CN114717209B - 具有增加的耐盐性的t4 dna连接酶变体 - Google Patents
具有增加的耐盐性的t4 dna连接酶变体 Download PDFInfo
- Publication number
- CN114717209B CN114717209B CN202210556291.7A CN202210556291A CN114717209B CN 114717209 B CN114717209 B CN 114717209B CN 202210556291 A CN202210556291 A CN 202210556291A CN 114717209 B CN114717209 B CN 114717209B
- Authority
- CN
- China
- Prior art keywords
- lys
- leu
- glu
- gly
- ile
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
- C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
- C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
Abstract
与野生型连接酶相比在高盐浓度下表现出增强的连接活性的多种T4DNA连接酶突变体，将其工程化、表征并经由来自标准连接测定的连接产物的凝胶电泳来选择。连接酶催化在双螺旋DNA的互补粘性末端或平末端的5'端和3'端之间形成磷酸二酯键，该过程对包括克隆和测序的许多分子生物学过程至关重要。
Description
背景技术
DNA连接酶是活生物体中不可或缺的修复蛋白，其催化双链DNA中单链断裂的修复。T4 DNA连接酶利用生物分子ATP的能量来修复带切口的双链DNA，还连接具有互补单链突出端（粘性末端）以及平末端片段的双链DNA，使其成为用于在体外组合较小DNA片段以产生双链DNA构建体的有用工具。
DNA连接酶在并置的5'磷酸和3'羟基末端的交叉处形成双螺旋核酸片段之间的磷酸二酯键。通过设计每个双链片段之间的互补突出端，连接可以是位置特异性连接和定向连接二者。这允许DNA或RNA片段特异性整合到更大的载体中，以满足分子生物学研究的需要。在可商购获得的连接酶中，T4 DNA连接酶是一种多功能的酶，其催化在突出端和平末端二者处连接双螺旋DNA或RNA的键，具有快速的连接速度，并经由连接修复带切口DNA中存在的错配。因为T4 DNA连接酶是许多分子生物学方案的支柱，并且被持续需要，因此由于它在分子生物学试剂市场中的重要性而使它为许多生命科学产品公司创造了很大份额的收入。
通常，野生型（WT）T4 DNA连接酶由于反应缓冲液中存在盐（诸如氯化钠）而受到抑制；在200 mM或以上的盐浓度下在大多数情况下可以看到连接被完全抑制。因为连接反应的插入物和载体样品中存在盐污染物并不罕见，因此在进行连接之前通常需要纯化步骤。此类增加的纯化步骤增加了实验的时间和复杂性，并且纯化程序通常会导致样品损失 -这可能是重大损失。另外，如果连接酶不受使用其他酶时通常需要的缓冲液中的盐的抑制，则有可能在一个反应容器中实现与连接组合的多个程序。对反应混合物中的盐具有增强耐受性的T4 DNA连接酶变体具有大量的分子生物学应用。因此，非常需要一种能够在高盐环境中操作的连接酶。
发明内容
本发明涉及与野生型T4 DNA连接酶相比，对反应混合物中盐（包括NaCl、KCl和其他盐）的存在表现出增加的耐受性的T4 DNA连接酶变体。以下T4 DNA连接酶突变体（在指定位置处具有取代，并且其中每种突变体的氨基酸序列是指定取代后面的序列标识号，并且其中每种这样的突变体的DNA序列是每种突变体的氨基酸序列标识号之前的序列表中的奇数编号的序列标识号）被鉴定为对NaCl的存在具有这种增加的耐受性：E88K（SEQ ID NO:4）、E132K（SEQ ID NO: 6）、E222K（SEQ ID NO: 8）、K306E（SEQ ID NO: 10）、D340R（SEQ IDNO: 12）、K365E（SEQ ID NO: 14）、D371A（SEQ ID NO: 16）、D371G（SEQ ID NO: 18）、D371H（SEQ ID NO: 20）、D371I（SEQ ID NO: 22）、D371K（SEQ ID NO: 24）、D371L（SEQ ID NO:26）、D371M（SEQ ID NO: 28）、D371R（SEQ ID NO: 30）、D371T（SEQ ID NO: 32）、D371Y（SEQID NO: 34）、E438K（SEQ ID NO: 36）、E440A（SEQ ID NO: 38）、E440H（SEQ ID NO: 40）、E440K（SEQ ID NO: 42）、E440N（SEQ ID NO: 44）、E440R（SEQ ID NO: 46）、E440S（SEQ IDNO: 48）、E440W（SEQ ID NO: 50）和D448R（SEQ ID NO: 52）。
这些变体中的每一种的完整序列在说明书描述性部分之后的序列表中示出。本发明还包括作为这些突变位点中的一个或多个的组合的变体，使得一些这样的变体可以具有这些突变位点中的两个，或这些突变位点中的三个或更多个。
本发明进一步包括具有至少一种上述突变的T4 DNA连接酶突变型氨基酸序列，但其中T4 DNA连接酶突变型氨基酸序列的其余部分仅具有保守取代，使得所述分子与序列表中相应的T4 DNA连接酶突变型氨基酸序列（以下称为“变体序列”）具有至少70%、80%、90%、95%、96%、97%、98%或99%的同一性。
本发明进一步包括上述突变体的每个氨基酸序列之前的DNA序列（即分别为从SEQID NO: 3至SEQ ID NO: 51的奇数编号的SEQ ID NO），并且进一步包括前述DNA序列和编码(i) 上述T4 DNA连接酶突变体中的每一种和 (ii) 所述变体序列中的任一种的氨基酸序列的其他简并核酸序列（统称为“简并核酸序列”）。
本发明进一步包括掺入有任何简并核酸序列的载体；以及用任何这样的载体或简并核酸序列转化并且能够表达任何上述T4 DNA连接酶突变型氨基酸序列或变体序列的细胞。
本发明进一步包括一种组合物或试剂盒，其包含任何上述T4 DNA连接酶突变型氨基酸序列或变体序列、简并核酸序列、或掺入有这种简并核酸序列的载体。本发明还包括一种扩增靶核酸的方法，其中在设计用于扩增靶核酸的反应混合物中采用任何上述T4 DNA连接酶突变体或变体序列，并且使试剂混合物经受用于扩增靶核酸的条件。
与野生型相比，上述突变型T4 DNA连接酶突变体在增加的盐浓度中、在扩增靶DNA序列方面具有更高的活性，并且预期所述变体序列也具有这种更高的活性。
序列表
<110> 武汉爱博泰克生物科技有限公司
<120> 具有增加的耐盐性的T4 DNA连接酶变体
<130> ABCL-T4SALT
<140> 17/675,278
<141> 2022-02-18
<160> 54
<170> PatentIn版本3.5
<210> 1
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 1
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 2
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 2
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 3
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 3
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat taaagaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 4
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 4
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Lys Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 5
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 5
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctaaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 6
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 6
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Lys Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 7
