其中EP15的突变为：Lys180Ser、Asn607Arg、Asp616Asn、Lys660Arg和Lys671Arg突变，EP16的突变为：Lys180Ser、Asn607Arg、Lys613Val、Asp616Asn、Asn617Arg和Lys660Arg。

2)根据拟突变的氨基酸序列进行设计引物，引物由北京擎科新业生物技术有限公司合成，所用引物见表1：

表1用于获得突变蛋白的引物

利用所得的引物进行定点突变PCR，模板为pET28TEV-FnCpf1质粒，所用酶为Q5高保真酶(NEB公司，货号M0491)。具体配置体系如下：

反应程序为：98℃，2min；反应98℃，10s；退火，10s；60℃，5min；72℃，5min；4℃，保存；运行25个循环。

对PCR产物进行胶回收(北京天根生化科技有限公司，货号DP214)，随后利用限制性内切酶Dpn I(NEB公司，货号R0176L)去除模板(37℃，2h)，将上述所得反应液转化大肠杆菌感受态细胞(DH5α)中，涂布于LB(含50μg/mL卡那抗生素)平板中，置于37℃培养箱中过夜。挑取平板中的单克隆，测序(北京擎科新业生物技术有限公司)，获得含有目标突变(目标片段如)的正确克隆。

2、野生型及突变体蛋白的表达纯化

蛋白纯化的步骤为：

1)将表达质粒转化至E.coli BL21(DE3)；

2)挑单克隆加到3ml含有50ug/ml kanamycin的LB液体培养基中37℃，220rpm过夜培养16小时；

3)将过夜培养菌液按1：1000加到含有50ug/ml kanamycin的LB液体培养基中，在37℃,200rpm培养至OD600＝0.2左右，随后将培养箱降温至20℃,继续220rpm培养直到OD600达到0.6-0.8；

4)加入IPTG诱导剂，随后进行20℃,220rpm过夜培养；

5)将过夜菌液4℃,3800rpm离心10分钟收菌，弃上清，将装有菌块的离心瓶置于冰上，用30ml含有1mM DTT和1mM PMSF的预冷1x Cpf1lysis buffer重悬菌块；

6)将重悬后的菌液转入50ml国产BD管中，置于冰上，随后使用sonicator进行超声破菌(参数设置为30％强度，开3秒，停6秒，超声15分钟)；

7)将细菌裂解液转入高速塑料离心管中，进行40C,18000rpm,30分钟高速离心；

8)高速离心期间，用5-10CV预冷的1x Cpf1washing buffer对镍柱进行平衡；

9)将7)中的上清液转入50ml BD管中，并加入8)中平衡过的镍胶与其混合，在层析柜中旋转混合一个小时使his-tag与镍结合；

10)将9中的蛋白镍胶混合液重新转入空柱中，用250ml预冷的1x Cpf1washingbuffer洗掉未与镍胶结合的杂蛋白，收集第一批穿出样本用于跑胶的阴性对照；

11)随后加入20ml预冷的1x Cpf1elution buffer洗脱目标蛋白,保留少量洗脱样本用于跑胶；

12)使用nanodrop测量蛋白浓度，然后按照1mg的TEV酶去切100mg的目标蛋白的比例加TEV酶进行过夜剪切；

13)使用millipore离心浓缩管将TEV酶过夜酶切的目标蛋白样本浓缩至0.2-0.5ml，随后加入50ml预冷的1x Cpf1washing buffer稀释样本中的咪唑浓度；

14)样本浓缩期间，用5-10CV预冷的1x Cpf1washing buffer对镍柱进行平衡，为反挂镍做准备；

15)将13)和14)混合，按照9进行操作；

16)将15)中的样本重新转入空柱中，收集含有切掉his-tag的目标蛋白的穿出液，随后用1x elution buffer洗脱镍胶，洗脱液做为TEV酶的酶切效果的阴性对照；

