CN110317813B - 三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用 - Google Patents
三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用 Download PDFInfo
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- CN110317813B CN110317813B CN201910644129.9A CN201910644129A CN110317813B CN 110317813 B CN110317813 B CN 110317813B CN 201910644129 A CN201910644129 A CN 201910644129A CN 110317813 B CN110317813 B CN 110317813B
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- portunus trituberculatus
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Abstract
本发明属于分子生物学技术领域,具体是一种三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用。本发明利用转录组测序获得的unigene和RACE技术从三疣梭子蟹中扩增得到PtCLec2基因cDNA,发现重组的PtCLec2蛋白具有显著的抑菌、菌凝集及菌清除活性。重组蛋白PtCLec2对革兰氏阴性菌(溶藻弧菌、铜绿假单胞菌)和革兰氏阳性菌(金黄色葡萄球菌、藤黄微球菌)具有明显的抑制效果。在Ca2+存在下,重组蛋白PtCLec2对溶藻弧菌、铜绿假单胞菌、金黄色葡萄球菌、藤黄微球菌均具有明显的凝集效果。同时,对溶藻弧菌具有明显的清除作用。
Description
技术领域
本发明属于分子生物学技术领域,具体的说是一种三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用。
背景技术
无脊椎动物缺乏适应性免疫系统,它识别“非己”物质主要是依靠模式识别受体(PRRs)识别病原相关分子模式(PAMPs)来完成的。无脊椎动物模式识别受体主要包括:肽聚糖识别蛋白、革兰氏阴性菌结合蛋白、含硫酯键蛋白、清道夫受体、硫依赖型凝集素、血素、Toll样受体和C型凝集素。C型凝集素是一类钙离子依赖蛋白,该类蛋白均具有一个能够识别并结合糖类的糖类识别结构域(CRD)。CRD结构域中一般含有非常保守的四个半胱氨酸,并通过它们形成两对二硫键,以维持整个结构域的构象。CRD结构域含有4个Ca2+结合位点,其中Ca2+结合位点2的作用最重要,主要参与对糖类底物的结合。Ca2+结合位点2是由两个高保守的基序组成,第一个基序是EPN或QPD,分别特异性结合甘露糖或半乳糖;另一个基序是WND,主要参与钙离子的结合并维持疏水核心。
三疣梭子蟹是一种重要的海产经济蟹类,由于其营养价值丰富、生长迅速等优点,已成为我国重要海水养殖品种。近年来,三疣梭子蟹受弧菌等病原菌的影响病害频发,严重制约梭子蟹养殖业的发展。甲壳动物缺乏抗体和特异性免疫细胞,主要依靠先天免疫清除病原微生物,进行免疫防御。目前关于三疣梭子蟹C型凝集素的研究尚少,其Ca2+结合位点2基序的变异对基因功能的影响尚不清楚。因此,模式识别受体C型凝集素的发掘和利用对理解三疣梭子蟹的免疫防御机制、进行病害防治具有重要的理论和实践意义。
发明内容
本发明旨在提供一种三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用。