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 7
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
cataaacaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 8
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 8
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Lys Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 9
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 9
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctgaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 10
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 10
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Glu Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 11
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 11
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattcgt 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 12
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 12
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Arg Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 13
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 13
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt atgaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 14
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 14
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Glu Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 15
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 15
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gcagttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 16
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 16
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Ala Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 17
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 17
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt ggtgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 18
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 18
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Gly Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 19
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 19
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt catgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 20
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 20
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile His Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 21
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 21
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt attgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 22
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 22
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Ile Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 23
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 23
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt aaagttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 24
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 24
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Lys Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 25
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 25
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt ttggttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 26
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 26
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Leu Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 27
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 27
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt atggttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 28
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 28
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Met Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 29
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 29
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt cgtgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 30
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 30
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Arg Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 31
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 31
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt actgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 32
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 32
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Thr Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 33
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 33
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt tacgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 34
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 34
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Tyr Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 35
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 35
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct aaaatgcgaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 36
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 36
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Lys Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 37
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 37
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgca 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 38
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 38
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Ala Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 39
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 39
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgccat 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 40
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 40
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys His Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 41
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 41
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcaaa 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 42
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 42
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 43
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 43
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcaac 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 44
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 44
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Asn Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 45
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 45
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgccgt 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 46
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 46
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Arg Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 47
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 47
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgctct 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 48
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 48
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Ser Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 49
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 49
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgctgg 1320
tgcaacggtt ggttaaaatc tgatggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 50
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 50
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Trp Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 51
<211> 1500
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多核苷酸
<400> 51
atgattctta aaattctgaa cgaaatagca tctattggtt caactaaaca gaagcaagca 60
attcttgaaa agaataaaga taatgaattg cttaaacgag tatatcgtct gacttattct 120
cgtgggttac agtattatat caagaaatgg cctaaacctg gtattgctac ccagagtttt 180
ggaatgttga ctcttaccga tatgcttgac ttcattgaat tcacattagc tactcggaaa 240