17)测量16)中收集的穿出液，按照浓度进行进一步的浓缩；

18)进行12％SDG-PAGE检测蛋白纯度；

19)制备50％(v/v)甘油Cpf1蛋白储存样本，速冻并保存在-80℃中。

采用上述方法，制备得到野生型FnCpf1及突变体蛋白P15和P16，采用电泳等方式验证的确得到了野生型FnCpf1及突变体蛋白P15和P16(如图1)。

二、酶活的验证

由于FnCpf1识别的PAM为3位，故设计含有PAM为5’-NNN-spacer-3’的DNA底物，用于酶活的验证。根据反应需要，合成与底物对应的crRNA。

1、CrRNA合成

1)转录的模板DNA制备：根据crRNA需要，设计合成转录的模板DNA对应的引物如下表2：

表2用于PAM测试的crRNA制备引物

利用所得的引物进行DNA富集PCR，具体配置体系如下：

反应程序为：98℃，2min；反应98℃，10s；退火，10s；50℃，7s；72℃，5min；4℃，保存；运行35个循环。

PCR反应液采用DNA纯化试剂盒回收(Tiangen,Beijing,China)。

1)crRNA的合成：RNA的合成参照HiScribe^TMT7High Yield RNA Synthesis Kit(NEB)的标准操作方案。制备好的RNA采用RNA Clean&Concentrator-5kit(Zymo Research,CA,USA)回收。浓度测量采用NanoDrop 2000C(Thermo Fisher Scientific,Massachusetts,USA)。RNA稀释至20μM备用；

2)底物DNA样品制备

为了制备具有对比性的DNA模板，我们首先制备了一系列包含同一spacer不同PAM的质粒库(表3)。然后利用PCR的方式富集得到64种PAM不同的1300bp的DNA底物；PCR引物如(表4)

表3 PAM验证质粒库

表4 PAM测试DNA底物扩增引物

利用所得的引物进行DNA富集PCR，具体配置体系如下：

反应液采用DNA纯化试剂盒回收(Tiangen,Beijing,China),浓度测量采用NanoDrop 2000C(Thermo Fisher Scientific,Massachusetts,USA)。模板DNA(对应表3标记为1-64)稀释至100ng/μl备用；

2、酶切反应及检测

表5反应加样

反应条件：37℃反应3h,然后75℃处理10min失活；然后向反应液加入适宜比例的loading dye,NEB,(6X)；

凝胶成像采用Gel Doc^TM XR+with Image Lab^TM Software(BIO-RAD,California,USA)，并通过对应软件分析各条带的体积值，通过公式计算得到PAM体外效率。

[E＝100X(((1-(1-(b+c)/(a+b+c))^^1/2)]。

3、结果

凝胶成像结果如图2～图4所示，对应分析得到剪切效率统计为表6：

表6 PAM鉴定结果

由表6可以看出，野生型FnCpf1对TTA等9种序列的识别效率高，而经过本发明改进后的FnCpf1突变体EP15对TTA等18种序列的识别效果率均非常高，FnCpf1突变体EP16对24种序列的识别效果率均非常高。

实验结果说明，本发明改进后的突变体对序列的识别范围广，可以用于更多序列的编辑，应用范围更广，实际应用前景更优良。

SEQUENCE LISTING

<110> 四川大学华西医院

<120> 一种FnCpf1突变体

<130> GY026-2019P013441CC

<150> 201810393544.7

<151> 2018-04-27

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1300

<212> PRT

<213> Francisella novicida（野生型FnCpf1的氨基酸序）

<400> 1

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Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

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Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

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Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

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Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

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Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

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Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

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Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

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Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

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Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

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Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

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Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

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Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

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Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

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Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

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Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

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Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

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Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

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Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

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Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

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Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

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Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

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His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

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Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

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Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

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Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

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Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

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Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

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Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

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Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

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Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

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Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

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Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

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Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

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Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

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Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

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Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

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Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

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Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

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Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

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Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

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Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 2

<211> 1300

<212> PRT

<213> Artificial Sequence

<220>

<223> EP15的氨基酸序列

<400> 2

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Ser Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Arg Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asn Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Arg Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Arg Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 3