为实现上述目的,本发明采用的技术方案为:
一种三疣梭子蟹C型凝集素PtCLec2基因,三疣梭子蟹C型凝集素PtCLec2基因为序列表SEQ ID No.1中的碱基序列所示。
一种三疣梭子蟹C型凝集素PtCLec2基因编码蛋白,所述PtCLec2基因编码蛋白为序列表SEQ ID No.2中的氨基酸序列所示。
一种三疣梭子蟹C型凝集素PtCLec2基因编码蛋白的应用,所述三疣梭子蟹C型凝集素PtCLec2基因的重组表达产物可制备为抗菌药物、免疫活性剂、饲料添加剂、防腐剂或保鲜剂。
所述三疣梭子蟹C型凝集素PtCLec2基因的重组表达产物可用于制备革兰氏阴性菌或革兰氏阳性菌的抗菌药物或凝集制剂。
所述革兰氏阴性菌为溶藻弧菌或铜绿假单胞菌;革兰氏阳性菌为金黄色葡萄球菌或藤黄微球菌。
所述三疣梭子蟹C型凝集素PtCLec2基因的重组表达产物可用于制备菌清除制剂。
所述菌为溶藻弧菌。
本发明所具有的优点:
本发明利用转录组测序获得的unigene和RACE技术从三疣梭子蟹中克隆到PtCLec2基因cDNA全长序列,通过PCR技术扩增编码PtCLec2成熟肽的基因片段并将其克隆到pET32a(+)表达载体中,在大肠杆菌BL21(DE3)-plysS实现体外重组表达。重组蛋白PtCLec2经TALON柱纯化和透析后,对革兰氏阴性菌(溶藻弧菌、铜绿假单胞菌)、革兰氏阳性菌(金黄色葡萄球菌、藤黄微球菌)均具有凝集活力,尤其是对革兰氏阴性菌的效果十分明显,而对真菌(毕赤酵母菌)无明显凝集效果。重组蛋白PtCLec2对溶藻弧菌、铜绿假单胞菌、金黄色葡萄球菌和藤黄微球菌均有明显的抑制作用,最小抑菌浓度分别为0.86~1.74μM、1.74~3.50μM、0.43~0.86μM、0.86~1.74μM。
本发明的PtCLec2基因及其重组蛋白可生产抑菌药物、菌凝集制剂、菌清除制剂等,应用于水产养殖过程中相关虾蟹疾病的治疗,或用于饲料添加剂、防腐剂或保鲜剂的生产等,另外还可以为进一步研究三疣梭子蟹免疫防御机制提供基础,并为三疣梭子蟹的病害防治和基因辅助选育提供参考。
附图说明
图1为本发明实例提供的三疣梭子蟹C型凝集素PtCLec2基因的核苷酸和氨基酸序列(红色下划线:起始密码子和终止密码子;灰色阴影部分:信号肽;黑框:Ca2+结合位点;黑色阴影:氨基酸保守基序)。
图2为本发明实例提供的纯化后三疣梭子蟹C型凝集素PtCLec2编码成熟肽的基因扩增产物(其中,M:DNA marker,1、2:成熟肽的基因扩增产物)。
图3为本发明实例提供的诱导和纯化后三疣梭子蟹C型凝集素PtCLec2重组蛋白(其中M:蛋白marker;1:IPTG诱导后pET-32a空质粒表达的蛋白;2:未诱导PtCLec2菌体中表达的蛋白;3:IPTG诱导后PtCLec2表达的蛋白;4:纯化的PtCLec2重组蛋白)。
图4为本发明实例三疣梭子蟹C型凝集素PtCLec2重组蛋白对细菌和真菌凝集活性作用图(检测荧光标记的溶藻弧菌、铜绿假单胞菌、藤黄微球菌、金黄色葡萄球菌和毕赤酵母菌,rPtCLec1、rTrx、CaCl2和EDTA分别是25nmol L-1、25nmol L-1、10mmol L-1和10mmol L-1,PBS和rTrx分别作为空白和阴性对照)。
图5为本发明实例三疣梭子蟹C型凝集素PtCLec2重组蛋白对溶藻弧菌清除活性作用图(只注射溶藻弧菌为对照)。
具体实施方式
下面的实施例中将本发明作进一步阐述,但本发明不限于此。
本发明中的三疣梭子蟹C型凝集素PtCLec2的cDNA序列克隆包括下列步骤:
a)三疣梭子蟹总RNA的提取及mRNA的检测;
b)三疣梭子蟹cDNA文库构建;
c)三疣梭子蟹转录组测序及分析;
d)三疣梭子蟹unigene序列的同源性分析及PtCLec2基因片段的筛选;
e)RACE扩增获得PtCLec2的全长序列以及全长序列的验证。
实施例1.
