CN101508992A - 三疣梭子蟹抗菌肽基因及其克隆方法 - Google Patents
三疣梭子蟹抗菌肽基因及其克隆方法 Download PDFInfo
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Abstract
三疣梭子蟹抗菌肽基因及其克隆方法,所说的三疣梭子蟹crustin抗菌肽基因cDNA不包括polyA共有565nt,其中含有5’端非编码区(5'UTR)长75nt,1个阅读框长330nt,3’非编码区(3'UTR)长160nt,其GenBank序列号为FJ467931。本发明成功克隆到三疣梭子蟹I型crustin cDNA全长序列,可通过基因工程方式构建原核或真核基因工程菌株,高效表达具生理活性的重组蛋白,大量生产。作为饲料免疫添加剂可替代传统饲料中的某些抗生素,用于海水养殖业,实现绿色养殖,减少乃至消除抗生素在水产品中的蓄积,提高水产品质量;也可以作为某些耐药性抗生素的有效替代品,用于水产养殖业中耐药性细菌的防治药物。
Description
技术领域
本发明涉及一种从三疣梭子蟹(Portunus trituberculatus)中克隆的I型Crustin抗菌肽基因,属生物技术领域。
背景技术
中国是全球最大的水产养殖大国,水产品总产量(捕捞+养殖)占全球35%,其中水产品养殖产量约占全球的70%,2005年水产品产量达5181万吨。其中海水产品2854万吨,占总产量的55%,捕捞约1022万吨,人工养殖产品约1888万吨。随着全球对水产品的消费逐年增加,国内消费增长势头迅猛,另一方面全球海洋捕捞资源衰退,供给逐步转向以养殖水产品为主,中国水产养殖业在全球消费中越来越重要。人工养殖比例逐年提高,1978年人工养殖比例占26%,2005年人工养殖比例达61%。随着水产养殖业的蓬勃发展,由于高密度养殖、养殖水域富营养化、生态环境恶化等原因使得水产养殖品种频频发生各种疾病,经济损失严重,已成为21世纪水产养殖业发展的重要制约因素。据初步统计,目前危害水产养殖生物的病害已达400~500种,大多数病害是由病毒、细菌、真菌等微生物所引起的。而传统的抗微生物药物及饲料添加剂——抗生素,由于耐药性病原菌的形成,以及食品安全性等公共卫生上问题的提出,目前在世界各国养殖业中的使用都受到严格限制,如欧盟自2006年1月1日起,全面禁止食品动物使用抗生素促生长饲料添加剂,因此寻找安全的抗生素替代物、开发安全高效的新型抗微生物免疫增强剂或能增强养殖品种机体免疫力的饲料添加剂成为研究的热点,也是我国养殖业、饲料行业健康、持续发展首要解决的课题之一。
近年来,随着对养殖动物的自身免疫机制的深入研究,发现动物体液中富含多种体液免疫因子如凝集素、防御素、抗菌肽及多种行使调控作用的蛋白因子等。养殖品种自身来源的体液免疫因子由于其特殊的作用机制,不存在食品安全问题与环境问题,并对养殖品种具有促生长、增强免疫力的功能,还可通过基因工程技术大量发酵生产,不存在药源问题,成为替代传统抗生素作为绿色饲料添加剂及抗感染药剂的理想选择,是当前饲料添加剂领域的研究热点。Crustins最早由Relf等在青蟹(Carcinus maenas)中分离鉴定,后续研究表明Crustins普遍存在于甲壳类(蟹、对虾、龙虾、螯虾等)中,主要由甲壳类血淋巴细胞合成,分子量7~14kDa,等电点pI7.0~8.7。甲壳类Crustins以其C末端的WAP(whey acidic protein)结构域为标志,与甲壳类中发现的其它富含Cys的抗菌肽区分开来,如同样富含Cys但不含WAP结构域的甲壳类抗菌肽defensins、penaeidins等。WAP结构域是由8个保守的Cys位点形成四个二硫键,构成一个紧密结构,这种结构又被称作4DSC结构(four-disulphide core)。含有WAP结构域的蛋白广泛存在于其它生物类群中,如哺乳动物的antileukoproteinases、elafins、trappins等,这些含WAP结构域蛋白一般都是小分子分泌蛋白,具有蛋白酶抑制剂、个体生长调节、组织分化调节等生理功能。目前发现的Crustins均由信号肽和成熟肽两部分组成,信号肽区域长16~24Aa,与其它类型抗菌肽如cathelicidins、defensins的保守性相比,Crustins信号肽保守性较差,而成熟肽区域相对保守些。根据信号肽与成熟肽C末端之间结构的差别,可将Crustins分为三个亚型:
I型:信号肽与WAP之间存在一个序列长度多变、富含Cys的结构域,但结构域中Cys的数量不超过6个,因此仅能形成一个4DSC类似结构域,而不能形成完整的4DSC结构域,该区域的一致框架一般为:—C—X(3)—C—X(8—12)—C—C—X(16—17)—C—X(6)—C—X(9—10)—。I型Crustins主要存在于蟹、龙虾和螯虾中。
