本发明涉及新硫酸软骨素酶及表达硫酸软骨素酶的乳酸乳球菌,属于生物工程技术领域。
背景技术:
硫酸软骨素酶(chondrosulphatase,简称chsase)是能够将硫酸软骨素(cs)、硫酸皮肤素和透明质酸等糖胺聚糖降解成不饱和二糖及寡糖的1种裂解酶。chsase根据其作用底物的不同分为chsaseabc、chsaseac、chsaseb及chsasec等。硫酸软骨素酶在生物化工及医药领域具有重要的应用价值,在基础研究中,它可以作为工具酶,用于硫酸软骨素质量研究和高效制备具有多种生物活性的硫酸软骨素低聚糖;ryecs等人报道了小分子的cs,以硫酸软骨素为主体构成的蛋白聚糖在软骨基质中起着一种“分子弹簧”的作用,具有止痛、促进软骨再生的功效,可从根本改善关节问题(elucidationofthemechanismofpolysaccharidebychondroitinaclyasefromflavobacteriumheparinum.jamchemsoc,2002,124(33):9756–9767)。且熊双丽等人也报道了低分子量的cs,无论是口服或是注射用,吸收效果均优于高分子量cs(不同分子量硫酸软骨素的制备和抗氧化活性探讨.中成药,2006,28(9):1343–1346.)。
然而大多数微生物来源的硫酸软骨素裂解酶为胞内酶且酶活力较低。陶科等人(硫酸软骨素裂解酶abc产生菌的筛选及发酵工艺研究[j].中国抗生素杂志,2004,(29)3:138-140.)曾报道过利用一株彭氏变形杆菌发酵生产chsase,该过程工艺较为复杂,而且在发酵后期,chsase的酶活下降较为明显,整个过程的最高酶活仅为322.17u/l。li等人(expression,purificationandcharacterizationofgapdh-chsaseabcifromproteusvulgarisinescherichiacoli[j].proteinexpressionandpurification,2016,(128):36-41.)也报道了将来源于变形杆菌的chsase在大肠杆菌中进行异源表达,为提高chsase的表达量,报道中共表达了甘油醛-3-磷酸脱氢酶,但是纯化后的酶活却降低了3.1倍,因此不利于工业放大。目前国内没有产品供应,几乎全部依靠进口,价格非常昂贵,且目前暂无利用乳酸乳球菌进行表达得到硫酸软骨素酶及其突变体的文献报导,限制了硫酸软骨素酶在制备低分子硫酸软骨素和食品、医药、化妆品等方面的应用,特别是在食品领域的应用,因此,筛选能在乳酸乳球菌中进行表达得到高酶活的硫酸软骨素酶具有重要的意义。
技术实现要素:
本发明的第一个目的是提供如(a)或(b)的硫酸软骨素酶突变体,其中:
(a)氨基酸序列如seqidno.2、seqidno.6、seqidno.7、seqidno.8、seqidno.9任一所示;
(b)氨基酸序列与(a)具有90%以上同源性,并具有第180位甘氨酸、第233位组氨酸和/或第296位精氨酸的硫酸软骨素酶。
本发明的第二个目的是提供所述硫酸软骨素酶突变体的编码基因。
在一种实施方式中,所述基因的核苷酸序列如seqidno.5所示。
本发明的第三个目的是提供携带所述编码基因的表达载体。
在一种实施方式中,所述表达载体为质粒pnz8148,携带所述编码基因的表达载体命名为pnz8148-chsase(180g)
本发明的第四个目的是提供表达所述硫酸软骨素酶突变体的微生物细胞。
在一种实施方式中,所述微生物细胞是由所述表达载体转化制备的重组工程菌。
在一种实施方式中,所述微生物细胞为乳酸乳球菌,因为该菌种安全性更高,应用范围更广泛。
本发明的第五个目的是提供一种提高硫酸软骨素酶酶活的方法,是将具有seqidno.1所示氨基酸序列的硫酸软骨素酶做如下至少一种突变:
(1)将第180位由缬氨酸突变成甘氨酸或丙氨酸,
(2)将第233位由丝氨酸突变成组氨酸;
(3)将第296位由脯氨酸突变成精氨酸。
在一种实施方式中,所述硫酸软骨素酶突变体基因进行优化表达。
本发明的第六个目的是提供一种生产硫酸软骨素酶的方法,是将所述微生物细胞在培养基中,厌氧培养产酶。
在一种实施方式中,所述培养基为gm17培养基。
在一种实施方式中,所述培养是在28~30℃厌氧培养至od600为0.4-0.8,再用质量浓度1ng/ml的乳酸菌肽nisin诱导5~20h。
本发明还提供所述的硫酸软骨素酶突变体在食品、医药、化妆品等领域制备含低分子量硫酸软骨素的产品中的应用。
在一种实施方式中,所述应用是:以硫酸软骨素为原料,用硫酸软骨素酶水解生产低分子量的硫酸软骨素。
有益效果:本发明将野生型的硫酸软骨素酶氨基酸序列第180位、233位、296位氨基酸进行一个或多个突变,获得了硫酸软骨素酶的酶活提高的突变体。本发明构建的硫酸软骨素酶的突变体在乳酸乳球菌中表达的酶活可达35800u/l,酶活表达水平较高,且发酵时间明显缩短,而且整个工艺和后处理相对更简单,且乳酸乳球菌为食品安全菌株,可广泛用于食品、医药、化妆品等领域制备含低分子量硫酸软骨素,具有潜在而广泛的应用价值。
附图说明
图1所示为硫酸软骨素酶突变体优化表达的乳酸乳球菌载体构建示意图。
具体实施方式
硫酸软骨素酶的酶活测定方法:将发酵液超声破碎细胞,12000rpm4℃离心20min取上清作为粗酶液;粗酶液经过mbp柱纯化(10mm的麦芽糖洗脱)。将40微升适当稀释的酶液加到960微升的底物溶液中(2g/l的4-硫酸软骨素cs溶于20mm的tris-hcl)基于动力学的酶活测定方式取1min内的初始反应速率。酶活定义:单位时间内每消耗1微摩尔的底物所需要的酶量定义为一个酶活单位。
实施例1表达系统的构建
表达硫酸软骨素酶的乳酸乳球菌构建如附图1所示;以人工合成csdna为模板,使用引物p-8148-f/p-8148-r将所得的seqidno.5所示的csdnapcr产物与骨架pnz8148组装,以产生pnz8148-chsase(180g)重组质粒。