三疣梭子蟹C型凝集素PtCLec2基因为SEQ ID No.1中所示的碱基序列。
参见图1,序列表SEQ ID No.1为:
AAATAATAGTGGTTGCGGAACATTCCCTCTTGTCTCCCCGAGAATGCGAATCTACTTGCTTCTGGCGGTGGCGCTGGCGGTGATCTGCTCCGTAGCAGCCCAAGGCCGTGTGTTGGCGTTGCCGGAAATAGAACTATGTGATAATCGCCCAAAGCAGTGGAAGTTCCGCAACCACTATTATTTCTTCTCGTGGGACCAGGATGGCCCAGACTTCAAGGAAGTAAATCCTAAAACAGGACAACTGGAAGGTAGCAAGGTTGACTGGCTGAAAGCTCGCAACTTGTGCCGTCAGCGATGCATGGACGCTGTCGGCATGGAGAGCGAAGAGGAGAACAACATGATTTTCGACTTTATCAAAAGACGCAACATCACGTACATCTGGACGTCTGGCCGCCTCTGTGACTTCAAGGGGTGCGATGAGCGCGAGGACCTGAAGCCCATCAGTGTCAAGGGATGGTTCTTCTCCAACACCAACACTAAGATGGCCCCGACCAACGCATCGCCACCAGGCTGGAAGTACCAGCCATGGAGCGACAAGGGCCACACCGGTGGACCGCAGCCAGACAACGCTGAGTTCGATATCAACCAGACATCGGAGTCTTGTCTCGGCGTACTCAACAATCTGTACAATGACGGCATCAAATGGCACGACATTGCCTGCTACCACAAGAAGCCCTTCATTTGTGAGGACAGTGATGAACTACTTCGGTACATCGAGGGACAAAAGCAGCAACTGCAACAACAAAACCGCCCAGGGAACCAGGGACAGGGAAACCGTGGACAAGGAAACCGTGGACAAGGGAATAACGACAACGCCAACCAGCGCGCACGAGCACATTTTGGTTAAGTTTTCACCACAACCCTTGAAACTTTTCCATTCTCCCTTGTGCAAAACTTTGTTAGTTAAGTTTACCACACTGTCCATCTTTGAAGACTGATACAACCCCATCAACACAGCCATACAACCGGCAAGGCCATGAACATCTTAGTGGCGGCCTTGCTCCAGTCTTCTGCTTATTTATTGTTAATACGACGCACTAGCCCCGTATGTGGCTGCTGCTCACAGTAAACACGGCACTCAGCCCGTTATTTATTGTAACACCACGATAATTGTGCTGTTTTCATTAATAAAACGCGATTGTTACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
(a)序列特征
●长度:1175bp(有效长度44~847bp)
●类型:碱基序列
●链型:单链
●拓扑结构:线性
(b)分子类型:双链DNA
(c)假设:否
(d)反义:否
(e)最初来源:三疣梭子蟹(Portunus trituberculatus)
(f)特异性名称:CDS
构建具体操作如下:
1.三疣梭子蟹总RNA的提取及mRNA纯化:利用Invitrogen公司的Trizol试剂提取三疣梭子蟹成体组织的总RNA。琼脂糖凝胶电泳分析RNA降解程度以及是否有污染,Nanodrop检测RNA的纯度,Qubit对RNA浓度进行精确定量,Agilent 2100精确检测RNA的完整性。
2.三疣梭子蟹cDNA文库构建:mRNA样品检测合格后,用带有Oligo(dT)的磁珠富集mRNA。随后加入fragmentation buffer将mRNA打断成短片段,以mRNA为模板,用六碱基随机引物合成一链cDNA,然后加入缓冲液、dNTPs和DNA polymerase I和RNase H合成二链cDNA,再用AMPure XP beads纯化双链cDNA。纯化的双链cDNA先进行末端修复、加A尾并连接测序接头,再用AMPure XP beads进行片段大小选择。最后进行PCR扩增,并用AMPure XP beads纯化PCR产物,得到最终的文库。