II型:II型Crustins主要发现于对虾中,不仅有一个富含Cys结构域,而且邻近信号肽区还有一个大约40~80Aa的Gly富集区。Gly富集区在不同种中Gly数量变化较大,一般在20~50个之间,在对虾中一般以5~8个VGGGLG重复结构出现,但在Penaeus monodon中发现的一个Type II crustin(EF523614)中,其Gly富集区的22个Gly残基不是以VGGGLG重复结构出现的。
III型:III型Crustins不仅缺乏II型的Gly富集区,而且也没有I型和II型中的Cys富集区,因此某些文献中不把它归为Crustins,而称之为SWD(single-whey domain)蛋白、chelonianin-like蛋白或antileukoproteinase-like蛋白。有关III型Crustins目前研究的还较少,仅发现于P.Monodon,L.Vannamei,Marsupenaeusjaponicus,Fenneropenaeus chinensis等对虾中。
用已报道的三种类型Crustins及其它一些非Crustins具WAP结构域蛋白构建系统树显示:I型和II型各独立聚合为分枝,而III型Crustins与非甲壳类WAP结构域蛋白,如人分泌型亮氨酸蛋白酶抑制剂(SLPI,X04502)、人抗白血球蛋白酶抑制剂(ALPI,P03973)、鼠SLPI(AF151982)等聚为一枝。
Crustins作为甲壳类直接抗菌防御因子的证据来源于研究Crustins在不同组织中的表达以及实验感染研究。研究表明大多数Crustins在血淋巴细胞中是高水平的组成型表达,在其它器官,如腮、心脏、肠也检测到编码Crustins转录体表达,但这些器官中有丰富的血管分布,因此在这些器官中检测到Crustins转录体是否是由于血淋巴干扰所造成的目前还不清楚;同时研究表明Crustins一般均有较好的抗菌活性。例如Relf等报道C.maenas Carcinin对龙虾致病菌A.viridansvar homari、两株海洋菌Planococcus spp.和一株耐盐菌M.luteus均具有抗菌活性;Supungul P等(2008)报道重组P.monodon Crustin(D766060)对S.aureus、S.iniae有很强的抑菌活性而对A.viridans、M.luteus无效,但另一种重组P.monodon Type II Crustins(EF654658)对G+菌和G-菌均有很强的抗菌活性,包括A.viridans var homari以及另两种致病G-菌E.coli 363、V.harveyi;Haug等(2006)报道蜘蛛蟹H.araneus Crustins对谷氨酸棒杆菌C.glutamicum的半致死浓度仅为3mM;Zhang(2007)等报道中国对虾F.chinensis重组crustins对S.aureus、M.luteus及三种Bacillus的半致死浓度也仅为2~8mM;Type III Crustins功能报道较少,仅见Amparyup P等(2008)黑虎虾P.monodon III型crustin重组蛋白具有抗菌活性,同时也是蛋白酶subtilisin A的竞争性抑制剂。就目前的研究结果显示:Crustins广泛的抗菌谱似乎与序列多态性有关,由于不同Crustins异构体抑菌差异没有被研究,不同异构体间是否有协同增效作用还不清楚。虽然Crustins的抗菌机制目前不明,但WAP结构域与抗菌活性密切相关,如Zhang等报道的一个Type II型F.chinensis Crustin没有WAP,无抗菌活性;当蛇毒WAP抗菌蛋白Cys残基被还原和烷化后,也丢失抗菌活性,因此WAP完整结构可能是Crustins抗菌活性必需的。哺乳动物WAP与丝氨酸蛋白酶抑制剂活性相关,通过将其抑制环插入蛋白酶活性位点口袋干扰催化亚基,WAP蛋白酶抑制剂活性位点特征为4DSC结构第二个Cys紧连着一个Met,而在脊椎动物具有抗菌活性的WAP蛋白中,Met被碱性氨基酸或疏水氨基酸所取代,并且Met也极少出现在Crustins的WAP结构域中。另外,甲壳类Crustins除了抗菌及免疫调节功能之外,在甲壳类再生组织中也检测到Crustins的表达,可能与机体修复功能相关。Stoss等报道小龙虾(P.argus)嗅觉器官再生上皮组织中检测到PET-15的表达;Durica等报道在招潮蟹(C.pugilator)再生断肢中检测到Crustins(DW176897)的表达。综上所述,甲壳类Crustins由于其功能、结构上的优势,极具应用开发价值,因而近年来研究报道也较多。
三疣梭子蟹隶属于甲壳纲、十足目、梭子蟹科、梭子蟹属,分布于中国、日本及朝鲜等海域,是一种大型海产经济蟹类,因其生长快,肉质鲜美、营养丰富而深受国内外消费者喜爱。沿海地区仅浙江省自上世纪八十年代末开始养殖以来,发展就异常迅猛,目前在浙江的舟山、宁波等地,已初步建立了梭子蟹围塘养殖示范园区和梭子蟹产业化养殖基地,2007年全省三疣梭子蟹养殖面积达到58320亩,产量26404吨,产值超过20亿元,已成为浙江省主导养殖品种之一。梭子蟹养殖业己在当地渔业结构调整、渔民转产、实现渔农民增收中发挥重要作用。但随着梭子蟹养殖业的发展,梭子蟹病害也和其它养殖品种一样,呈现逐年上升趋势,如弧菌病、烂鳃病、甲壳溃疡病、牛奶病等,给梭子蟹养殖业造成重大损失,成为制约产业健康发展的一种重要因素。因此,急需开发无公害梭子蟹自身免疫因子来源的免疫增强剂或饲料添加剂。
