CN112375131A - Seroin蛋白的截短体及其应用 - Google Patents

Seroin蛋白的截短体及其应用 Download PDF

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CN112375131A
CN112375131A CN202011280285.0A CN202011280285A CN112375131A CN 112375131 A CN112375131 A CN 112375131A CN 202011280285 A CN202011280285 A CN 202011280285A CN 112375131 A CN112375131 A CN 112375131A
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董照明
赵萍
夏庆友
诸鸿韬
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Abstract

本发明公开了Seroin蛋白的截短体及其应用,Seroin蛋白的截短体为Seroin1、Seroin2或Seroin3的N端或C端片段;截短后的多肽具有抗菌活性,能够用于抗革兰氏阳性菌和革兰氏阴性菌,通过截短大大缩短了蛋白质的分子量,从而降低了通过化学法合成多肽的成本与难度,与此前完整蛋白的生产技术,本发明具有生产成本低、活性高、稳定性强、简便快捷等明显优点。

Description

Seroin蛋白的截短体及其应用
技术领域
本发明涉及生物技术领域,具体涉及Seroin蛋白的截短体,还涉及该蛋白截短体的应用。
背景技术
家蚕(Bombyx mori)是鳞翅目昆虫的典型代表,起源于中国,由原始的野桑蚕(Bombyx mandarina)经人为驯化而来,具有重要的农业和经济意义(Sutherland et al.,2010)。作为一种完全变态昆虫,家蚕在变态成蛹的过程中不进食也不运动,仅以茧包裹自身来抵御外界危害。构成蚕茧的蚕丝是一种外部纺成的纤维蛋白分泌物。蚕丝主要由丝素蛋白(fibroin)和丝胶蛋白(sericin)构成(向仲怀,2005)。通过质谱等分子生物学研究技术对蚕丝的组分进行研究分析,发现蚕丝中除丝素和丝胶外,还有许多低分子量蛋白质,主要包括各种蛋白酶抑制剂和seroin蛋白(Zhang et al.,2015,Dong et al.,2013)。Seroin蛋白是鳞翅目泌丝昆虫所特有的一类蛋白,1998年,Zurovec等人在大蜡螟(Galleriamellorella)的丝中发现一种由中部丝腺和后部丝腺共同分泌合成的小分子丝蛋白seroin(Zurovec et al.,2013)。2001年在家蚕中也鉴定到了两种seroin蛋白,同样由中部丝腺和后部丝腺共同分泌,并将其命名为seroin1和seroin2,并通过后续研究发现,家蚕的seorin蛋白在细菌诱导后的蚕体中上调表达,进一步研究发现seroin蛋白对细菌具有较强的抑制活性,其中seroin1对革兰氏阳性细菌藤黄微球菌的抑制活性较强,而seroin2对革兰氏阴性细菌大肠杆菌和革兰氏阳性细菌藤黄微球菌的抑制活性都很强(Nirmala et al.,2001,Singh et al.,2014)。近年来,通过搜索NCBI蛋白质序列库,表达序列标签(EST)库和转录组数据库,研究人员在分属于13个鳞翅目家族的32个物种中,共鉴定到了64个seroin蛋白。其中,在家蚕中鉴定到了除seroin1和seroin2外的另一个seroin蛋白,将其命名为seroin3,三个seroin蛋白的分子量都在10kDa左右。通过比较不同seroin的序列,发现seroin蛋白的C端序列较为保守,而N端序列富含脯氨酸(Dong et al.,2016)。
Seroin蛋白具有的抗菌活性使其能够作为一种天然防腐剂、抗菌剂,在农业、食品、医药、美容化妆品等多个领域都具有潜在的应用价值。解决大量合成制备seroin蛋白的方法是实现seroin蛋白应用的首要前提。目前人工制备蛋白质主要有两种方法:基因工程合成法和化学合成法。此前对于seroin蛋白主要通过原核表达的方式,即基因工程合成法,获得重组蛋白(Singh et al.,2014)。原核表达需要使用大肠杆菌(Escherichia coli)等细菌作为表达菌株,而seroin作为一类抑菌活性较强的蛋白,通过原核表达的方式来获得其重组蛋白存在一个无法避免的问题:seroin蛋白对宿主具有毒性或生长抑制性。这一问题可能导致菌株生长缓慢甚至无法表达,或仅能表达折叠不完全、功能受限的重组蛋白。因此,通过原核表达来获得seroin蛋白的方式具有很大的不确定性,无法保证生产效率、产物的产量和产物的活性。化学合成法目前主要用于合成小于10kDa的小分子蛋白,但是对于10kDa以上的蛋白,合成成本高,且合成难度大,因此研究具有生物学活性的seroin截短片段对开发Seroin抗菌产品具有重要意义。