ttgactggaa atgcagcaat tgaggaatta actggatata tcaccgatgg taaaaaagat 300
gatgttgaag ttttgcgtcg agtgatgatg cgagaccttg aatgtggtgc ttcagtatct 360
attgcaaaca aagtttggcc aggtttaatt cctgaacaac ctcaaatgct cgcaagttct 420
tatgatgaaa aaggcattaa taagaatatc aaatttccag cctttgctca gttaaaagct 480
gatggagctc ggtgttttgc tgaagttaga ggtgatgaat tagatgatgt tcgtctttta 540
tcacgagctg gtaatgaata tctaggatta gatcttctta aggaagagtt aattaaaatg 600
accgctgaag cccgccagat tcatccagaa ggtgtgttga ttgatggcga attggtatac 660
catgagcaag ttaaaaagga gccagaaggc ctagattttc tttttgatgc ttatcctgaa 720
aacagtaaag ctaaagaatt cgccgaagta gctgaatcac gtactgcttc taatggaatc 780
gccaataaat ctttaaaggg aaccatttct gaaaaagaag cacaatgcat gaagtttcag 840
gtctgggatt atgtcccgtt ggtagaaata tacagtcttc ctgcatttcg tttgaaatat 900
gatgtacgtt tttctaaact agaacaaatg acatctggat atgataaagt aattttaatt 960
gaaaaccagg tagtaaataa cctagatgaa gctaaggtaa tttataaaaa gtatattgac 1020
caaggtcttg aaggtattat tctcaaaaat atcgatggat tatgggaaaa tgctcgttca 1080
aaaaatcttt ataaatttaa agaagtaatt gatgttgatt taaaaattgt aggaatttat 1140
cctcaccgta aagaccctac taaagcgggt ggatttattc ttgagtcaga gtgtggaaaa 1200
attaaggtaa atgctggttc aggcttaaaa gataaagccg gtgtaaaatc gcatgaactt 1260
gaccgtactc gcattatgga aaaccaaaat tattatattg gaaaaattct agagtgcgaa 1320
tgcaacggtt ggttaaaatc tcgtggccgc actgattacg ttaaattatt tcttccgatt 1380
gcgattcgtt tacgtgaaga taaaactaaa gctaatacat tcgaagatgt atttggtgat 1440
tttcatgagg taactggtct aggttctggc agttcaggtc atcaccacca tcatcactaa 1500
<210> 52
<211> 499
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
多肽
<400> 52
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Arg
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Ser Gly Ser Ser Gly His His His
485 490 495
His His His
<210> 53
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
肽
<400> 53
Gly Leu Gly Ser Gly Ser Ser Gly
1 5
<210> 54
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的描述：合成
6xHis标签
<400> 54
His His His His His His
1 5
附图说明
图1A至1C示出了描绘DNA底物连接的琼脂糖凝胶（1.2%）图像，其来自在零浓度和然后七种不同NaCl浓度（即NaCl浓度，其中0 mM、50 mM、100 mM、200 mM、250 mM、300 mM、400 mM和500 mM）下连接酶活性的顺序测试，其中反应混合物在16°C下孵育60分钟。图1A示出了WT T4 DNA连接酶和T4 DNA连接酶变体E88K（SEQ ID NO: 4）、E132K（SEQ ID NO: 6）、E222K（SEQ ID NO: 8）、K306E（SEQ ID NO: 10）、D340R（SEQ ID NO: 12）、K365E（SEQ IDNO: 14）、D371A（SEQ ID NO: 16）和D371G（SEQ ID NO: 18）。图1B示出了WT T4 DNA连接酶和T4 DNA连接酶变体D371H（SEQ ID NO: 20）、D371I（SEQ ID NO: 22）、D371K（SEQ ID NO:24）、D371L（SEQ ID NO: 26）、D371M（SEQ ID NO: 28）、D371R（SEQ ID NO: 30）、D371T（SEQID NO: 32）和D371Y（SEQ ID NO: 34）。图1C示出了WT T4 DNA连接酶和T4 DNA连接酶变体E438K（SEQ ID NO: 36）、E440A（SEQ ID NO: 38）、E440H（SEQ ID NO: 40）、E440K（SEQ IDNO: 42）、E440N（SEQ ID NO: 44）、E440R（SEQ ID NO: 46）、E440S（SEQ ID NO: 48）、E440W（SEQ ID NO: 50）和D448R（SEQ ID NO: 52）。
实施方式
术语“生物活性片段”是指T4 DNA连接酶突变体的任何片段、衍生物、同源物或类似物，其具有生物分子特有的
体内或体外活性；包括例如连接酶活性，或经由连接修复带切口DNA中存在的错配。在一些实施方案中，突变型T4 DNA连接酶的生物活性片段、衍生物、同源物或类似物在任何感兴趣的
体内或体外测定中均具有突变型T4 DNA连接酶的任何程度的生物活性。
在一些实施方案中，生物活性片段可以任选地包括突变型T4 DNA连接酶的任何数量的连续氨基酸残基。本发明还包括编码任何这种生物活性片段的多核苷酸。
生物活性片段可以来自转录后加工或可替代剪接RNA的翻译，或者可替代地可以通过工程化、批量合成或其他合适的操作产生。生物活性片段包括在天然或内源细胞中表达的片段，以及在表达系统（例如像细菌、酵母、植物、昆虫或哺乳动物细胞）中产生的片段。
如本文所用，短语“保守氨基酸取代”或“保守突变”是指一个氨基酸被另一个具有共同特性的氨基酸替换。定义单个氨基酸之间共同特性的功能性方法是分析同源生物体的相应蛋白质之间氨基酸变化的归一化频率（Schulz (1979) Principles of ProteinStructure [蛋白质结构原理], Springer-Verlag）。根据此类分析，氨基酸组可以定义为组内的氨基酸优先相互交换，因此在它们对整个蛋白质结构的影响方面彼此最相似（前述Schulz (1979)）。以这种方式定义的氨基酸组的实例可以包括：“带电荷/极性组”，包括Glu、Asp、Asn、Gln、Lys、Arg和His；“芳族或环状组”，包括Pro、Phe、Tyr和Trp；以及“脂族组”，包括Gly、Ala、Val、Leu、Ile、Met、Ser、Thr和Cys。在每个组内，还可以鉴定亚组。例如，带电荷/极性氨基酸的组可以细分为多个亚组，包括：“带正电荷的亚组”，包括Lys、Arg和His；“带负电荷的亚组”，包括Glu和Asp；以及“极性亚组”，包括Asn和Gln。在另一个实例中，芳族或环状组可以细分为多个亚组，包括：“氮环亚组”，包括Pro、His和Trp；“苯基亚组”，包括Phe和Tyr。在另一个进一步的实例中，脂族组可以细分为多个亚组，包括：“大脂族非极性亚组”，包括Val、Leu和Ile；“脂族弱极性亚组”，包括Met、Ser、Thr、和Cys；以及“小残基亚组”，包括Gly和Ala。保守突变的实例包括上述子组内氨基酸的氨基酸取代，诸如但不限于：Lys取代Arg，反之亦然，使得可以保持正电荷；Glu取代Asp，反之亦然，使得可以保持负电荷；Ser取代Thr，反之亦然，使得可以保持游离-OH；以及Gln取代Asn，反之亦然，使得可以保持游离-NH2。“保守变体”是包括一个或多个氨基酸的多肽，所述一个或多个氨基酸已被取代以用具有共同特性的氨基酸（例如，属于如上所述的相同氨基酸组或子组）替换参考多肽（例如，其序列在出版物或序列数据库中公开，或其序列已通过核酸测序确定的多肽）的一个或多个氨基酸。