发明内容
本发明的目的旨在提供一种从三疣梭子蟹(Portunus trituberculatus)中克隆的I型crust in抗菌肽基因。该基因可与酿酒酵母等构建真核表达载体建立高效表达I型crustin的基因工程菌株,体外大量生产三疣梭子蟹I型crustin抗菌肽,作为饲料免疫添加剂可替代传统饲料中的某些抗生素,还可以作为替代某些耐药性抗生素的有效抗菌药物。
本发明的目的是通过如下技术方案实现的。
三疣梭子蟹的I型crustin抗菌肽基因,所说的三疣梭子蟹crustin抗菌肽基因cDNA不包括polyA共有565nt,其中含有5’端非编码区(5’UTR)长75nt,1个阅读框长330nt,3’非编码区(3’UTR)长160nt,其GenBank序列号为FJ467931,全长cDNA序列如下:
10 20 30 40 50
60
gaaaagagca gaacaagaca ctgtggtgga cacacctgct ctcaccaacg
tcttcctcaa
70 80 90 100 110
120
gaatctatag agataatgaa gatgcagact gtaatagcca tggcagttgt
ggctaccatt
130 140 150 160 170
180
gtggccatga cagaagcatc cctagtactt ccatacccag gtctggattg
taagtactgg
190 200 210 220 230
240
tgcaaggaca actacgataa acactactgc cgtggcccac caggacgtac
ctatccacct
250 260 270 280 290
300
tataccgagc gctctggtaa atgtcctccg gtccgtgcta catgtactgg
tgtcaggtca
310 320 330 340 350
360
cgcctaccaa agttgtgtcc ccatgatggt gcttgtgact ttccaagcaa
gtgctgttat
370 380 390 400 410
420
aatcattgaa
430 440 450 460 470
480
ccagactgaa gatgaaaatt tttcaccaat tttgcttaca aactttacag
agatgatatt
490 500 510 520 530
540
ctcttaaaac attgatttat ggttatgcta ttctatattc atttgtaaac
aatataacaa
550 560 570 580
aataataaac gcaattgtat ccgtgaaaaa aaaaaaaaaa
所说的三疣梭子蟹的I型crustin抗菌肽cDNA推导编码氨基酸序列如下:
10 20 30 40 50 60
70 80
MKMQTVIAMA VVATIVAMTE ASLVLPYPGL DCKYWCKDNY DKHYCRGPPG RTYPPYTERS
GKCPPVRATC TGVRSRLPKL
90 100 110
CPHDGACDFP SKCCYDACVE HHVCKTPDFY*
所说的三疣梭子蟹的I型crustin抗菌肽cDNA读码框及推导蛋白质氨基酸序列如下:
gaaaagagca gaacaagaca ctgtggtgga cacacctgct ctcaccaacg tcttcctcaa
60
gaatctatag agata aag atg cag act gta ata gcc atg gca gtt gtg 111
Met Lys Met Gln Thr ValIle Ala Met Ala Val Val 12
gct acc att gtg gcc atg aca gaa gca tcc cta gta ctt cca tac cca 159
Ala Thr Ile Val Ala Met Thr Glu Ala Ser Leu Val Leu Pro Tyr Pro 28
ggt ctg gat tgt aag tac tgg tgc aag gac aac tac gat aaa cac tac 207
Gly Leu Asp Cys Lys Tyr Trp Cys Lys Asp Asn Tyr Asp Lys His Tyr 44
tgc cgt ggc cca cca gga cgt acc tat cca cct tat acc gag cgc tct 255