重组菌的构建:向乳酸乳球菌nz9000中,转入重组质粒pnz8148-chsase(180g),得到重组乳酸乳球菌pnz8148-chsase(180g)。
引物信息如下:5’-3’方向
以按照相同转化方法向乳酸乳球菌中转入pnz8148空质粒的菌株作为对照菌,挑取上述构建的重组菌株及对照菌,单克隆接种于5mllb培养基,根据需要添加相应的抗生素(加入终浓度为25ug/ml的氯霉素),在30℃厌氧培养至od600为0.4,再用质量浓度1ng/ml的乳酸菌肽nisin诱导20h,最终得到具有seqidno.2所示的氨基酸序列的硫酸软骨素酶突变体,硫酸软骨素酶的酶活高达31200u/l。
实施例2乳酸乳球菌发酵生产硫酸软骨素酶突变体v180g
将重组菌的单菌落接种于5mllb培养基加入终浓度为25ug/ml的氯霉素,在30℃厌氧培养至od600为0.8,再用质量浓度1ng/ml的乳酸菌肽nisin诱导20h,最终得到具有seqidno.2所示的氨基酸序列的硫酸软骨素酶突变体的酶活为35800u/l。
实施例3乳酸乳球菌发酵生产硫酸软骨素突变体v180a
具体实施方式同实施例2,区别在于,将第180位的缬氨酸突变为丙氨酸(氨基酸序列详见seqidno.9),结果显示,发酵相同时间,酶活仅为25050u/l。
实施例4乳酸乳球菌发酵生产硫酸软骨素酶突变体s233h
在seqidno.1序列的基础上,设计引物,将chsase酶第233位突变成组氨酸得到seqidno.6,向乳酸乳球菌中,转入pnz8148-chsase(233h)重组质粒,向乳酸乳球菌中,转入重组质粒pnz8148-chsase(233h),得到重组菌。接种于5mllb培养基,加入终浓度为25ug/ml的氯霉素,在30℃厌氧培养至od600为0.4,再用质量浓度1ng/ml的乳酸菌肽nisin诱导20h,最终得到具有seqidno.6所示的氨基酸序列的硫酸软骨素酶突变体,经测定,酶活为5700u/l。
实施例5乳酸乳球菌发酵生产硫酸软骨素酶突变体p296r
在seqidno.1序列的基础上将296位突变成精氨酸得到seqidno.7,向乳酸乳球菌中,转入pnz8148-chsase(296r)重组质粒,向乳酸乳球菌中,转入重组质粒pnz8148-chsase(p296r),得到重组菌。接种于5mllb培养基,加入终浓度为25ug/ml的氯霉素,在30℃厌氧培养至od600为0.4,再用质量浓度1ng/ml的乳酸菌肽nisin诱导20h,最终得到具有seqidno.7所示的氨基酸序列的硫酸软骨素酶突变体,最终得到的硫酸软骨素酶突变体的酶活为12230u/l。
实施例6乳酸乳球菌发酵生产硫酸软骨素酶突变体v180g/s233h/p296r
在seqidno.1序列的基础将180位突变成甘氨酸,233位突变成组氨酸,296位突变成精氨酸得到seqidno.8,向乳酸乳球菌中,转入pnz8148-chsase(180g/233h/296r)重组质粒,向乳酸乳球菌中,转入重组质粒pnz8148-chsase(180g/233h/296r),得到重组菌。接种于5mllb培养基,加入终浓度为25ug/ml的氯霉素,在30℃厌氧培养至od600为0.4,再用质量浓度1ng/ml的乳酸菌肽nisin诱导20h,最终得到具有seqidno.8所示的氨基酸序列的硫酸软骨素酶突变体,最终得到的硫酸软骨素酶突变体的酶活为15030u/l。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
sequencelisting
<110>南京汉欣医药科技有限公司
<120>新硫酸软骨素酶及表达硫酸软骨素酶的乳酸乳球菌
<130>baa201626a
<160>9
<170>patentinversion3.3
<210>1
<211>998
<212>prt
<213>人工序列
<400>1
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnvalserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealaserileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnprogluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995
<210>2
<211>998
<212>prt
<213>人工序列
<400>2
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnglyserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealaserileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnprogluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995
<210>3
<211>27
<212>dna
<213>人工序列
<400>3
ataacgcgagcataataaacggctctg27
<210>4
<211>28
<212>dna
<213>人工序列
<400>4
ttgagtaaccccaatgatgagtcgtgac28
<210>5
<211>2994
<212>dna
<213>人工序列
<400>5
atggcgacgtcaaatccagcgtacgacgcgaagaatcttttacaatccgagatctatcac60
tttgcacagaacaatcctctggctgactttagcgcaagcaagaattcaatacttactctg120
agcgacaaacggagcatcatgggaaatcagtctctgttgtggaagtggcaacggggcagc180