3.转录组测序及分析:cDNA文库检验合格后,把不同文库按照有效浓度及目标下机数据量的需求pooling后进行Illumina HiSeq/MiSeq测序。对于无参考基因组物种的转录组分析,先将测序所得的序列拼接成转录本,以转录本为参考序列,进行后续分析。高通量测序得到的原始图像数据文件经CASAVA碱基识别分析转化为原始测序序列,我们称之为Raw Data或Raw Reads。然而,原始测序序列中含有带接头的、低质量的reads。为了保证信息分析质量,必须对raw reads过滤得到cleanreads,后续分析都基于clean reads。获得clean reads后,采用Trinity转录组拼接软件对clean reads进行拼接。取每条基因中最长的转录本作为unigene,以此进行后续的分析。后续分析包括基因功能注释(基因功能注释数据库包括Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,GO)、CDS预测、基因表达水平分析、直系同源基因筛选等生物信息学分析。
4.三疣梭子蟹unigene序列的同源性分析及PtCLec2基因片段的筛选:在三疣梭子蟹转录组中获得1条PtCLec2相关的unigene,将其在数据库中进行BLASTn和BLASTx分析,结果显示该序列与拟穴青蟹CTL序列相似性很高,确定为三疣梭子蟹PtCLec2基因的unigene序列。
5.三疣梭子蟹PtCLec2基因cDNA ORF序列的克隆:根据与PtCLec2基因同源的unigene
序列设计特异引物F1(5'CCGAGAATGCGAATCTACTTGC 3')和R1(5'TGTTTACTGTGAGCAGCAGCCAC 3'),分别利用载体通用引物M13F(5'TGTAAAACGACGGCCAGT 3')和M13R(5'CAGGAAACAGCTATGACC 3')进行扩增PtCLec2的ORF。PCR产物用1%琼脂糖凝胶电泳进行检测,用Axygen胶回收试剂盒进行PCR产物回收和纯化,再与pMD-19T载体(山东赛恩斯科技有限公司)连接,然后转化大肠杆菌感受态DH5α(北京全式金生物技术有限公司),挑选阳性克隆用载体引物M13进行测序,所得结果经SeqMan软件进行拼接,得到三疣梭子蟹PtCLec2基因ORF cDNA序列见SEQ ID No.1。
6.三疣梭子蟹PtCLec2基因cDNA全长序列的扩增:在测序拼接的PtCLec2全长序列上设计扩增5'端的两条特异引物5P1(5'AGCCTGGTGGCGATGCGTTGG 3')和5P2(5'TCAGCCAGTCAACCTTGCTACCTTCC 3'),同理设计扩增3'端的两条特异引物3P1(5'CGACCAACGCATCGCACCAG 3')和3P2(5'CGACATTCGCATCGCCACCAG 3'),特异引物5P1和3P1分别与100×UPM(5'CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3')进行5'和3'端的第一次扩增,以cDNA为模板进行全长的验证。特异引物5P2和3P2分别与Nup(5'AAGCAGTGGTAACAACGCAGAGT 3')进行5'和3'端的第二次扩增,以第一次扩增的结果为模板进行全长的验证。测序及分析同5。
ORF扩增反应体系及反应条件:
25μL反应体系:
反应在TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc.)中进行,反应条件为:94℃变性3min;94℃变性30s,55℃退火50s,72℃延伸2min,35个循环;72℃延伸10min。
5'RACE和3'RACE第一次扩增反应体系及反应条件:
25μL反应体系:
反应在TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc.)中进行,反应条件为:98℃变性2min;98℃变性20s,64℃退火30s,72℃延伸2min,5个循环;98℃变性20s,61℃退火30s,72℃延伸2min,8个循环;98℃变性20s,58℃退火30s,72℃延伸2min,25个循环;72℃延伸10min。
5'RACE和3'RACE第二次扩增反应体系及反应条件:
50μL反应体系:
反应在TaKaRa PCR Thermal Cycler Dice Model TP600(Takara Bio Inc.)中进行,反应条件为:98℃变性2min;98℃变性20s,64℃退火30s,72℃延伸2min,5个循环;98℃变性20s,61℃退火30s,72℃延伸2min,8个循环;98℃变性20s,58℃退火30s,72℃延伸2min,25个循环;72℃延伸10min。
序列表SEQ ID No.1从三疣梭子蟹中克隆到PtCLec2基因cDNA全长1175bp,其中开放阅读框804bp,5'非翻译区43bp,3'非翻译区328bp,有多聚腺苷酸加尾信号(AATAAA)和多聚腺苷酸尾巴。
实施例2.