Cys Arg Gly Pro Pro Gly Arg Thr Tyr Pro Pro Tyr Thr Glu Arg Ser 60
ggt aaa tgt cct ccg gtc cgt gct aca tgt act ggt gtc agg tca cgc 303
Gly Lys Cys Pro Pro Val Arg Ala Thr Cys Thr Gly Val Arg Ser Arg 76
cta cca aag ttg tgt ccc cat gat ggt gct tgt gac ttt cca agc aag 351
Leu Pro Lys Leu Cys Pro His Asp Gly Ala Cys Asp Phe Pro Ser Lys 92
tgc tgt tat gac gcc tgt gtg gag cac cac gta tgc aag act cct gat 399
Cys Cys Tyr Asp Ala Cys Val Glu His His Val Cys Lys Thr Pro Asp 108
448
Phe Tyr ***
110
attttgctta caaactttac agagatgata ttctcttaaa acattgattt atggttatgc
508
tattctatat tcatttgtaa acaatataac aaaataataa acgcaattgt atccgtgaaa
568
aaaaaaaaaa aa
580
所说的3’-RACE有义链简并引物crustin F1为包括起始密码子ATG的起始密码子后+238碱基起共17bp。
所说的5’-RACE反义链特异引物crustinR1为终止密码子TAA后第45个碱基起共18bp。
所说的5’-RACE反义链特异引物crustinR2为终止密码子TAA前第20个碱基开始,即从基因+310位开始共23bp。
三疣梭子蟹的I型crustin抗菌肽基因的克隆方法,步骤为:
(1)设计有义链简并引物crustin F1:5’-GTN TGY GCN CAY GAY GG-3’,与逆转录引物上的M13Primer M4:5’-GTT TTC CCA GTC ACG AC-3’配合,以三疣梭子蟹血淋巴细胞总RNA模板,通过3’-RACE技术克隆三疣梭子蟹的I型crustin抗肽基菌因mRNA3’端209nt基因片段,不包括引物及PolyA;
(2)根据获得的I型crustin抗肽基菌因3’端209nt基因片段设计特异引物反义链引物crustinR1:5′-CTG TAA AGT TTG TAA GCA-3′和crustinR2:5′-TTAGTAGAAATCAGGAGTCTTGC-3′,5’端有义链引物使用大连宝生物公司5’-Full RACEkit试剂盒自带的:5′RACEOuter Primer:5′-CATGGCTACATGCTGACAGCCTA-3′,5′RACEInner Primer:5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′,以三疣梭子蟹血淋巴细胞总RNA模板,按5’-Full RACE kit操作总RNA去磷酸化处理、“去帽子”反应、5′RACE Adaptor的连接、反转录反应、嵌套PCR,从三疣梭子蟹血淋巴细胞中扩增出三疣梭子蟹的I型crustin抗菌肽基因完整5’UTR及编码区在内的408nt的目的片段;
(3)将3’-RACE及5′RACE的测序结果进行拼接和碱基校对,最终得到三疣梭子蟹的I型crustin抗菌肽基因的全长cDNA序列。
本发明以三疣梭子蟹血淋巴细胞总RNA为模板,参考已报道的甲壳类crustins的基因序列,通过优化选择设计简并引物,利用3’RACE、5’RACE等分子生物学技术手段,成功克隆到三疣梭子蟹I型crustin cDNA全长序列,该基因属于甲壳类crustins基因家族的新组员。该基因其前体序列与Imjongjirak等报道的青蟹(Scylla paramamosain,Genbank accession:ABY20727)、Burgess报道的岸蟹(Carcinus maenas,Genbank accession:CAH25401)crustins基因氨基酸序列同源性分别达到69%和63%。且前体中信号肽与成熟肽之间的剪切位点一致,为TEA—S/RL;推导的三疣梭子蟹I型crustin成熟肽与其它I型crustins一样,由N端富含Cys结构域和C端完整WAP结构域组成。通过文献检索证实该类结构crustin多肽具有抗G+、G-菌作用,且具蛋白酶抑制剂及免疫调节功能活性,对动物无毒,可通过基因工程方式构建原核或真核基因工程菌株,高效表达具生理活性的重组蛋白,大量生产。作为饲料免疫添加剂可替代传统饲料中的某些抗生素,用于海水养殖业,实现绿色养殖,减少乃至消除抗生素在水产品中的蓄积,提高水产品质量;也可以作为某些耐药性抗生素的有效替代品,用于水产养殖业中耐药性细菌的防治药物。
附图说明