tcgtttactctgcaccgtaaaatcttggttcctacggataaagaagcttcgaaagcttgg240
ggcagatcatccacaccagtttttagcttttggctgtacaacgaaaagccgattgacggc300
tatcttacgattgattttggtgaaaaattgattagcacatccgaggcacaagcgggattt360
aaagtcaaattggactttacggggtggcgcgcagttggtgtttcactgaatatggacctt420
gaaaataaacaggaattaactaatgcaacaaatacttcgagtgacggtactcaggattcc480
atcggacggtcactcggtgcgaaagtagacagcatccgcttcaaagcaccgtctaacggc540
tcacagggagaaatttacatcgacagaataatgttcagcgttgacgacgcacgttaccaa600
tgggccgattatcaagttaaattgcgcctgtcggaaccggaacttaatttccatacggtc660
acaccgcaaatccctgtaaccccagagaatatcgcgtcgatcgatttactgcggcaacgc720
ttgattaacgagttcgtcggagcggaaaaagatacaaacacagcaatacagattaacgtg780
caaaaactcatccaggaatatgaagcattgaacattgaactgaccgccaatggcgcgact840
caaggacgccatattctgacggaaaaacaagtaatattataccagcctgaaaacttaaat900
tcacaagaaaaacagctttttgaaaattacgttatattggggaattatacgactttaatg960
ttcaacattagcagagcgtatgtcctgcaaaaagaaccgactcaaaaagctcaattgaag1020
caaatgtatatactgatgacaaaacatattttagatcaaggctttgtgaaaagtggagcc1080
ttagttcttactcataaatgggggtattcttctcgctattggtatatttccacacttaca1140
atgtcggatgctcttaaagaagcgaatctccaaacccaagtttatgattctctcttatgg1200
tatagtcgggaatttaaagcttctttcgatatgaaggtatcggcggattctgcagatctg1260
gactatttcaacacgttatcaagacaacatttggctttgacacttatcgaaccggacgat1320
caaaaacgtatcaatcttgtgaacacattcagccattatattacagcaggattgacgcaa1380
gtacctcctggcgccagagatgctttgcgccctgatggaaccgcctggagacatgaggga1440
aactatccgggatatgcttttccagcttttaaagatgcgtctcagcttatttatctgctt1500
aaagatacgccgttctcggtagctcagtctggttggaataatttaaaaagagctatggtg1560
gcgagttggatctattctaatccacaagtgggattgccgcttgcaggaaaacatccgttt1620
aattcaccattgtccaagagcgtggcacaaggctaccagtggttggccatgtcagcgaag1680
gctagtcctgataaaacgttatcagcgatctatctggcactttcggataaaactcagaac1740
caatcaacggctctgtttggagaaacaattacgccggcttcattacctcaaggattttat1800
gcttttaacggagccgcgtttggaatccatcggtggcaggataaaatggttacgcttaag1860
gcatataacacaaattgggtggcctcggaaatttacaacaaagacatgcgctatgggaga1920
tatcaaagccacggtgtggcgcaaatcgtgtcgaatggaagccagctttctcaaggctat1980
cagcaagaaggctgggaatggaaccgcatggagggagcgactatactccacctgccgtta2040
aaagatatcgaaagccctaagccgcacacgataaatcaaagaggagaacgaggtttttcg2100
ggcactgcttcgcttgaggcccaatatggtatgaattcattcaacatactgtatccggcc2160
aatctggagcgatttgaccctaattttacagctaagagatcagtattgtcggctgataat2220
catttgatcttcattagcggaaatatcatggccagtgacaaaaaaaacccggtagaaact2280
accattttccaacatgctatcactccgactattaatcttacatggatcaatggacagaaa2340
ctggaaaacatgccataccaaacgcttatacaggatggcgaatggctgattgattctatg2400
ggtaatgcctatatattgttgcaagctgaaaaagtcaatatctcgcgccaacatcaggtc2460
agcgcccaaaatcgtaatcgtcaacctacacaggccaatttcgcgtctgcgtggatcgat2520