三疣梭子蟹C型凝集素序列表SEQ ID No.1所述碱基序列,所述的氨基酸序列如序列表SEQ ID No.2所述。
序列表SEQ ID No.2为:
MRIYLLLAVALAVICSVAAQGRVLALPEIELCDNRPKQWKFRNHYYFFSWDQDGPDFKEVNPKTGQLEGSKVDWLKARNLCRQRCMDAVGMESEEENNMIFDFIKRRNITYIWTSGRLCDFKGCDEREDLKPISVKGWFFSNTNTKMAPTNASPPGWKYQPWSDKGHTGGPQPDNAEFDINQTSESCLGVLNNLYNDGIKWHDIACYHKKPFICEDSDELLRYIEGQKQQLQQQNRPGNQGQGNRGQGNRGQGNNDNANQRARAHFG
其具有完整的编码蛋白含有267个氨基酸,该编码序列有信号肽(1-19),预测的分子量为30.65kDa,等电点为7.53。成熟肽含有248个氨基酸具有典型的CRD结构域(32-215),其中包含4个半胱氨酸残基,能够形成两个二硫键。CRD结构域中缺少典型的脊椎动物和无脊椎动物中识别甘露糖的基序EPN(Glu-Pro-Asn)但含有识别半乳糖的基序QPD(Gln-Pro-Asp),同时Ca2+结合位点2的第二个基序不是保守的WND(Trp-Asn-Asp)而是WHD(Trp-His-Asp)。
三疣梭子蟹C型凝集素PtCLec2重组蛋白的获得,具体操作如下:
根据SEQ ID No.2对应的cDNA序列,设计含有限制性内切酶BamHI和XhoI酶切位点的特异性引物F2(5'CGCGGATCCCAAGGCCGTGTGTTGGCG 3')和R2(5'CCGCTCGAGACCAAAATGTGCTCGTGCGCG 3'),通过PCR技术扩增编码PtCLec2成熟肽的基因片段(参见图2),反应在TaKaRa PCR ThermalCycler Dice Model TP600(Takara Bio Inc.)中进行,反应条件为:94℃变性3min;94℃变性30s,55℃退火50s,72℃延伸2min,35个循环;最后72℃延伸10min。然后通过酶切将其克隆到pET32a(+)表达载体中,转化到大肠杆菌BL21(DE3)-plysS,测序确认表达框正确后,接种阳性克隆到LB培养基中,37℃振荡培养至OD600nm=0.4~0.6,加入IPTG至终浓度为1mM诱导4h后离心收集菌体。菌体在冰浴条件下用超声波180W处理30min(每次2s,间隔2s),离心去掉上清,收集沉淀。沉淀以8M的尿素溶解后,利用Clontech公司的TALON柱纯化重组产物。将纯化的重组蛋白转移到透析袋中,4℃条件下,在含有2mM还原谷胱苷肽、0.2mM氧化谷胱苷肽、1mM EDTA、50mM Tris-HCl、50mM NaCl、10%甘油和1%甘氨酸的及梯度降低的尿素(6、5、4、3、2、1、0M)的透析复性液(pH=8.0)中透析,使重组蛋白复性,最后在50mM Tris-HCl(pH=8.0)的缓冲液透析2次,以除去溶液中其它成分。透析复性后的重组蛋白经PALL公司的Microsep Advance超滤离心浓缩管进行浓缩,用碧云天公司的BCA蛋白浓度测定试剂盒测得三疣梭子蟹C型凝集素PtCLec2重组蛋白的浓度为1.65mg/mL(参见图3)。
实施例3.