图1是三疣梭子蟹血淋巴细胞总RNA电泳图;
图2是三疣梭子蟹血淋巴3’-RACE电泳图;
图3是三疣梭子蟹血淋巴5’-RACE电泳图。
具体实施方式
1.三疣梭子蟹血淋巴细胞总RNA的提取
选健康三疣梭子蟹断肢采血10ml,加入10ml抗凝剂(NaCl 8.2g、葡萄糖19.8g、柠檬酸6.3g、柠檬酸钠7.6g、EDTA 3.7g,pH7.4,加蒸留水定容至1L,121℃灭菌15min),3000rpm离心5min,弃上清,用marine saline洗涤沉淀一次(NaCl 33.9g、CaCl2 2.95g、KCl 0.90g、Na2HPO4 0.2g、Tris-碱6.05g,pH7.4,加水定容至1L,121℃灭菌15分钟),加入0.5ml Trizol,用枪头抽吸混匀,用1ml针筒,6#针头抽吸匀浆液两次以剪切基因组DNA后移入1.5ml Eppendoff管(DEPC处理),加100ul氯仿/异戊醇(24∶1),剧烈震荡混匀30sec,12,000rpm室温离心5min,将上清液(约450ul)转移到1.5ml离心管中,加150ul无水乙醇,混匀后转移到UNIQ-10柱中,室温放置2min,8,000rpm室温离心1min,弃去收集管中的废液,将柱放回收集管中,加入450ul的RPE Solution,10,000rpm室温离心30sec,再用450ul的RPE Solution洗涤一次,10,000rpm室温离心15sec,小心取出柱,放到无菌RNAase-free的1.5ml离心管里,在柱内膜的中央小心加入50ul DEPC-H2O,室温或55~80℃放置2min,10,000rpm,室温离心1min。离心管内的溶液为RNA样品,测OD260和OD260/280,于1%的琼脂糖凝胶检测,如图1所示。
2.3’RACE
2.1 cDNA第一链合成
取总RNA1ug,加入试剂盒自带的Oligo dT-Adaptor Primer(5uM)1ul,5×M-MLV buffer 2ul,dNTP Mixture(各10mM)1ul,RNase Inhibitor(40U/ul)0.25ul,Reverse Transcriptase M-MLV(RNaseH-)(200U/ul)0.25ul,加RNase FreedH2O至总体积10ul。42℃反应60min,75℃ 15min灭活Reverse TranscriptaseM-MLV,置于冰上。
2.2 PCR
根据已报道的甲壳类crustin氨基酸序列和核酸序列ClustalW结果,选取靠近C端的比较保守区域设计有义链简并引物crustin F1:5’-GTN TGY GCN CAYGAY GG-3’。反义链引物用试剂盒自带引物M13Primer M4:5’-GTT TTC CCAGTC ACG AC-3’。DNA聚合酶使用大连宝生物公司ExTaq酶。反应体系如下:上述1的反转录反应液2ul,1×cDNADilution Buffer II 8ul,10×PCR Buffer 4ul,MgCl(25mM)3ul,引物crustin F1(10uM)2ul,引物M13 Primer M4(10uM)2uM,TaKaRa Ex Taq(5U/μl)0.5ul,dH2O 28.5ul。反应条件,94℃预变性3min;94℃变性1min,40℃退火1min,72℃延伸1min,5个循环;94℃变性1min,55℃退火1min,72℃延伸1min,35个循环;72℃后延伸10min。1%琼脂糖凝胶电泳检测得到约260nt的特异cDNA扩增带,如图2所示。
2.3 目的片断割胶回收与T载体克隆测序
PCR产物跑1%琼脂糖凝胶电泳,割取目的带,用小量胶回收试剂盒(上海华舜生物试剂公司,W5212)进行纯化回收。将PCR回收产物连接入pMD19-TVector(大连宝生物公司)。连接产物转入以E.coli DH5α菌株制备的感受态细胞中,用X-gal、IPTG、Amp系统筛选目的克隆子,PCR法检测重组成功克隆,扩大培养后用柱式小量质粒提取试剂盒(上海生工)抽提质粒。提取的质粒作为模板,引物B0012:5’-CGCCAGGGTTTTCCCAGTCACGAC-3’、B0014:5’-AGCGGATAACAATTTCACACAGGA-3’进行双向测序。测序结果校正后翻译成氨基酸序列,在NCBI网站(http://www.ncbi.nlm.nih.gov/)通过blast检索同源基因,结果显示与与青蟹Scylla paramamosain crustins蛋白C末端同源性最高,达到84%。
3.5’RACE
3.1 全长mRNA 5’加接头及反转录反应
3.1.1 三疣梭子蟹总RNA去磷酸化处理