<211> 1300

<212> PRT

<213> Artificial Sequence

<220>

<223> EP16的氨基酸序列

<400> 3

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Ser Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Arg Gly

595 600 605

Trp Asp Lys Asn Val Glu Pro Asn Arg Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Arg Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 4

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> EP15的核苷酸序列

<400> 4

atgagcatct atcaggagtt cgtgaataag tacagcctgt ccaagaccct gcggtttgag 60

ctgatccccc agggcaagac actggagaac atcaaggcca ggggcctgat cctggacgat 120

gagaagcgcg ccaaggacta taagaaggcc aagcagatca tcgataagta ccaccagttc 180

tttatcgagg agatcctgag cagcgtgtgc atctctgagg atctgctgca gaattacagc 240

gacgtgtatt tcaagctgaa gaagtctgac gatgacaacc tgcagaagga cttcaagagc 300

gccaaggaca ccatcaagaa gcagatcagc gagtatatca aggactccga gaagtttaag 360

aatctgttca accagaatct gatcgatgcc aagaagggcc aggagtccga cctgatcctg 420

tggctgaagc agtctaagga caatggcatc gagctgttca aggccaactc tgatatcacc 480

gatatcgacg aggccctgga gatcatcaag agctttaagg gctggaccac atactttagc 540

ggcttccacg agaacaggaa gaacgtgtac agcagcaacg acatccctac aagcatcatc 600

taccgcatcg tggatgacaa tctgccaaag ttcctggaga acaaggccaa gtatgagtcc 660

ctgaaggaca aggcccccga ggccatcaat tacgagcaga tcaagaagga tctggccgag 720

gagctgacct tcgatatcga ctataagaca tccgaggtga accagcgggt gttttctctg 780

gacgaggtgt ttgagatcgc caatttcaac aattacctga accagtccgg catcaccaag 840

ttcaatacaa tcatcggcgg caagtttgtg aacggcgaga ataccaagag aaagggcatc 900

aacgagtaca tcaatctgta tagccagcag atcaacgaca agaccctgaa gaagtacaag 960

atgagcgtgc tgttcaagca gatcctgtcc gatacagagt ctaagagctt tgtgatcgat 1020

aagctggagg atgactctga cgtggtgacc acaatgcaga gcttttatga gcagatcgcc 1080

gccttcaaga ccgtggagga gaagtctatc aaggagacac tgagcctgct gttcgatgac 1140

ctgaaggccc agaagctgga cctgtctaag atctacttca agaacgataa gtccctgacc 1200

gacctgtctc agcaggtgtt tgatgactat agcgtgatcg gcaccgccgt gctggagtac 1260

atcacacagc agatcgcccc aaagaacctg gataatccct ctaagaagga gcaggagctg 1320

atcgccaaga agaccgagaa ggccaagtat ctgagcctgg agacaatcaa gctggccctg 1380

gaggagttca ataagcaccg ggatatcgac aagcagtgca gatttgagga gatcctggcc 1440

aacttcgccg ccatccccat gatctttgat gagatcgccc agaacaagga caatctggcc 1500

cagatctcca tcaagtacca gaaccagggc aagaaggacc tgctgcaggc ctctgccgag 1560

gatgacgtga aggccatcaa ggatctgctg gaccagacca acaatctgct gcacaagctg 1620

aagatcttcc acatctccca gtctgaggat aaggccaata tcctggataa ggacgagcac 1680

ttttatctgg tgttcgagga gtgttacttc gagctggcca acatcgtgcc cctgtacaac 1740

aagatcagaa attatatcac acagaagcct tactccgacg agaagtttaa gctgaacttc 1800