1.三疣梭子蟹C型凝集素PtCLec2重组蛋白的体外抑菌试验
微生物的培养及制备:溶藻弧菌用TSB培养基28℃培养,铜绿假单胞菌用TSB培养基37℃培养,金黄色葡萄球菌用LB培养基37℃培养,藤黄微球菌用LB培养基37℃培养,毕赤酵母用YPD培养基28℃培养,上述各菌株用摇床220rpm/min培养使菌浓度达到对数生长期时,分别用50mM Tris-HCl(pH=8.0)缓冲液稀释菌体,使其每毫升菌液中的菌落数约为1×103个。
重组蛋白PtCLec2抑菌活性测定:利用上述实施例所得重组蛋白PtCLec2用Tris-HCl(50mM,pH=8.0)梯度稀释(1、1/2、1/4、1/8、1/16、1/32、1/64)后,取50μL加到无菌平底96孔板(Costar,Fisher)中,50μL Tris-HCl(50mM,pH=8.0)设为对照,然后加入50μL稀释好的菌悬液,只加入50μL菌液的孔为空白孔。96孔板在菌液的培养温度下孵育2小时后,加入相应的培养基150μL,空白孔加入培养基至200μL,孵育过夜。加溶藻弧菌、铜绿假单胞菌、毕赤酵母的96孔板在波长为560nm下读数测吸光值,加金黄色葡萄球菌和藤黄微球菌的96孔板在波长为600nm下测量。最小抑菌浓度为微生物可以生长的最大重组蛋白浓度和微生物被完全抑制生长的最低浓度之间的范围。发现上述实施例重组蛋白PtCLec2对革兰氏阴性菌溶藻弧菌和铜绿假单胞菌、革兰氏阳性菌金黄色葡萄球菌和藤黄微球菌有明显的抑制作用,最小抑菌浓度分别为0.86~1.74μM、1.74~3.50μM、0.43~0.86μM、0.86~1.74μM,而对真菌毕赤酵母没有明显的抑制作用。
2.三疣梭子蟹C型凝集素PtCLec2重组蛋白的体外微生物凝集实验
FITC染色:分别取上述培养至对数生长期的细菌、真菌1.0mL,于4℃4000rpm离心5min收集菌体,弃去培养基后用PBS洗菌3次。加入终浓度为0.1mg mL-1的FITC,暗处慢摇染色过夜。于4℃4000rpm离心5min收集菌体,弃去培养基后用PBS洗菌3次,以去除残留的FITC。
凝菌实验:使用无菌PBS将FITC标记的菌重悬,调整菌浓度至2.5×109个mL-1。实验组中,取25μL PtCLec2重组蛋白与20μL FITC标记的菌悬液于1.5mL离心管中混匀;对照组中,取25μL rTrx与20μLFITC标记的菌悬液混匀。为观察Ca2+是否对凝集活性有影响,在实验组和对照组中分别加入10mM CaCl2和用10mM EDTA螯合Ca2+。将样品置于暗处28℃慢摇孵育2h,取5μL于荧光显微镜下观察(参见图4)。发现上述实施例重组蛋白PtCLec2在Ca2+存在下对革兰氏阴性菌溶藻弧菌和铜绿假单胞菌、革兰氏阳性菌金黄色葡萄球菌和藤黄微球菌均有显著的凝集作用,对毕赤酵母菌无明显凝集作用。
3.三疣梭子蟹C型凝集素PtCLec2重组蛋白的细菌清除活性鉴定实验
将100μL OD540nm=0.6~0.7(浓度为3.2×108个/mL)的溶藻弧菌与100μL PtCLec2重组蛋白于4℃孵育1h。将孵育后的重组蛋白与溶藻弧菌的结合物于4℃4000rpm离心10min,去上清,将沉淀用Tris-HCl缓冲液洗2遍,然后用Tris-HCl缓冲液将结合物稀释成1×105个/mL的蛋白结合菌液。用Tris-HCl缓冲液将溶藻弧菌菌液(即浓度为3.2×108个/mL)稀释至1×105个/mL。分别取试验组100μL蛋白结合菌液和对照组100μL溶藻弧菌菌液(浓度为1×105个/mL)注射到健康三疣梭子蟹(体重为250g)体内,每组试验设置3个平行。分别在注射菌液后的15、30、90min,抽出100μL血淋巴(抽取时不加抗凝剂),迅速涂布到LB琼脂培养基上,于28℃培养24h,分别记录下在不同抽血涂板的时间点形成的单菌落数(见图5)。发现上述实施例重组蛋白PtCLec2与溶藻弧菌孵育15、30min后,蟹血细胞中的弧菌数量与对照组相比,明显下降,分别下降了68%和57%,在注射后90min时无明显差异。说明重组蛋白PtCLec2能有效地清除溶藻弧菌。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 中国科学院海洋研究所
<120> 三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1175
<212> DNA
<213> 三疣梭子蟹(Portunus trituberculatus)
<400> 1
aaataatagt ggttgcggaa cattccctct tgtctccccg agaatgcgaa tctacttgct 60
tctggcggtg gcgctggcgg tgatctgctc cgtagcagcc caaggccgtg tgttggcgtt 120
gccggaaata gaactatgtg ataatcgccc aaagcagtgg aagttccgca accactatta 180
tttcttctcg tgggaccagg atggcccaga cttcaaggaa gtaaatccta aaacaggaca 240