当提及基因时，“突变体”意指相对于天然或野生型基因，所述基因具有至少一个碱基（核苷酸）改变、缺失或插入。突变（一个或多个核苷酸的改变、缺失和/或插入）可以在基因的编码区中，或者可以在内含子、3' UTR、5' UTR或启动子区域中。作为非限制性实例，突变基因可以是在启动子区域内具有插入的基因，所述插入可以增加或减少所述基因的表达；可以是具有缺失的基因，导致无功能蛋白、截短蛋白、显性阴性蛋白或无蛋白的产生；或者，可以是具有一个或多个点突变的基因，导致所编码蛋白质的氨基酸改变或导致基因转录物的异常剪接。
术语“本发明的突变型T4 DNA连接酶”和“突变型T4 DNA连接酶”在该“具体实施方式”部分中使用时，根据上下文，共同或单独地是指在足以显著降低野生型T4 DNA连接酶的连接活性的盐浓度存在下测试并表现出显著连接活性的突变型T4 DNA连接酶多肽，它们是：E88K（SEQ ID NO: 4）、E132K（SEQ ID NO: 6）、E222K（SEQ ID NO: 8）、K306E（SEQ IDNO: 10）、D340R（SEQ ID NO: 12）、K365E（SEQ ID NO: 14）、D371A（SEQ ID NO: 16）、D371G（SEQ ID NO: 18）、D371H（SEQ ID NO: 20）、D371I（SEQ ID NO: 22）、D371K（SEQ ID NO:24）、D371L（SEQ ID NO: 26）、D371M（SEQ ID NO: 28）、D371R（SEQ ID NO: 30）、D371T（SEQID NO: 32）、D371Y（SEQ ID NO: 34）、E438K（SEQ ID NO: 36）、E440A（SEQ ID NO: 38）、E440H（SEQ ID NO: 40）、E440K（SEQ ID NO: 42）、E440N（SEQ ID NO: 44）、E440R（SEQ IDNO: 46）、E440S（SEQ ID NO: 48）、E440W（SEQ ID NO: 50）和D448R（SEQ ID NO: 52）。
和/或变体序列和/或简并核酸序列，这些术语在发明内容部分中定义。
“天然存在”或“野生型”是指在自然界中发现的形式。例如，天然存在或野生型多肽或多核苷酸序列是存在于生物体中的序列，其没有被人为操作有意修饰。
关于核酸或多肽序列的术语“同一性百分比”或“同源性”定义为在排列序列以获得最大同一性百分比并引入缺口（如果需要）以实现最大同源性百分比之后，候选序列中与已知多肽相同的核苷酸或氨基酸残基的百分比。N-末端或C-末端插入或缺失不应被解释为影响同源性。核苷酸或氨基酸序列水平上的同源性或同一性可以通过BLAST（基本局部比对搜索工具（Basic Local Alignment Search Tool））分析来确定，所述分析使用由程序blastp、blastn、blastx、tblastn和tblastx采用的算法（Altschul (1997), NucleicAcids Res [核酸研究]. 25, 3389-3402和Karlin (1990), Proc. Natl. Acad. Sci.USA [美国国家科学院院刊]87, 2264-2268），所述程序是为序列相似性搜索定制的。BLAST程序使用的方法是首先考虑查询序列和数据库序列之间的相似片段（有或没有缺口），然后评估被鉴定的所有匹配的统计显著性，最后仅总结满足预先选择的显著性阈值的那些匹配。关于序列数据库相似性搜索中基本问题的讨论，参见Altschul (1994), NatureGenetics [自然遗传学]6, 119-129。直方图、描述、比对、期望（即，用于报告与数据库序列匹配的统计显著性阈值）、截止、矩阵和过滤器（低复杂度）的搜索参数可以是默认设置。blastp、blastx、tblastn和tblastx使用的默认评分矩阵是BLOSUM62矩阵（Henikoff(1992), Proc. Natl. Acad. Sci. USA [美国国家科学院院刊]89, 10915-10919），推荐用于长度超过85个单位（核苷酸碱基或氨基酸）的查询序列。
在一些实施方案中，本发明涉及用于进行连接反应的方法（以及相关试剂盒、系统、设备和组合物），所述方法包括或由以下组成：在一种或多种核苷酸存在下，使突变型T4DNA连接酶或其生物活性片段与核酸模板接触，并使用突变型T4 DNA连接酶或其生物活性片段连接所述一种或多种核苷酸中的至少一种。
在一些实施方案中，所述方法可以包括将双链RNA或DNA多核苷酸链连接到环状分子中。在一些实施方案中，所述方法可以进一步包括通过使用传感器检测指示连接的信号。在一些实施方案中，传感器是ISFET。在一些实施方案中，传感器可以包括连接反应中的可检测标记或可检测试剂。
在一些实施方案中，本发明涉及用于进行核酸的滚环扩增（参见美国专利号5,714,320，通过引用并入）的方法（以及相关的试剂盒、系统、设备和组合物），所述方法使用突变型T4 DNA连接酶作为扩增过程的连接步骤中的酶。扩增包括在溶液中扩增核酸，以及在固体支持物（诸如存在于固体支持物表面上的核酸珠、流动池、核酸阵列或孔）上克隆扩增核酸。
本发明的突变型T4 DNA连接酶可以在任何合适的宿主系统中表达，所述宿主系统包括细菌、酵母、真菌、杆状病毒、植物或哺乳动物宿主细胞。对于细菌宿主细胞，用于指导本公开核酸构建体转录的合适启动子包括从以下获得的启动子：大肠杆菌（
E.coli）乳糖操纵子、天蓝色链霉菌琼脂糖酶基因（dagA）、枯草芽孢杆菌果聚糖蔗糖酶基因（sacB）、地衣芽孢杆菌α-淀粉酶基因（amyL）、嗜热脂肪芽孢杆菌麦芽糖淀粉酶基因（amyM）、解淀粉芽孢杆菌α-淀粉酶基因（amyQ）、地衣芽孢杆菌青霉素酶基因（penP）、枯草芽孢杆菌xylA和xylB基因和原核β-内酰胺酶基因（Villa-Kamaroff等人, 1978, Proc. Natl Acad. Sci. USA[美国国家科学院院刊]75: 3727-3731），以及tac启动子（DeBoer等人, 1983, Proc. NatlAcad. Sci. USA [美国国家科学院院刊]80: 21-25）。
对于丝状真菌宿主细胞，用于指导本公开核酸构建体转录的合适启动子包括从以下酶的基因获得的启动子：米曲霉TAKA淀粉酶、米黑根毛霉天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定α-淀粉酶、黑曲霉或泡盛曲霉葡糖淀粉酶（glaA）、米黑根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、构巢曲霉乙酰胺酶和尖孢镰孢胰蛋白酶样蛋白酶（WO 96/00787），以及NA2-tpi启动子（来自黑曲霉中性α-淀粉酶和米曲霉磷酸丙糖异构酶的基因的启动子的杂交体），及其突变、截短和杂交启动子。
在酵母宿主中，有用的启动子可以来自以下酶的基因：酿酒酵母（
Saccharomyces
cerevisiae）烯醇化酶（ENO-1）、酿酒酵母半乳糖激酶（GAL1）、酿酒酵母乙醇脱氢酶/甘油醛-3-磷酸脱氢酶（ADH2/GAP）和酿酒酵母3-磷酸甘油酸激酶。酵母宿主细胞的其他有用的启动子由Romanos等人, 1992, Yeast [酵母] 8:423-488描述。
对于杆状病毒表达，来源于鳞翅目（蛾和蝴蝶）的昆虫细胞系，诸如草地贪夜蛾被用作宿主。基因表达受强启动子（
例如，pPolh）的控制。
植物表达载体基于根癌土壤杆菌（Agrobacterium tumefaciens）的Ti质粒，或基于烟草花叶病毒（TMV）、马铃薯X病毒或豇豆花叶病毒。植物表达载体中常用的组成型启动子是花椰菜花叶病毒（CaMV）35S启动子。
对于哺乳动物表达，培养的哺乳动物细胞系诸如中国仓鼠卵巢（CHO）、COS，包括人细胞系诸如HEK和HeLa，可以用于产生突变型T4 DNA连接酶。哺乳动物表达载体的实例包括腺病毒载体、pSV和pCMV系列质粒载体、牛痘病毒和逆转录病毒载体以及杆状病毒。巨细胞病毒（CMV）和SV40的启动子通常用于哺乳动物表达载体中以驱动基因表达。非病毒启动子，诸如延伸因子(EF)-1启动子也是已知的。
用于表达的控制序列也可以是合适的转录终止子序列，即，由宿主细胞识别以终止转录的序列。终止子序列可操作地连接至编码所述多肽的核酸序列的3'末端。可以使用在所选择宿主细胞中具有功能性的任何终止子。
例如，用于丝状真菌宿主细胞的示例性转录终止子可以从以下酶的基因获得：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡糖苷酶和尖孢镰孢胰蛋白酶样蛋白酶。
用于酵母宿主细胞的示例性终止子可以从以下酶的基因获得：酿酒酵母烯醇化酶、酿酒酵母细胞色素C（CYC1）和酿酒酵母甘油醛-3-磷酸脱氢酶。
用于昆虫、植物和哺乳动物宿主细胞的终止子也是熟知的。