cattctaccaaaccgaaagacgcgagttatgaatatatggtttttatcgatgcaacccca2580
gaaaaaatgggagagatggcgcagaagtttcgcgaaaacaacggattgtatcaggttctg2640
agaaaggataaggacgtccatattatattggataaattgtcaaacgttactggttatgca2700
ttttatcagcctgcatcaattgaagataaatggatcaaacgtgtaaataaaccagcgatc2760
gttatgacgcacaaacaacgagatacaatacttgtcgcgtctgttaccccagatttaaac2820
atgactcgtcaaaaagcttcgacgccagttaccatcaatttgactttgaatgggaagtgg2880
caggctagtgataaaaactcagaggttcggtaccaagtatctggtgataacacagaatta2940
acattcacgtcctactttggtatacctcaagaaattaaactctctcctttacct2994
<210>6
<211>998
<212>prt
<213>人工序列
<400>6
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnvalserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealahisileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnprogluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995
<210>7
<211>998
<212>prt
<213>人工序列
<400>7
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnvalserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealaserileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnarggluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995
<210>8
<211>998
<212>prt
<213>人工序列
<400>8
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnglyserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealahisileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnarggluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995
<210>9
<211>998
<212>prt
<213>人工序列
<400>9
metalathrserasnproalatyraspalalysasnleuleuglnser
151015
gluiletyrhisphealaglnasnasnproleualaasppheserala
202530
serlysasnserileleuthrleuserasplysargserilemetgly
354045
asnglnserleuleutrplystrpglnargglyserserphethrleu
505560
hisarglysileleuvalprothrasplysglualaserlysalatrp
65707580
glyargserserthrprovalpheserphetrpleutyrasnglulys
859095
proileaspglytyrleuthrileasppheglyglulysleuileser
100105110
thrserglualaglnalaglyphelysvallysleuaspphethrgly
115120125
trpargalavalglyvalserleuasnmetaspleugluasnlysgln
130135140
gluleuthrasnalathrasnthrserseraspglythrglnaspser
145150155160
ileglyargserleuglyalalysvalaspserileargphelysala
165170175
proserasnalaserglnglygluiletyrileaspargilemetphe
180185190
servalaspaspalaargtyrglntrpalaasptyrglnvallysleu
195200205
argleusergluprogluleuasnphehisthrvalthrproglnile
210215220
provalthrprogluasnilealaserileaspleuleuargglnarg
225230235240
leuileasngluphevalglyalaglulysaspthrasnthralaile
245250255
glnileasnvalglnlysleuileglnglutyrglualaleuasnile
260265270
gluleuthralaasnglyalathrglnglyarghisileleuthrglu
275280285
lysglnvalileleutyrglnprogluasnleuasnserglnglulys
290295300
glnleuphegluasntyrvalileleuglyasntyrthrthrleumet