使用Alkaline Phosphatase(CIAP)对Total RNA中裸露的5′磷酸基团进行去磷酸反应,按下列组份配制去磷酸反应液:总RNA 2ug,RNase Inhibitor(40U/μl)1μl,10×Alkaline Phosphatase Buffer(MgCl2Free)5μl,Alkaline Phosphatase(Calfintestine)(16U/μl)0.6μl,加RNase Free dH2O至总体积50μl。50℃反应1小时后向反应液中加入20μl的3M CH3COONa(pH5.2),130μl的RNase FreedH2O充分混匀,加入200μl的苯酚/氯仿/异戊醇(25:24:1),充分混匀后13,000×g室温离心5分钟,将上层水相转移至新的EP管中,加入200μl的氯仿,充分混匀后13,000×g室温离心5分钟,将上层水相转移至新的EP管中,加入2μl的NA Carrier后均匀混合,加入200μl的异丙醇,充分混匀后,冰上冷却10分钟,13,000×g4℃离心20分钟,弃上清,加入500μl的70%冷乙醇(RNase Free dH2O配制)漂洗,13,000×g4℃离心5分钟,弃上清后干燥,加入7μl的RNase Free dH2O溶解沉淀,得到CIAP-treated RNA。
3.1.2“去帽子”反应
使用Tobacco Acid Pyrophosphatase(TAP)去掉mRNA的5′帽子结构,保留一个磷酸基团,按下列组份配制“去帽子”反应液:CIAP-treated RNA 7μl,RNaseInhibitor(40U/μl)1μl,10×TAP Reaction Buffer 1μl,Tobacco AcidPyrophosphatase(0.5U/μl)1μl,总体积10ul。37℃反应1小时,此反应液为CIAP/TAP-treated RNA。取5μl用于5′RACE Adaptor连接反应,剩余的5μl保存于-80℃。
3.1.3 5′RACE Adaptor的连接
首先配制下列反应液:CIAP/TAP-treated RNA 5μl,5′RACE Adaptor(15μM)1μl,RNase Free dH2O 4μl,65℃保温5分钟后冰上放置2分钟,然后加入下列试剂:RNase Inhibitor(40U/μl)1μl,5×RNA Ligation Buffer 8μl,40% PEG#600020μl,T4 RNA Ligase(40U/μl)1μl,16℃反应1小时后向反应液中加入20μl的3M CH3COONa(pH5.2),140μl的RNase Free dH2O充分混匀,加入200μl的苯酚/氯仿/异戊醇(25:24:1),充分混匀后13,000×g室温离心5分钟,将上层水相转移至新的EP管中,加入200μl的氯仿,充分混匀后13,000×g室温离心5分钟,将上层水相转移至新的EP管中,加入2μl的NA Carrier后均匀混合,加入200μl的异丙醇,充分混匀后,冰上冷却10分钟,13,000×g 4℃离心20分钟,弃上清。加入500μl的70%冷乙醇(RNase Free dH2O配制)漂洗,13,000×g 4℃离心5分钟,弃上清后干燥,加入6μl的RNase Free dH2O溶解沉淀,得到LigatedRNA。
3.1.4 反转录反应
按下列组份配制反转录反应液:Ligated RNA 6μl,oligo(dT)primer(50μM)0.5μl,5×M-MLV Buffer 2μl,dNTP(10mM each)1μl,RNase Inhibitor(40U/μl)0.25μl,Reverse Transcriptase M-MLV(RNase H-)(200U/μl)0.25μl,总体积10ul。反转录反应条件如下:30℃ 10min,42℃ 1hr,70℃ 15min,反应结束后可以进行下一步实验,或将反应液保存于-20℃。
3.2 PCR反应
根据获得的三疣梭子蟹crustin基因片段,设计两个反义链引物crustinR1:5′-CTG TAA AGT TTG TAA GCA-3′(位于crustin 3’-UTR末端)和crustinR 2:5′-TTAGTAGAAATCAGGAGTCTTGC-3′(位于crustin C末端),有义链引物为试剂盒自带的5′RACE Outer Primer:5′-CATGGCTACATGCTGACAGCCTA-3′,5′RACE Inner Primer:5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′。
3.2.1 Outer PCR反应
按下列组份配制Outer PCR反应液:反转录反应液2μl,1×cDNA Dilution BufferII 10μl,10×PCR Buffer(Mg2+Free)4μl,MgCl2(25mM)3μl,TaKaRa Ex Taq(5U/μl)0.25μl,crustinR1(10μM)2μl,5′RACE Outer Primer(10μM)2μl,加dH2O至总体积50μl。反应条件:94℃预变性3min;94℃变性30sec,55℃退火30sec,72℃延伸30sec,20个循环;72℃后延伸10min。