控制序列也可以是合适的前导序列，即对由宿主细胞进行的翻译很重要的mRNA的非翻译区。前导序列可操作地连接至编码所述多肽的核酸序列的5'末端。可以使用在所选择宿主细胞中具有功能性的任何前导序列。用于丝状真菌宿主细胞的示例性前导序列从米曲霉TAKA淀粉酶和构巢曲霉磷酸丙糖异构酶的基因获得。用于酵母宿主细胞的合适的前导序列从以下酶的基因获得：酿酒酵母烯醇化酶（ENO-1）、酿酒酵母3-磷酸甘油酸激酶、酿酒酵母α-因子、以及酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶（ADH2/GAP）。
控制序列还可以是多腺苷酸化序列，一种可操作地连接至所述核酸序列的3'末端并且当转录时由宿主细胞识别为将多腺苷酸残基添加至所转录的mRNA的信号的序列。在所选择宿主细胞中具有功能性的任何多腺苷酸化序列均可以用于本发明中。用于丝状真菌宿主细胞的示例性多腺苷酸化序列可以来自以下酶的基因：米曲霉TAKA淀粉酶、黑曲霉葡糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、尖孢镰孢胰蛋白酶样蛋白酶和黑曲霉α-葡糖苷酶。
控制序列还可以是编码与多肽的氨基末端连接的氨基酸序列并指导所编码多肽进入细胞的分泌途径的信号肽编码区。核酸序列的编码序列的5'端本身可以含有在翻译阅读框中与编码分泌的多肽的编码区的区段天然连接的信号肽编码区。可替代地，编码序列的5'端可以含有对编码序列是外源的信号肽编码区。在编码序列天然地不含有信号肽编码区的情况下，可能需要外源信号肽编码区。
可替代地，外源信号肽编码区可以单纯地替代天然信号肽编码区以便增强多肽的分泌。然而，可以使用指导已表达多肽进入所选择宿主细胞的分泌途径的任何信号肽编码区。
用于细菌宿主细胞的有效信号肽编码区是从以下酶的基因获得的信号肽编码区：芽孢杆菌NCIB 11837麦芽糖淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣芽孢杆菌枯草杆菌蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶（nprT、nprS、nprM）、以及枯草芽孢杆菌prsA。进一步的信号肽由Simonen和Palva, 1993, Microbiol Rev [微生物评论] 57: 109-137描述。
用于丝状真菌宿主细胞的有效信号肽编码区可以是从以下酶的基因获得的信号肽编码区：米曲霉TAKA淀粉酶、黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米黑根毛霉天冬氨酸蛋白酶、特异腐质霉纤维素酶、以及疏棉状腐质霉脂肪酶。
用于酵母宿主细胞的有用的信号肽可以来自酿酒酵母α-因子和酿酒酵母转化酶的基因。用于其他宿主细胞系统的信号肽也是熟知的。
控制序列还可以是编码位于多肽的氨基末端处的氨基酸序列的前肽编码区。所得的多肽被称为前体酶（proenzyme）或多肽原（或在一些情况下被称为酶原（zymogen））。多肽原通常是无活性的并且可以通过催化切割或自身催化切割来自多肽原的前肽而转化为成熟的活性多肽。前肽编码区可以从以下酶的基因获得：枯草芽孢杆菌碱性蛋白酶（aprE）、枯草芽孢杆菌中性蛋白酶（nprT）、酿酒酵母α-因子、米黑根毛霉天冬氨酸蛋白酶、以及嗜热毁丝霉乳糖酶（WO 95/33836）。
在信号肽和前肽区二者都存在于多肽的氨基末端处的情况下，所述前肽区位于紧邻多肽的氨基末端，并且所述信号肽区位于紧邻前肽区的氨基末端。
还可期望的是添加调节序列，其允许相对于宿主细胞的生长调节突变型T4 DNA连接酶的表达。调节系统的实例是响应于化学或物理刺激（包括调节化合物的存在）而引起基因表达被开启或关闭的那些。在原核宿主细胞中，合适的调节序列包括lac、tac和trp操纵子系统。在酵母宿主细胞中，合适的调节系统包括例如，ADH2系统或GAL1系统。在丝状真菌中，合适的调节序列包括TAKA α淀粉酶启动子、黑曲霉葡糖淀粉酶启动子和米曲霉葡糖淀粉酶启动子。用于其他宿主细胞的调节系统也是熟知的。
调节序列的其他实例是允许基因扩增的那些序列。在真核系统中，这些包括在甲氨蝶呤存在下扩增的二氢叶酸还原酶基因以及用重金属扩增的金属硫蛋白基因。在这些情况下，编码本发明KRED多肽的核酸序列将与调节序列可操作地连接。
另一个实施方案包括重组表达载体，其包含编码工程化突变型T4 DNA连接酶或其变体的多核苷酸，和一个或多个表达调节区诸如启动子和终止子，以及复制起点，这取决于它们将被引入的宿主的类型。上述各种核酸和控制序列可以连接在一起以产生重组表达载体，所述重组表达载体可以包括一个或多个便利的限制位点以允许编码突变型T4 DNA连接酶的核酸序列在此类位点处的插入或取代。可替代地，可以通过将核酸序列或包含所述序列的核酸构建体插入用于表达的适当载体中来表达突变型T4 DNA连接酶的核酸序列。在产生表达载体时，编码序列位于载体中，使得编码序列与用于表达的适当控制序列可操作地连接。
重组表达载体可以是可以方便地经受重组DNA程序并且可以引起突变型T4 DNA连接酶多核苷酸序列表达的任何载体（例如，质粒或病毒）。载体的选择将通常取决于载体与待引入载体的宿主细胞的相容性。载体可以是直链质粒或闭合环状质粒。
表达载体可以是自主复制载体，即作为染色体外实体存在的载体，其复制不依赖于染色体复制，例如质粒、染色体外元件、微染色体或人工染色体。载体可以含有用于确保自我复制的任何手段。可替代地，载体可以是这样的载体，当它引入宿主细胞中时被整合到基因组中并与其中已整合了它的一个或多个染色体一起被复制。此外，可以使用单独的载体或质粒或共同含有待引入宿主细胞基因组的总DNA的两个或更多个载体或质粒，或可以使用转座子。
本文表达载体优选地含有一个或多个选择性标记，其允许容易地选择转化的细胞。选择性标记是一种基因，其产物提供了杀生物剂抗性或病毒抗性、对重金属抗性、对营养缺陷型的原营养等。细菌选择性标记的实例是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或赋予抗生素抗性（诸如氨苄青霉素、卡那霉素、氯霉素（实施例1）或四环素抗性）的标记。用于酵母宿主细胞的合适标记是ADE2、HIS3、LEU2、LYS2、MET3、TRP1、和URA3。用于丝状真菌宿主细胞中的选择性标记包括但不限于amdS（乙酰胺酶）、argB（鸟氨酸氨甲酰基转移酶）、bar（草丁膦乙酰转移酶）、hph（潮霉素磷酸转移酶）、niaD（硝酸还原酶）、pyrG（乳清酸核苷-5'-磷酸脱羧酶）、sC（硫酸腺苷酰基转移酶）和trpC（邻氨基苯甲酸合酶）、以及其等效物。用于曲霉属细胞中的实施方案包括构巢曲霉或米曲霉的amdS和pyrG基因，以及吸水链霉菌的bar基因。用于昆虫、植物和哺乳动物细胞的选择性标记也是熟知的。
本发明的表达载体优选地含有允许载体整合到宿主细胞的基因组中或载体在细胞中不依赖于基因组的自主复制的一个或多个元件。对于整合到宿主细胞基因组中，载体可以依赖于编码所述多肽的核酸序列或用于通过同源或非同源重组将载体整合到基因组中的所述载体的任何其他元件。
可替代地，表达载体可以含有另外的核酸序列，用于指导通过同源重组整合到宿主细胞的基因组中。另外的核酸序列使得载体能够整合到宿主细胞基因组中一个或多个染色体的一个或多个精确位置处。整合元件可以是与宿主细胞基因组内的靶序列同源的任何序列。此外，整合元件可以是非编码或编码核酸序列。另一方面，载体可以通过非同源重组整合到宿主细胞的基因组中。
为了自主复制，载体可以进一步包含复制起点，该复制起点使得载体能够在讨论中的宿主细胞中自主复制。细菌复制起点的实例是P15A ori，或允许在大肠杆菌中复制的质粒pBR322、pUC19、pACYC177（所述质粒具有P15A ori）或pACYC184的复制起点、以及允许在芽孢杆菌中复制的质粒pUB110、pE194、pTA1060或pAM31的复制起点。用于酵母宿主细胞中的复制起点的实例是2微米复制起点，ARS1，ARS4，ARS1与CEN3的组合，以及ARS4与CEN6的组合。复制起点可以是具有使其在宿主细胞中的功能对温度敏感的突变的复制起点（参见例如，Ehrlich, 1978, Proc Natl Acad Sci. USA [美国国家科学院院刊]75:1433）。
可以将突变型T4 DNA连接酶的多于一个拷贝的核酸序列插入宿主细胞中，以增加基因产物的产生。通过将序列的至少一个另外的拷贝整合到宿主细胞基因组中或者通过包括与所述核酸序列一起的可扩增的选择性标记基因可以获得核酸序列的增加的拷贝数目，其中通过在适当的选择性试剂的存在下培养细胞可以选择含有选择性标记基因的经扩增的拷贝以及由此所述核酸序列的额外拷贝的细胞。
用于突变型T4 DNA连接酶多核苷酸的表达载体可商购获得。合适的商业表达载体包括来自圣路易斯的西格玛奥德里奇化学公司（Sigma-Aldrich Chemicals, St. LouisMo.）的p3xFLAGTM表达载体，其包括用于在哺乳动物宿主细胞中表达的CMV启动子和hGH聚腺苷酸化位点，以及用于在大肠杆菌中扩增的pBR322复制起点和氨苄青霉素抗性标记物。合适的其他表达载体是可从加利福尼亚州拉荷亚（LaJolla Calif.）的Stratagene公司商业获得的pBluescriptII SK(-)和pBK-CMV，以及来源于pBR322（Gibco BRL）、
pUC（GibcoBRL）、
pREP4、
pCEP4（Invitrogen）或pPoly（Lathe等人, 1987, Gene [基因]57:193-201）的质粒。