gagaacagca ccctggccag aggctgggat aagaataagg agcctaacaa cacagccatc 1860

ctgttcatca aggatgacaa gtactatctg ggcgtgatga ataagaagaa caataagatc 1920

ttcgatgaca aggccatcaa ggagaacaag ggcgagggct acaagaagat cgtgtatagg 1980

ctgctgcccg gcgccaataa gatgctgcct agggtgttct tttccgccaa gtctatcaag 2040

ttctacaacc catccgagga catcctgcgg atcagaaatc actccaccca cacaaagaac 2100

ggctctcccc agaagggcta tgagaagttt gagttcaata tcgaggattg ccggaagttt 2160

atcgacttct acaagcagag catctccaag caccctgagt ggaaggattt tggcttcagg 2220

tttagcgaca cccagcggta caactccatc gacgagttct acagagaggt ggagaatcag 2280

ggctataagc tgacatttga gaacatctct gagagctaca tcgacagcgt ggtgaatcag 2340

ggcaagctgt acctgttcca gatctataac aaggacttca gcgcctattc caagggccgg 2400

ccaaacctgc acaccctgta ctggaaggcc ctgttcgatg agagaaatct gcaggacgtg 2460

gtgtataagc tgaacggcga ggccgagctg ttttacagga agcagtccat ccctaagaag 2520

atcacacacc cagccaagga ggccatcgcc aacaagaata aggacaatcc taagaaggag 2580

agcgtgttcg agtacgatct gatcaaggac aagcggttca ccgaggataa gttctttttc 2640

cactgtccaa tcacaatcaa cttcaagtcc tctggcgcca acaagtttaa tgacgagatc 2700

aatctgctgc tgaaggagaa ggccaacgat gtgcacatcc tgagcatcga ccggggcgag 2760

agacacctgg cctactatac cctggtggat ggcaagggca atatcatcaa gcaggatacc 2820

ttcaacatca tcggcaatga caggatgaag acaaactacc acgataagct ggccgccatc 2880

gagaaggata gggactccgc ccgcaaggac tggaagaaga tcaacaatat caaggagatg 2940

aaggagggct atctgtctca ggtggtgcac gagatcgcca agctggtcat cgagtacaat 3000

gccatcgtgg tgttcgagga tctgaacttc ggctttaaga ggggccgctt taaggtggag 3060

aagcaggtgt atcagaagct ggagaagatg ctgatcgaga agctgaatta cctggtgttt 3120

aaggataacg agttcgacaa gaccggaggc gtgctgaggg cataccagct gaccgccccc 3180

tttgagacat tcaagaagat gggcaagcag acaggcatca tctactatgt gccagccggc 3240

ttcacctcca agatctgccc cgtgacaggc tttgtgaacc agctgtaccc taagtatgag 3300

tccgtgtcta agagccagga gtttttcagc aagttcgata agatctgtta taatctggac 3360

aagggctact tcgagttttc cttcgattat aagaactttg gcgacaaggc cgccaagggc 3420

aagtggacca tcgcctcttt cggcagccgg ctgatcaact ttagaaattc cgataagaac 3480

cacaattggg acacccggga ggtgtaccca acaaaggagc tggagaagct gctgaaggac 3540

tacagcatcg agtatggcca cggcgagtgc atcaaggccg ccatctgtgg cgagagcgat 3600

aagaagtttt tcgccaagct gacctccgtg ctgaatacaa tcctgcagat gcggaacagc 3660

aagaccggca cagagctgga ctacctgatc tcccccgtgg ccgatgtgaa cggcaacttc 3720

ttcgacagca gacaggcccc caagaatatg cctcaggatg ccgacgccaa cggcgcctat 3780

cacatcggcc tgaagggcct gatgctgctg ggcaggatca agaacaatca ggagggcaag 3840

aagctgaacc tggtcatcaa gaacgaggag tactttgagt tcgtgcagaa ccgcaacaat 3900

tga 3903

<210> 5