发明内容
有鉴于此,本发明的目的之一在于提供一种Seroin蛋白的截短体;本发明的目的之二在于提供所述Seroin蛋白的截短体在制备抗菌剂中的应用。
为达到上述目的,本发明提供如下技术方案:
1、Seroin蛋白的截短体,所述Seroin蛋白的截短体为Seroin1、Seroin2或Seroin3的N端或C端片段;
Seroin1的N端氨基酸序列如SEQ ID NO.7所示,Seroin1的C端氨基酸序列SEQ IDNO.8所示;
Seroin2的N端氨基酸序列如SEQ ID NO.9所示,Seroin1的C端氨基酸序列SEQ IDNO.10所示;
Seroin3的N端氨基酸序列如SEQ ID NO.11所示,Seroin1的C端氨基酸序列SEQ IDNO.12所示。
优选的,所述Seroin蛋白的截短体为Seroin2的N端或C端片段,Seroin2的N端氨基酸序列如SEQ ID NO.9所示,Seroin2的C端氨基酸序列SEQ ID NO.10所示。
2、所述Seroin蛋白的截短体在制备抗菌剂中的应用。
优选的,所述抗菌剂为抗革兰氏阴性菌或/和抗革兰氏阳性菌。
优选的,所述革兰氏阴性菌为大肠杆菌;所述抗革兰氏阳性菌为金黄色葡萄球菌。
本发明的有益效果在于:本发明公开了Seroin蛋白的截短体,Seroin蛋白截短体来源于三种Seroin蛋白的分子量为3.7kDa—4.8kDa的N端多肽和分子量为5.5kDa—6.2kDa的C端多肽,研究发现截短后的多肽具有抗菌活性,特别是三种seroin蛋白截短后3.7kDa—4.8kDa的N端多肽具有与其全长蛋白序列几乎一样高的抗菌活性,由于截短片段大大缩短了蛋白质的分子量,从而降低了通过化学法合成多肽的成本与难度。与此前完整蛋白的生产技术,本发明具有生产成本低、活性高、稳定性强、简便快捷等明显优点。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为家蚕seroin蛋白的序列比对;
图2为大肠杆菌与Seroin1全长及截短肽段孵育后的相对增长率;
图3为金黄色葡萄球菌与Seroin1全长及截短肽段孵育后的相对增长率;
图4为大肠杆菌与Seroin2全长及截短肽段孵育后的相对增长率;
图5为金黄色葡萄球菌与Seroin2全长及截短肽段孵育后的相对增长率;
图6为大肠杆菌与Seroin3全长及截短肽段孵育后的相对增长率;
图7为金黄色葡萄球菌与Seroin2全长及截短肽段孵育后的相对增长率。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1、seroin蛋白生物信息学分析
基于NCBI数据库(https://www.ncbi.nlm.nih.gov/)下载的seroin1(基因编号gi|19070653)、seroin2(基因编号gi|19070655)和seroin3(基因编号gi|512931752)的核苷酸和氨基酸序列,seroin1、seroin2和seroin3的氨基酸分别如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示;seroin1、seroin2和seroin3的核苷酸序列如SEQ ID NO.4、SEQ IDNO.5和SEQ ID NO.6所示。然后利用以下在线软件及生物学软件进行序列的分析:
蛋白质的信号肽预测:SignalP(http://www.cbs.dtu.dk/services/SignalP/);
蛋白质的分子量和等电点预测:ExPASy(http://www.expasy.org/tools/);
同源比对软件:Clustal X和GeneDoc;
氨基酸序列长度、信号肽、分子量和等电点的分析结果如表1所示。
表1、家蚕seroin蛋白的序列特征分析
Figure BDA0002780555300000031
Figure BDA0002780555300000041
序列比对信息如图1所示。
实施例2、seroin蛋白截短合成
基于此前研究结果,将3种家蚕seroin蛋白去除信号肽序列后划分为N端与C端两部分,所得序列如表2所示(Dong et al.,2016)。利用化学合成方式获得seroin蛋白全长与截短肽段,并分别命名为seroin1-N、seroin1-C、seroin2-N、seroin2-C、seroin3-N和seroin3-C,相关信息已在表1中说明。
表2、家蚕seroin蛋白的截短体氨基酸序列
Figure BDA0002780555300000042
实施例3、seroin蛋白截短肽段的抑菌活性验证
为评价不同seroin蛋白肽段的抑菌活性,本实验选取两种细菌:大肠杆菌(革兰氏阴性菌,G-)和金黄色葡萄球菌(Staphylococcus aureus,革兰氏阳性菌,G+)分别与各seroin蛋白肽段进行孵育,用分光光度法检测细菌的生长曲线,并计算其相对于对照组的细菌增长率。具体方法如下:
1)使用无菌磷酸缓冲液(PBS),溶解足量的Seroin蛋白肽段至蛋白浓度为1mg/mL;
2)使用LB液体培养基,将细菌培养至OD600=0.2-0.3;
3)取50μL蛋白溶液和150μL菌液,分别加入至96孔板每孔中,充分混匀,并以未添加蛋白的无菌PBS溶液组作为空白对照、以添加蛋白等量牛血清蛋白(BSA)溶液组作为阴性对照、以添加蛋白等量乙二胺四乙酸(EDTA)溶液组作为阳性对照;
4)将96孔板置于37℃,60rpm环境下孵育8小时,并利用紫外分光光度计,每隔1小时测量一次每孔OD600的吸光度并记录;
5)将记录所得数据根据公式计算其基于空白对照组的相对增长率,公式如下:
Figure BDA0002780555300000051
6)根据处理后的数据绘制各组相对增长率曲线,结果如图2-7所示。
实验结果表明,seroin蛋白的全长及截短肽段对大肠杆菌和金黄色葡萄球菌两种细菌均具有一定的抑制性,尤其是金黄色葡萄球菌(G+),且全长的抑制效果最好,N端的截短肽段具有与全长相似的抑制效果,而C端的截短肽段的抑菌效果较差。具体结果为,seroin1蛋白的全长及截短肽段对大肠杆菌(G-),具有约15%的抑制活性;对于金黄色葡萄球菌(G+),seroin1蛋白的全长及截短肽段具有约35%的抑制活性,seroin1全长及截短肽段的抑菌效果可持续约1小时,如图2-3。Seroin2蛋白的全长及seroin2-N对大肠杆菌(G-),具有约40-45%的抑制活性,而seroin2-C仅具有20%的抑制性;对于金黄色葡萄球菌(G+),seroin2蛋白的全长及seroin-N段具有约60%的抑制活性,而seroin2-C的抑制性仅有25%,seroin2的全长及截短肽段的抑菌效果可持续约3小时,如图4-5。Seroin3蛋白的全长及seroin3-N和seroin3-C对大肠杆菌(G-),分别具有25%、20%和15%的抑制活性;对于金黄色葡萄球菌(G+),seroin3蛋白的全长及seroin3-N和seroin3-C对具有约40%、30%和20%的抑制活性;seroin3全长及截短肽段的抑菌效果可持续约4小时,如图6-7。
综合来说,本发明通过多肽合成的方式,获得了3种seroin蛋白的全长及N端和C端的截短肽段,并在体外验证了肽段的抑菌活性。抑菌实验的结果表明,seroin蛋白对革兰氏阳性菌具有更好的抑制活性,并且N端的截短肽段能在体外达到与全长相似的抑制效果,而相对保守C端的抑制活性较差。3种seroin蛋白的全长及截短肽段的抑菌活性比较表明,seroin2具有最好的抑菌效果,seroin3次之,seroin1的抑菌效果最差。且在全长及截短肽段的抑菌效果持续性上,seroin2和seroin3同样比seroin1要好。相比于此前报道的通过原核表达技术获取seroin蛋白的方式,本发明完善了其稳定性差、效率低下、实验周期长等缺陷。并在不影响抑菌效果的前提下,通过对seroin蛋白进行截短,进一步降低了实验成本、提高了实验的成功率。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
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<120> Seroin蛋白的截短体及其应用
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ttcaagagta ctttccttcc actagtctga taatggcgtt tacgaagttt ctttttgtga 60
ttacattaat tacaatcgcg agcgctggct ttgtatggga ggacgatgac gatttgttcc 120
cgggattytc agatacgttc aagatgcgag agataccaga gataaagtct cttgaatttg 180
atgatattaa aactcacgtc gcgggagata acgagcaata taccggagag tctaagtcga 240
gttacagttc ctcatccacg gtcaacggca agacagtgag cagcggcgga gttagcgagt 300
tgacaaatga cggaaaagcc gtcgaagaaa aggtcatgga atacaaagat ggcgattaaa 360
taaatccagc gattattcac gaataaaaaa atttaagtca aaaatattat ttcacaaaag 420
gggattttaa attgttaaaa ataaaacgtt aatgtttttc atctctgcat tgtatagtca 480