3.2.2 inner PCR反应
按下列组份配制Inner PCR反应液:Outer PCR反应液1μl,10×PCR Buffer(Mg2+Free)5μl,MgCl2(25mM)4μl,dNTP Mixture(2.5mM each)4μl,TaKaRa Ex Taq(5U/μl)0.5μl,crustinR2(10μM)2μl,5′RACE Inner Primer(10μM)2μl,加dH2O至总体积50μl。反应条件:94℃预变性3min;94℃变性30sec,55℃退火30sec,72℃延伸30sec,30个循环;72℃后延伸10min。1%琼脂糖凝胶电泳检测得到约约400nt的特异cDNA扩增带,如图3所示。
3.3 目的片断割胶回收与T载体克隆测序
inner PCR反应液跑1%的琼脂糖回收胶,割目的带用上海生工小量胶回收试剂盒回收克隆进pMD19-T Vector(大连宝生物公司)测序。得到405nt的三疣梭子蟹crustin完整的5’UTR和CDS。
4.序列分析与基因确定
扩增获得的cDNA产物由上海invitrogen生物技术有限公司进行DNA核苷酸序列测定,为保证准确都挑三个克隆子正反双向测序。序列同源性比对和相似性搜索用NCBI BLAST在线进行;多序列比对用ClustalW;以blast2软件辅助进行序列拼接;信号肽预测用SignalP 3.0 Server在线分析。
Claims (7)
1.三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的三疣梭子蟹crustin抗菌肽基因cDNA不包括polyA共有565nt,其中含有5’端非编码区(5’UTR)长75nt,1个阅读框长330nt,3’非编码区(3’UTR)长160nt,其GenBank序列号为FJ467931,全长cDNA序列如下:
10 20 30 40 50
60
gaaaagagca gaacaagaca ctgtggtgga cacacctgct ctcaccaacg
tcttcctcaa
70 80 90 100 110
120
gaatctatag agataatgaa gatgcagact gtaatagcca tggcagttgt
ggctaccatt
130 140 150 160 170
180
gtggccatga cagaagcatc cctagtactt ccatacccag gtctggattg
taagtactgg
190 200 210 220 230
240
tgcaaggaca actacgataa acactactgc cgtggcccac caggacgtac
ctatccacct
250 260 270 280 290
300
tataccgagc gctctggtaa atgtcctccg gtccgtgcta catgtactgg
tgtcaggtca
310 320 330 340 350
360
cgcctaccaa agttgtgtcc ccatgatggt gcttgtgact ttccaagcaa
gtgctgttat
370 380 390 400 410
420
aatcattgaa
430 440 450 460 470
480
ccagactgaa gatgaaaatt tttcaccaat tttgcttaca aactttacag
agatgatatt
490 500 510 520 530
540
ctcttaaaac attgatttat ggttatgcta ttctatattc atttgtaaac
aatataacaa
550 560 570 580
aataataaac gcaattgtat ccgtgaaaaa aaaaaaaaaa
2.如权利要求1所述的三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的三疣梭子蟹的I型crustin抗菌肽cDNA推导编码氨基酸序列如下:
10 20 30 40 50 60
70 80
MKMQTVIAMA VVATIVAMTE ASLVLPYPGL DCKYWCKDNY DKHYCRGPPG RTYPPYTERS
GKCPPVRATC TGVRSRLPKL
90 100 110
CPHDGACDFP SKCCYDACVE HHVCKTPDFY*。
3.如权利要求1所述的三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的三疣梭子蟹的I型crustin抗菌肽cDNA读码框及推导蛋白质氨基酸序列如下:
gaaaagagca gaacaagaca ctgtggtgga cacacctgct ctcaccaacg tcttcctcaa
60
Met Lys Met Gln Thr Val Ile Ala Met Ala Val Val 12