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> EP16的核苷酸序列

<400> 5

atgagcatct atcaggagtt cgtgaataag tacagcctgt ccaagaccct gcggtttgag 60

ctgatccccc agggcaagac actggagaac atcaaggcca ggggcctgat cctggacgat 120

gagaagcgcg ccaaggacta taagaaggcc aagcagatca tcgataagta ccaccagttc 180

tttatcgagg agatcctgag cagcgtgtgc atctctgagg atctgctgca gaattacagc 240

gacgtgtatt tcaagctgaa gaagtctgac gatgacaacc tgcagaagga cttcaagagc 300

gccaaggaca ccatcaagaa gcagatcagc gagtatatca aggactccga gaagtttaag 360

aatctgttca accagaatct gatcgatgcc aagaagggcc aggagtccga cctgatcctg 420

tggctgaagc agtctaagga caatggcatc gagctgttca aggccaactc tgatatcacc 480

gatatcgacg aggccctgga gatcatcaag agctttaagg gctggaccac atactttagc 540

ggcttccacg agaacaggaa gaacgtgtac agcagcaacg acatccctac aagcatcatc 600

taccgcatcg tggatgacaa tctgccaaag ttcctggaga acaaggccaa gtatgagtcc 660

ctgaaggaca aggcccccga ggccatcaat tacgagcaga tcaagaagga tctggccgag 720

gagctgacct tcgatatcga ctataagaca tccgaggtga accagcgggt gttttctctg 780

gacgaggtgt ttgagatcgc caatttcaac aattacctga accagtccgg catcaccaag 840

ttcaatacaa tcatcggcgg caagtttgtg aacggcgaga ataccaagag aaagggcatc 900

aacgagtaca tcaatctgta tagccagcag atcaacgaca agaccctgaa gaagtacaag 960

atgagcgtgc tgttcaagca gatcctgtcc gatacagagt ctaagagctt tgtgatcgat 1020

aagctggagg atgactctga cgtggtgacc acaatgcaga gcttttatga gcagatcgcc 1080

gccttcaaga ccgtggagga gaagtctatc aaggagacac tgagcctgct gttcgatgac 1140

ctgaaggccc agaagctgga cctgtctaag atctacttca agaacgataa gtccctgacc 1200

gacctgtctc agcaggtgtt tgatgactat agcgtgatcg gcaccgccgt gctggagtac 1260

atcacacagc agatcgcccc aaagaacctg gataatccct ctaagaagga gcaggagctg 1320

atcgccaaga agaccgagaa ggccaagtat ctgagcctgg agacaatcaa gctggccctg 1380

gaggagttca ataagcaccg ggatatcgac aagcagtgca gatttgagga gatcctggcc 1440

aacttcgccg ccatccccat gatctttgat gagatcgccc agaacaagga caatctggcc 1500

cagatctcca tcaagtacca gaaccagggc aagaaggacc tgctgcaggc ctctgccgag 1560

gatgacgtga aggccatcaa ggatctgctg gaccagacca acaatctgct gcacaagctg 1620

aagatcttcc acatctccca gtctgaggat aaggccaata tcctggataa ggacgagcac 1680

ttttatctgg tgttcgagga gtgttacttc gagctggcca acatcgtgcc cctgtacaac 1740

aagatcagaa attatatcac acagaagcct tactccgacg agaagtttaa gctgaacttc 1800

gagaacagca ccctggccag aggctgggat aagaatgtgg agcctaacag aacagccatc 1860

ctgttcatca aggatgacaa gtactatctg ggcgtgatga ataagaagaa caataagatc 1920

ttcgatgaca aggccatcaa ggagaacaag ggcgagggct acaagaagat cgtgtatagg 1980

ctgctgcccg gcgccaataa gatgctgcct aaggtgttct tttccgccaa gtctatcaag 2040

ttctacaacc catccgagga catcctgcgg atcagaaatc actccaccca cacaaagaac 2100