aatttacgta atagttcttt ttttgttcaa atttgtgtta ttttgtgact gtttgtgcga 540
aaatataaat ttttttcatt cacaagtttt agatgtgaag aaaattaaag ttaaatcaag 600
tcattaattg tcttttttca aaacggacat tattatttca gcagtactta cgacctttga 660
gccgtgttag taaaatagct aaggaattca agaaatgtta tctgcgtagt ttttactatt 720
aaaattaaat taaatattat taaattaaaa ttctacaaaa aaaaaaaaa 769
<210> 5
<211> 1413
<212> DNA
<213> 家蚕(Bombyx mori L.)
<400> 5
gtgtcctaca gttggtttca acagtgctac acagctcgat ttttttattg ttgaagcctt 60
ttaaccgcct gataatggcg ttcacgaagt ttctttttat gttgtcatta atcaccatcg 120
cgagcgctgg ctttgtttgg caggacgata actttccggg attcccaagc gatatgtggc 180
cctctataca gatcccaacg attccgccat tcgatcccaa aattccgaac tttgcttttt 240
cgttcccctc tcctgacaac attaagaaaa ctaaaccaca acccggacaa acctatagcg 300
gcgtctacgt gagcagcaat ggagggaagg gaactatggt tgcaaacatc aatggggaag 360
tgatcgaaaa aaagtttgga gaggattcca aaaaatctaa gagcaagtcc tagaagatag 420
aagatttaaa attaagtaac cgtcatttct gacacccaca tttcgattta atttcattta 480
aaatattttt atgttaacaa atgaatacat tcattatata ataacaattt taagataatt 540
taaatgttat ttgatatagt ttaaatgttg tttttccttc cttcttttac cgatgcatta 600
cataacacat acaaaaccca cacaattaat taattatttt tttattttat caacttcagg 660
tcttcttcaa tgtaatttat tataaaattc aaatgttttt cacctctgca ctgtaccgta 720
aaattaacgt aatcgttttg atatttataa aactgaattc atgatttcat tcaaagtaac 780
aatctaaaag ctatgaaacg gatttgaatg tgaaccaatt taaacctaat ttacagcaag 840
aatctttttt ttaaaacata aattattatt tcagcagtag gtactacaac ctttgatctg 900
tgttatgtag attgccacag gaattcataa attattaact gctaggtttc atctgagcct 960
ctctctagct ttgatttcaa tattctaaca ttatttagat tagaacatgg tgactacaaa 1020
cacagcaatc aaagatctct cttccatcat ggcgattctg atgtctacta cgcccgtgta 1080
attaaaagtt tattgatcga ggaaaaaggt cctaatgatg tactactatt attcagttct 1140
cacttaatgt atggccatgt cgaataaacg tgtagtcaat gttcggaaag tgcggttact 1200
tattttggat ttacgtttta aacagtccga tttccgggct aatttcattt gtaatcttat 1260
atctttttat tttatatgtc tttcaaaaat taaagaaaat ttaactgttg taattcaaat 1320
tccaacgatt ttcatgtatg taatttaagt tcaaaaaggt ttatattgca gtaattaaca 1380
ttaataaatt atacataccg aaaaaaaaaa aaa 1413
<210> 6
<211> 1695
<212> DNA
<213> 家蚕(Bombyx mori L.)