用于表达编码突变型T4 DNA连接酶的多核苷酸的合适宿主细胞是本领域熟知的，并且包括但不限于：细菌细胞，诸如大肠杆菌、克菲尔乳杆菌（
Lactobacilluskefir）、短乳杆菌、微小乳杆菌、链霉菌和鼠伤寒沙门氏菌细胞；真菌细胞，诸如酵母细胞（例如，酿酒酵母或巴斯德毕赤酵母（ATCC登记号201178））；昆虫细胞，诸如果蝇S2和夜蛾Sf9细胞；动物细胞，诸如CHO、COS、BHK、293和鲍斯黑色素瘤细胞（Bowes melanoma cell）；以及植物细胞。用于上述宿主细胞的适当培养基和生长条件是本领域熟知的。
可以通过本领域已知的各种方法将用于表达突变型T4 DNA连接酶的多核苷酸引入细胞。这些技术包括电穿孔、生物射弹粒子轰击、脂质体介导的转染、氯化钙转染和原生质体融合。用于将多核苷酸引入细胞的各种方法是技术人员已知的。
可以根据已知的合成方法，通过标准固相方法来制备编码突变型T4 DNA连接酶的多核苷酸。在一些实施方案中，可以单独合成多达约100个碱基的片段，然后连接（
例如，通过酶促或化学连接方法，或聚合酶介导的方法）以形成任何所希望的连续序列。例如，可以使用例如由Beaucage等人, 1981, Tet Lett [四面体快报]22:1859-69描述的经典亚磷酰胺方法，或由Matthes等人, 1984, EMBO J.[欧洲分子生物学学报]3:801-05描述的方法（
例如，如其通常应用于自动合成方法中）而通过化学合成来制备多核苷酸。根据亚磷酰胺方法，
例如在自动DNA合成仪中合成寡核苷酸，将其纯化、退火、连接并克隆到合适的载体中。另外，基本上任何核酸都可以从各种商业来源获得，所述商业来源诸如德克萨斯州米德兰的Midland Certified Reagent Company公司、加利福尼亚州拉蒙纳的Great AmericanGene Company公司、伊利诺伊州芝加哥的ExpressGen公司和加利福尼亚州阿拉米达的Operon Technologies公司。
使用任何一种或多种熟知的蛋白质纯化技术，包括溶菌酶处理、超声处理、过滤、盐析、超速离心和色谱法，可以从细胞和/或培养基中回收在宿主细胞中表达的工程化突变型T4 DNA连接酶。用于从细菌（诸如大肠杆菌）裂解和高效提取蛋白质的合适溶液可从圣路易斯的西格玛奥德里奇公司（Sigma-Aldrich）以商品名CelLytic B.TM.商购获得。
用于分离突变型T4 DNA连接酶的色谱技术包括反相色谱、高效液相色谱、离子交换色谱、凝胶电泳和亲和色谱。纯化条件将部分取决于诸如净电荷、疏水性、亲水性、分子量、分子形状的因素，并且对于本领域技术人员将是显而易见的。
在一些实施方案中，亲和技术可以用于分离突变型T4 DNA连接酶。对于亲和色谱纯化，可以使用特异性结合突变型T4 DNA连接酶的任何抗体。为了产生抗体，各种宿主动物（包括但不限于兔、小鼠、大鼠等）可以通过注射化合物进行免疫。所述化合物可以通过侧链官能团或附接至侧链官能团的接头附接至合适的载体，诸如BSA。各种佐剂可以用于增强免疫应答，这取决于宿主种类，包括但不限于弗氏（Freund's）（完全和不完全）、矿物凝胶诸如氢氧化铝、表面活性物质诸如溶血卵磷脂、复合多元醇（pluronic polyols）、聚阴离子、肽、油乳剂、钥孔戚血蓝蛋白（keyhole limpet hemocyanin）、二硝基酚，以及潜在有用的人佐剂诸如BCG（卡介苗）和短小棒状杆菌。
通过常规反向PCR诱变产生T4 DNA连接酶突变体。对所有突变体进行测序验证，在大肠杆菌中表达并纯化。为了便于纯化，所有T4 DNA连接酶突变体和野生型都具有添加的六元C-末端His标签，其前面是序列：GLGSGSSG（SEQ ID NO: 53）。
通过以下程序制备连接底物：
所使用的DNA载体底物是pUC19（新英格兰生物实验室，目录号N3041S）。可商购获得的pUC19是2686个碱基对长的双链环质粒。用BsaI-HF®v2（新英格兰生物实验室，目录号R3733S）消化所述质粒，所述BsaI-HF®v2具有5'-GGTCTC(N1)/(N5)-3'的识别序列，并在质粒pUC19中产生一个切口，从而产生具有粘性末端的线性DNA。将5 µl浓度为1 mg/ml的puC19与2.5 µl 20,000个单位/ml的BsaI-HF®v2、5 µl 10X rCutSmartTM缓冲液（新英格兰生物实验室，目录号B6004S）（50 mM乙酸钾、20 mM Tris-乙酸盐、10 mM乙酸镁、100 µg/ml重组白蛋白）和35 µl水组合，在37°C下孵育1小时，然后在80°C下热灭活孵育20分钟。然后将最终产物用水稀释至浓度为10 ng/µl。
如下进行连接筛选程序。每个连接酶样品（野生型或变体）的储备浓度为100 ng/µl。初步筛选测试所有变体，比较在含有100 mM和200 mM NaCl的反应混合物中的突变型与野生型活性。
对于每个反应，将200 ng的酶一式两份移液到PCR板的单个孔中。向每个孔中添加主混合物，所述主混合物由1X T4 DNA连接酶反应缓冲液（新英格兰生物实验室，目录号B0202A：50 mM Tris-HCl、10 mM MgCl2、1 mM ATP、10 mM DTT）、5 ng底物（用BsaI-HF®v2消化的pUC19）、达到所希望最终盐浓度所需的一定体积的5M NaCl，以及足够的水（以使每个反应的总体积为20 µl）组成。将反应物在16°C下孵育30分钟。在30分钟的孵育期后，反应物接受80°C的热休克2分钟以停止任何进一步的活动。另外，向每个反应中添加4 µl终止液（120 mM EDTA、30%甘油、50 mM Tris-HCl pH 8.0、0.0125%溴酚蓝、0.1% SDS和5x凝胶红核酸染色剂（加利福尼亚州弗里蒙特的Biotium公司（Biotium, Fremont, CA）））。
使用1.2%琼脂糖凝胶在180V下运行35分钟进行凝胶电泳。每个凝胶一式两份展示野生型T4 DNA连接酶样品和47个变体T4 DNA连接酶样品。通过在任何浓度下比WT更有效地完成连接而表明具有更高耐盐性的变体被鉴定用于二次筛选和确认。
二次筛选包括用盐滴定挑战每个先前选择的突变体以及野生型。该滴定中测试的NaCl浓度为：0 mM、50 mM、100 mM、200 mM、250 mM、300 mM、400 mM和500 mM。每个反应含有400 ng总蛋白（4 µl储备蛋白），加上16 µl主混合物，所述主混合物由1X T4 DNA连接酶反应缓冲液（新英格兰生物实验室，目录号B0202A：50 mM Tris-HCl、10 mM MgCl2、1 mM ATP、10 mM DTT）、10 ng底物、实现每种盐浓度所需体积的5M NaCl和足够的水（以实现每个反应的总体积为20 µl）组成。将连接反应在16°C下孵育60分钟，然后在80°C下经受2分钟的热休克以停止任何进一步的活动。最后，向每个反应中添加6 µl终止液（120 mM EDTA、30%甘油、50 mM Tris-HCl pH 8.0、0.0125%溴酚蓝、0.1% SDS和5x凝胶红核酸染色剂（加利福尼亚州弗里蒙特的Biotium公司））。
使用1.2%琼脂糖凝胶在180V下运行35分钟进行凝胶电泳。每个凝胶展示野生型T4DNA连接酶样品和11个变体T4 DNA连接酶样品，每个样品在8个泳道上的盐范围为0-500mM。鉴定了与野生型T4 DNA连接酶相比，对反应混合物中盐的存在表现出增加的耐受性的变体，结果在图1中示出；并且还在表1中列出了每种突变体在保持活性的同时对盐浓度的耐受限值。这些变体是E88K、E132K、E222K、K306E、D340R、D371H、K365E、E438K、D448R、D371A、D371G、D371I、D371K、D371L、D371M、D371R、D371T、D371Y、E440A、E440H、E440K、E440N、E440R、E440S、E440W和D448R。
与野生型相比，显示变体的连接活性的合成琼脂糖凝胶图像显示于图1中。所述图像显示双链连接的超螺旋质粒为最低条带，未连接的线性化质粒为中间条带，并且微弱的上条带为超螺旋单链连接的带切口环状DNA。随着每个连接系列上盐浓度从左到右增加，连接的底物最终消失，并且由于反应缓冲液中存在过量NaCl使连接酶灭活，因此仅剩余未连接的底物和超螺旋的带切口环状DNA。
表1：工程化T4 DNA连接酶变体的摩尔盐浓度限值
本文所述的具体方法和组合物是优选实施方案的代表，并且是示例性的且不旨在限制本发明的范围。考虑到本说明书，本领域技术人员将想到其他目的、方面和实施方案，并且包含在由权利要求书的范围所限定的本发明的精神内。对于本领域技术人员来说，将显而易见的是，在不脱离本发明的范围和精神的情况下，可以对本文公开的发明进行各种替换和修改。可以在不存在本文没有明确公开为必要的任何一种或多种要素、或者任何一种或多种限制的情况下适当地实践本文说明性描述的本发明。因此，例如，在本文的每种情况下，在本发明的实施方案或实例中，术语“包含”、“包括”、“含有”等中的任一项都应该被广泛而无限制地阅读。本文中示例性描述的方法和过程可以不同的步骤顺序适当地实施，并且它们不必限于本文或权利要求中指示的步骤顺序。还应注意，除非上下文另外清楚地指出，否则如在本文以及在所附权利要求所用的，单数形式“一个/一种”以及“所述”包括复数指示物，并且复数包括单数形式。在任何情况下，都不能将本专利申请解释为限于本文具体公开的具体实例或实施方案或方法。在任何情况下，本专利申请都不应被解释为受专利商标局的任何审查员或任何其他官员或雇员所做的任何声明的限制，除非此声明由申请人在响应性书面材料中明确且无条件或专门保留地采用。