305310315320
pheasnileserargalatyrvalleuglnlysgluprothrglnlys
325330335
alaglnleulysglnmettyrileleumetthrlyshisileleuasp
340345350
glnglyphevallysserglyalaleuvalleuthrhislystrpgly
355360365
tyrserserargtyrtrptyrileserthrleuthrmetseraspala
370375380
leulysglualaasnleuglnthrglnvaltyraspserleuleutrp
385390395400
tyrserarggluphelysalaserpheaspmetlysvalseralaasp
405410415
seralaaspleuasptyrpheasnthrleuserargglnhisleuala
420425430
leuthrleuilegluproaspaspglnlysargileasnleuvalasn
435440445
thrpheserhistyrilethralaglyleuthrglnvalproprogly
450455460
alaargaspalaleuargproaspglythralatrparghisglugly
465470475480
asntyrproglytyralapheproalaphelysaspalaserglnleu
485490495
iletyrleuleulysaspthrpropheservalalaglnserglytrp
500505510
asnasnleulysargalametvalalasertrpiletyrserasnpro
515520525
glnvalglyleuproleualaglylyshispropheasnserproleu
530535540
serlysservalalaglnglytyrglntrpleualametseralalys
545550555560
alaserproasplysthrleuseralailetyrleualaleuserasp
565570575
lysthrglnasnglnserthralaleupheglygluthrilethrpro
580585590
alaserleuproglnglyphetyralapheasnglyalaalaphegly
595600605
ilehisargtrpglnasplysmetvalthrleulysalatyrasnthr
610615620
asntrpvalalasergluiletyrasnlysaspmetargtyrglyarg
625630635640
tyrglnserhisglyvalalaglnilevalserasnglyserglnleu
645650655
serglnglytyrglnglngluglytrpglutrpasnargmetglugly
660665670
alathrileleuhisleuproleulysaspilegluserprolyspro
675680685
histhrileasnglnargglygluargglypheserglythralaser
690695700
leuglualaglntyrglymetasnserpheasnileleutyrproala
705710715720
asnleugluargpheaspproasnphethralalysargservalleu
725730735
seralaaspasnhisleuilepheileserglyasnilemetalaser
740745750
asplyslysasnprovalgluthrthrilepheglnhisalailethr
755760765
prothrileasnleuthrtrpileasnglyglnlysleugluasnmet
770775780
protyrglnthrleuileglnaspglyglutrpleuileaspsermet
785790795800
glyasnalatyrileleuleuglnalaglulysvalasnileserarg
805810815
glnhisglnvalseralaglnasnargasnargglnprothrglnala
820825830
asnphealaseralatrpileasphisserthrlysprolysaspala
835840845
sertyrglutyrmetvalpheileaspalathrproglulysmetgly
850855860
glumetalaglnlysphearggluasnasnglyleutyrglnvalleu
865870875880
arglysasplysaspvalhisileileleuasplysleuserasnval
885890895
thrglytyralaphetyrglnproalaserilegluasplystrpile
900905910
lysargvalasnlysproalailevalmetthrhislysglnargasp
915920925
thrileleuvalalaservalthrproaspleuasnmetthrarggln
930935940
lysalaserthrprovalthrileasnleuthrleuasnglylystrp
945950955960
glnalaserasplysasnsergluvalargtyrglnvalserglyasp
965970975
asnthrgluleuthrphethrsertyrpheglyileproglngluile
980985990
lysleuserproleupro
995