<400> 6
atcgacttgt gtgcgagcat tagaacgcgt cgagccaccg acgttggtaa cgtgtcataa 60
ctatatccgc gttttccaag atttttgtta tttatttgat tgttttaact cattcgcatt 120
agcattatga agttggttct tgtaacagtg ctcatcgtgg tttccgttca agcggtccca 180
agttttccaa tcggtgatga gttgtttgat gctgcctctt ccggagattg ggaaaaagtt 240
cacgaattaa ttaacagtaa actcaacgag aacgatagtt ggaaaccagt gtcagcgggc 300
agtgtcaaaa gcttgaagcc aatacccgga ggccatgtgt acggtgaatc cgagtacaca 360
ttccattcat cgtcagacat caacggacag aagaccgaca aaagcggagg tcacaaaatt 420
atcaacgatg acggaaaagt ctacgagttc gatttcaatc ctaaagtaaa gggctattaa 480
gttcattata cattaaagta gtcattatag taggtggact gcgttgggag tgaagtcagt 540
attaaggata tttgggacgt agaaatagtg taaagaaaaa gtcacgttaa taaaagtaaa 600
cgtgggctgt tagttttaga cattgctcat ataacatgta acgagatcaa tgacaaatta 660
caaatcaaat ccataaggtg gtgttttgcc tataatacct ttatttgggc tcggctgtaa 720
tttctagact ctttctgagc gtctcggcct ctagcctata tatagttaat tacatagggt 780
atatcttcta gaaaatgatc agtcagatat agatattacc gattgcaatt taaaaagttc 840
taaggataga cattagagca tatggttatt tcttgaatta tttcgaacag acatcaaggg 900
ataggttcaa aaggctttgt aagatataaa aagtaaccct gactaaattt atttagtaaa 960
actaacagta ggttaacgta actcgatcga tgaaccagaa taaaaagctc aaagcgtgtc 1020
gaatcaattt acgttatcta aatgtgagta atgatttaat cacggccgat aaatagataa 1080
gcagtagata taacaatttt actgacgcag tacatcggaa cgtgttttat gataattacg 1140
tatcaaatca ttcataatat taagattttg ttttgaaata tgttttattt ttatttttaa 1200
taagcagtgc tcggaaataa tagcataatg ttttgtcatt cattagcttt tcattgttga 1260
tgttaatcta tgatatgtag tccaaaatgt ttttcaattg caagaggagt cataatttcg 1320
tattaattta tgtttttaac catccacata tccacagttc tttgtacatt ctgtggcaat 1380
cacttttaat tttataataa tggtaacaaa acttgtgatt gtaatgtatt aaagtatcaa 1440
aaatatattt taattttttt gttacctgat tatttgcttt tcgtgggaga atgactagcc 1500
caatgaataa atgactatta cactgcggca gatttaggca gcgatgaatg tctgattgga 1560
atattactaa aacaagattt ttttaattat tcatgaggct ttaatcattt gcatattatc 1620
tttttaacgc cgcttttaaa ttggtagatt taaaattaat tgtctgtaaa atatatataa 1680
aaactacatt ttaga 1695
<210> 7
<211> 31
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 7
Gly Phe Val Trp Glu Asp Asp Asp Asp Leu Phe Pro Gly Phe Ser Asp
1 5 10 15