gct acc att gtg gcc atg aca gaa gca tcc cta gta ctt cca tac cca 159
Ala Thr Ile Val Ala Met Thr Glu Ala Ser Leu Val Leu Pro Tyr Pro 28
ggt ctg gat tgt aag tac tgg tgc aag gac aac tac gat aaa cac tac 207
Gly Leu Asp Cys Lys Tyr Trp Cys Lys Asp Asn Tyr Asp Lys His Tyr 44
tgc cgt ggc cca cca gga cgt acc tat cca cct tat acc gag cgc tct 255
Cys Arg Gly Pro Pro Gly Arg Thr Tyr Pro Pro Tyr Thr Glu Arg Ser 60
ggt aaa tgt cct ccg gtc cgt gct aca tgt act ggt gtc agg tca cgc 303
Gly Lys Cys Pro Pro Val Arg Ala Thr Cys Thr Gly Val Arg Ser Arg 76
cta cca aag ttg tgt ccc cat gat ggt gct tgt gac ttt cca agc aag 351
Leu Pro Lys Leu Cys Pro His Asp Gly Ala Cys Asp Phe Pro Ser Lys 92
tgc tgt tat gac gcc tgt gtg gag cac cac gta tgc aag act cct gat 399
Cys Cys Tyr Asp Ala Cys Val Glu His His Val Cys Lys Thr Pro Asp 108
448
Phe Tyr ***
110
attttgctta caaactttac agagatgata ttctcttaaa acattgattt atggttatgc
508
tattctatat tcatttgtaa acaatataac aaaataataa acgcaattgt atccgtgaaa
568
aaaaaaaaaa aa
580
4.如权利要求1所述的三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的3’-RACE有义链简并引物crustin F1为包括起始密码子ATG的起始密码子后+238碱基起共17bp。
5.如权利要求1所述的三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的5’-RACE反义链特异引物crustinR1为终止密码子TAA后第45个碱基起共18bp。
6.如权利要求1所述的三疣梭子蟹的I型crustin抗菌肽基因,其特征在于,所说的5’-RACE反义链特异引物crustinR2为终止密码子TAA前第20个碱基开始,即从+310位开始共23bp。
7.如权利要求1~6的任意一项所述的三疣梭子蟹的I型crustin抗菌肽基因的克隆方法,其特征在于,步骤为:
(1)设计有义链简并引物crustin F1:5’-GTN TGY GCN CAY GAY GG-3’,与逆转录引物上的M13 Primer M4:5’-GTT TTC CCA GTC ACG AC-3’配合,以三疣梭子蟹血淋巴细胞总RNA模板,通过3’-RACE技术克隆三疣梭子蟹的I型crustin抗肽基菌因mRNA3’端209nt基因片段,不包括引物及polyA;
(2)根据获得的I型crustin抗肽基菌因3’端209nt基因片段设计特异引物反义链引物crustinR1:5′-CTG TAA AGT TTG TAA GCA-3′和crustinR2:5′-TTAGTAGAAATCAGGAGTCTTGC-3′,5’端有义链引物使用大连宝生物公司5’-Full RACE kit试剂盒自带的:5′RACE Outer Primer:5′-CATGGCTACATGCTGACAGCCTA -3′,5′RACE Inner Primer:5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′,以三疣梭子蟹血淋巴细胞总RNA模板,按5’-Full RACE kit操作总RNA去磷酸化处理、“去帽子”反应、5′RACE Adaptor的连接、反转录反应、嵌套PCR,从三疣梭子蟹血淋巴细胞中扩增出三疣梭子蟹的I型crustin抗菌肽基因完整5’UTR及编码区在内的408nt的目的片段;
(3)将3’-RACE及5′RACE的测序结果进行拼接和碱基校对,最终得到三疣梭子蟹的I型crustin抗菌肽基因的全长cDNA序列。
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