已经在本文中宽泛且概括地对本发明进行了描述。落在类属公开之内的更窄的种类和亚属的分类中的每一个也形成了本发明的一部分。已经采用的术语和表达被用作描述的术语且没有限制性，并且并非旨在使用此类术语和表达而将所示出和描述的特征的任何等效物或其部分排除在外，但是将认识到的是提出要求保护的本发明范围内的不同修改是可能的。因此，应当理解，虽然已经通过优选实施方案和任选特征具体地公开了本发明，但本领域技术人员可以采用本文所公开概念的修改和变更，包括但不限于变体序列，并且认为此类修改和变更在由所附权利要求限定的本发明范围内。
Claims (7)
1.一种突变型T4 DNA连接酶，其氨基酸序列如SEQ ID NO: 6、SEQ ID NO: 8、SEQ IDNO: 12中的任一项所示，且不包括在其C-末端处的SEQ ID NO:53和SEQ ID NO:54所示序列。
2.一种多核苷酸，其编码如权利要求1所述的突变型T4 DNA连接酶之一的氨基酸序列。
3.一种载体，其掺入有如权利要求2所述的多核苷酸。
4.一种细胞，其用如权利要求2所述的多核苷酸转化并表达所述多核苷酸。
5.一种在不同多核苷酸之间进行多核苷酸连接或通过连接多核苷酸的5'端和3'端来进行多核苷酸连接以产生环状多核苷酸的方法，其中所述多核苷酸具有平末端或粘性末端，所述方法包括：
提供连接混合物，其包含待连接的多核苷酸和如权利要求1所述的突变型T4 DNA连接酶，其中所述连接混合物中盐的浓度大于或等于250 mM；和
将所述连接混合物置于发生连接的温度下。
6.如权利要求5所述的方法，其中所述盐是NaCl。
7.如权利要求5所述的方法，其中所述连接混合物包含Tris-HCl、MgCl2、ATP、二硫苏糖醇和水。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US17/675,278 | 2022-02-18 | ||
| US17/675,278 US11685913B1 (en) | 2022-02-18 | 2022-02-18 | T4 DNA ligase variants with increased resistance to salt |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114717209A CN114717209A (zh) | 2022-07-08 |
| CN114717209B true CN114717209B (zh) | 2023-04-18 |
Family
ID=82230323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210556291.7A Active CN114717209B (zh) | 2022-02-18 | 2022-05-19 | 具有增加的耐盐性的t4 dna连接酶变体 |
Country Status (2)
| Country | Link |
|---|---|
| US (2) | US11685913B1 (zh) |
| CN (1) | CN114717209B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11788082B2 (en) * | 2022-02-22 | 2023-10-17 | Abclonal Science, Inc. | T4 DNA ligase variants with increased thermostability |
| US20230295707A1 (en) * | 2022-03-21 | 2023-09-21 | Abclonal Science, Inc. | T4 DNA Ligase Variants with Increased Ligation Efficiency |
| CN115896047B (zh) * | 2022-12-12 | 2023-06-16 | 南京诺唯赞生物科技股份有限公司 | 重组t4 dna连接酶突变体、融合蛋白及其应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG11201909957TA (en) * | 2017-05-08 | 2019-11-28 | Codexis Inc | Engineered ligase variants |
| US10837009B1 (en) | 2017-12-22 | 2020-11-17 | New England Biolabs, Inc. | DNA ligase variants |
| EP3867373A1 (en) * | 2018-10-19 | 2021-08-25 | New England Biolabs, Inc. | Improved ordered assembly of multiple dna fragments |
| CN113774032B (zh) * | 2021-11-12 | 2022-03-01 | 翌圣生物科技(上海)股份有限公司 | 重组t4连接酶突变体、编码dna及ngs建库方法 |
-
2022
- 2022-02-18 US US17/675,278 patent/US11685913B1/en active Active
- 2022-05-19 CN CN202210556291.7A patent/CN114717209B/zh active Active
-
2023
- 2023-05-10 US US18/195,702 patent/US20240067951A1/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| US20240067951A1 (en) | 2024-02-29 |
| US11685913B1 (en) | 2023-06-27 |
| CN114717209A (zh) | 2022-07-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114717209B (zh) | 具有增加的耐盐性的t4 dna连接酶变体 | |
| US8569029B2 (en) | DNase expression in recombinant host cells | |
| US20240124854A1 (en) | Mutant Taq Polymerase Providing Faster Amplification | |
| KR20200003903A (ko) | 조작된 리가제 변이체 | |
| US11168312B2 (en) | Mutant taq polymerase for amplification in increased salt concentration or body fluids | |
| CN114555812A (zh) | 用于蛋白质产生的材料和方法 | |
| CN114854699B (zh) | 具有提高的热稳定性的t4 dna连接酶变体 | |
| CN114934026B (zh) | 具有增加的连接效率的t4 dna连接酶变体 | |
| US20230212550A1 (en) | T4 DNA Ligase Variants with Increased Ligation Efficiency | |
| EP3883973A1 (en) | Polypeptides having lipase activity and use thereof for wheat separation | |
| EP2078078B1 (en) | Selection of well-expressed synthetic genes | |
| CN115044569B (zh) | 一种Taq DNA聚合酶突变体及其应用 | |
| US20230332117A9 (en) | Mutant Taq Polymerase for Increased Salt Concentration or Body Fluids | |
| CN117778348A (zh) | 一种具有逆转录酶活性的Pfu DNA聚合酶突变体及其应用 | |
| CN117965497A (zh) | 双标记的粘质沙雷氏菌核酸酶以及使用其分解核酸的方法 | |
| CN115433725A (zh) | 甘油单-二酰酯脂肪酶突变体及其应用 |
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 |