Thr Phe Lys Met Arg Glu Ile Pro Glu Ile Lys Ser Leu Glu Phe
20 25 30
<210> 8
<211> 59
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 8
Asp Asp Ile Lys Thr His Val Ala Gly Asp Asn Glu Gln Tyr Thr Gly
1 5 10 15
Glu Ser Lys Ser Ser Tyr Ser Ser Ser Ser Thr Val Asn Gly Lys Thr
20 25 30
Val Ser Ser Gly Gly Val Ser Glu Leu Thr Asn Asp Gly Lys Ala Val
35 40 45
Glu Glu Lys Val Met Glu Tyr Lys Asp Gly Asp
50 55
<210> 9
<211> 42
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 9
Gly Phe Val Trp Gln Asp Asp Asn Phe Pro Gly Phe Pro Ser Asp Met
1 5 10 15
Trp Pro Ser Ile Gln Ile Pro Thr Ile Pro Pro Phe Asp Pro Lys Ile
20 25 30
Pro Asn Phe Ala Phe Ser Phe Pro Ser Pro
35 40
<210> 10
<211> 52
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 10
Asp Asn Ile Lys Lys Thr Lys Pro Gln Pro Gly Gln Thr Tyr Ser Gly
1 5 10 15
Val Tyr Val Ser Ser Asn Gly Gly Lys Gly Thr Met Val Ala Asn Ile
20 25 30
Asn Gly Glu Val Ile Glu Lys Lys Phe Gly Glu Asp Ser Lys Lys Ser
35 40 45
Lys Ser Lys Ser
50
<210> 11
<211> 41
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 11
Val Pro Ser Phe Pro Ile Gly Asp Glu Leu Phe Asp Ala Ala Ser Ser
1 5 10 15
Gly Asp Trp Glu Lys Val His Glu Leu Ile Asn Ser Lys Leu Asn Glu
20 25 30
Asn Asp Ser Trp Lys Pro Val Ser Ala
35 40
<210> 12
<211> 60
<212> PRT
<213> 人工序列(Artificial sequence )
<400> 12
Gly Ser Val Lys Ser Leu Lys Pro Ile Pro Gly Gly His Val Tyr Gly
1 5 10 15
Glu Ser Glu Tyr Thr Phe His Ser Ser Ser Asp Ile Asn Gly Gln Lys
20 25 30
Thr Asp Lys Ser Gly Gly His Lys Ile Ile Asn Asp Asp Gly Lys Val
35 40 45
Tyr Glu Phe Asp Phe Asn Pro Lys Val Lys Gly Tyr
50 55 60

Claims (5)

1.Seroin蛋白的截短体,其特征在于:所述Seroin蛋白的截短体为Seroin1、Seroin2或Seroin3的N端或C端片段;
Seroin1的N端氨基酸序列如SEQ ID NO.7所示,Seroin1的C端氨基酸序列SEQ ID NO.8所示;
Seroin2的N端氨基酸序列如SEQ ID NO.9所示,Seroin1的C端氨基酸序列SEQ IDNO.10所示;
Seroin3的N端氨基酸序列如SEQ ID NO.11所示,Seroin1的C端氨基酸序列SEQ IDNO.12所示。
2.根据权利要求1所述Seroin蛋白的截短体,其特征在于:所述Seroin蛋白的截短体为Seroin2的N端或C端片段,Seroin2的N端氨基酸序列如SEQ ID NO.9所示,Seroin2的C端氨基酸序列SEQ ID NO.10所示。
3.权利要求1或2所述Seroin蛋白的截短体在制备抗菌剂中的应用。
4.根据权利要求3所述的应用,其特征在于:所述抗菌剂为抗革兰氏阴性菌或/和抗革兰氏阳性菌。
5.根据权利要求4所述的应用,其特征在于:所述革兰氏阴性菌为大肠杆菌;所述抗革兰氏阳性菌为金黄色葡萄球菌。
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