ggctctcccc agaagggcta tgagaagttt gagttcaata tcgaggattg ccggaagttt 2160

atcgacttct acaagcagag catctccaag caccctgagt ggaaggattt tggcttcagg 2220

tttagcgaca cccagcggta caactccatc gacgagttct acagagaggt ggagaatcag 2280

ggctataagc tgacatttga gaacatctct gagagctaca tcgacagcgt ggtgaatcag 2340

ggcaagctgt acctgttcca gatctataac aaggacttca gcgcctattc caagggccgg 2400

ccaaacctgc acaccctgta ctggaaggcc ctgttcgatg agagaaatct gcaggacgtg 2460

gtgtataagc tgaacggcga ggccgagctg ttttacagga agcagtccat ccctaagaag 2520

atcacacacc cagccaagga ggccatcgcc aacaagaata aggacaatcc taagaaggag 2580

agcgtgttcg agtacgatct gatcaaggac aagcggttca ccgaggataa gttctttttc 2640

cactgtccaa tcacaatcaa cttcaagtcc tctggcgcca acaagtttaa tgacgagatc 2700

aatctgctgc tgaaggagaa ggccaacgat gtgcacatcc tgagcatcga ccggggcgag 2760

agacacctgg cctactatac cctggtggat ggcaagggca atatcatcaa gcaggatacc 2820

ttcaacatca tcggcaatga caggatgaag acaaactacc acgataagct ggccgccatc 2880

gagaaggata gggactccgc ccgcaaggac tggaagaaga tcaacaatat caaggagatg 2940

aaggagggct atctgtctca ggtggtgcac gagatcgcca agctggtcat cgagtacaat 3000

gccatcgtgg tgttcgagga tctgaacttc ggctttaaga ggggccgctt taaggtggag 3060

aagcaggtgt atcagaagct ggagaagatg ctgatcgaga agctgaatta cctggtgttt 3120

aaggataacg agttcgacaa gaccggaggc gtgctgaggg cataccagct gaccgccccc 3180

tttgagacat tcaagaagat gggcaagcag acaggcatca tctactatgt gccagccggc 3240

ttcacctcca agatctgccc cgtgacaggc tttgtgaacc agctgtaccc taagtatgag 3300

tccgtgtcta agagccagga gtttttcagc aagttcgata agatctgtta taatctggac 3360

aagggctact tcgagttttc cttcgattat aagaactttg gcgacaaggc cgccaagggc 3420

aagtggacca tcgcctcttt cggcagccgg ctgatcaact ttagaaattc cgataagaac 3480

cacaattggg acacccggga ggtgtaccca acaaaggagc tggagaagct gctgaaggac 3540

tacagcatcg agtatggcca cggcgagtgc atcaaggccg ccatctgtgg cgagagcgat 3600

aagaagtttt tcgccaagct gacctccgtg ctgaatacaa tcctgcagat gcggaacagc 3660

aagaccggca cagagctgga ctacctgatc tcccccgtgg ccgatgtgaa cggcaacttc 3720

ttcgacagca gacaggcccc caagaatatg cctcaggatg ccgacgccaa cggcgcctat 3780

cacatcggcc tgaagggcct gatgctgctg ggcaggatca agaacaatca ggagggcaag 3840

aagctgaacc tggtcatcaa gaacgaggag tactttgagt tcgtgcagaa ccgcaacaat 3900

tga 3903

Claims

1.一种Cpf1突变体，其特征在于：所述Cpf1突变体的氨基酸序列为SEQ ID NO：2或3所示。

2.编码权利要求1所述Cpf1突变体的核苷酸序列，核苷酸序列如SEQ ID NO：4或5所示。

3.一种重组载体，其特征在于：它包含编码权利要求1所述Cpf1突变体的核苷酸序列；所述的重组载体是原核载体。

4.一种重组菌，其特征在于：它包含权利要求3所述的重组载体。

5.权利要求4所述的重组菌，其特征在于：所述菌为大肠杆菌BL21 (DE3)菌株。

6.制备权利要求1所述Cpf1突变体的方法。

7.权利要求1所述Cpf1突变体、权利要求2所述的核苷酸序列、权利要求3所述的重组载体，或权利要求4或5所述的重组菌在基因编辑中的用途。

8.根据权利要求7所述的用途，其特征在于：所述基因编辑包含基因敲除、基因突变、靶向基因激活/抑制、DNA连接、DNA多片段组装、DNA片段插入、DNA片段替换、碱基替换。