actggaaggt agcaaggttg actggctgaa agctcgcaac ttgtgccgtc agcgatgcat 300
ggacgctgtc ggcatggaga gcgaagagga gaacaacatg attttcgact ttatcaaaag 360
acgcaacatc acgtacatct ggacgtctgg ccgcctctgt gacttcaagg ggtgcgatga 420
gcgcgaggac ctgaagccca tcagtgtcaa gggatggttc ttctccaaca ccaacactaa 480
gatggccccg accaacgcat cgccaccagg ctggaagtac cagccatgga gcgacaaggg 540
ccacaccggt ggaccgcagc cagacaacgc tgagttcgat atcaaccaga catcggagtc 600
ttgtctcggc gtactcaaca atctgtacaa tgacggcatc aaatggcacg acattgcctg 660
ctaccacaag aagcccttca tttgtgagga cagtgatgaa ctacttcggt acatcgaggg 720
acaaaagcag caactgcaac aacaaaaccg cccagggaac cagggacagg gaaaccgtgg 780
acaaggaaac cgtggacaag ggaataacga caacgccaac cagcgcgcac gagcacattt 840
tggttaagtt ttcaccacaa cccttgaaac ttttccattc tcccttgtgc aaaactttgt 900
tagttaagtt taccacactg tccatctttg aagactgata caaccccatc aacacagcca 960
tacaaccggc aaggccatga acatcttagt ggcggccttg ctccagtctt ctgcttattt 1020
attgttaata cgacgcacta gccccgtatg tggctgctgc tcacagtaaa cacggcactc 1080
agcccgttat ttattgtaac accacgataa ttgtgctgtt ttcattaata aaacgcgatt 1140
gttacaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1175
<210> 2
<211> 267
<212> PRT
<213> 三疣梭子蟹(Portunus trituberculatus)
<400> 2
Met Arg Ile Tyr Leu Leu Leu Ala Val Ala Leu Ala Val Ile Cys Ser
1 5 10 15
Val Ala Ala Gln Gly Arg Val Leu Ala Leu Pro Glu Ile Glu Leu Cys
20 25 30
Asp Asn Arg Pro Lys Gln Trp Lys Phe Arg Asn His Tyr Tyr Phe Phe
35 40 45
Ser Trp Asp Gln Asp Gly Pro Asp Phe Lys Glu Val Asn Pro Lys Thr
50 55 60
Gly Gln Leu Glu Gly Ser Lys Val Asp Trp Leu Lys Ala Arg Asn Leu
65 70 75 80
Cys Arg Gln Arg Cys Met Asp Ala Val Gly Met Glu Ser Glu Glu Glu
85 90 95
Asn Asn Met Ile Phe Asp Phe Ile Lys Arg Arg Asn Ile Thr Tyr Ile
100 105 110
Trp Thr Ser Gly Arg Leu Cys Asp Phe Lys Gly Cys Asp Glu Arg Glu
115 120 125
Asp Leu Lys Pro Ile Ser Val Lys Gly Trp Phe Phe Ser Asn Thr Asn
130 135 140
Thr Lys Met Ala Pro Thr Asn Ala Ser Pro Pro Gly Trp Lys Tyr Gln
145 150 155 160
Pro Trp Ser Asp Lys Gly His Thr Gly Gly Pro Gln Pro Asp Asn Ala
165 170 175
Glu Phe Asp Ile Asn Gln Thr Ser Glu Ser Cys Leu Gly Val Leu Asn
180 185 190
Asn Leu Tyr Asn Asp Gly Ile Lys Trp His Asp Ile Ala Cys Tyr His
195 200 205
Lys Lys Pro Phe Ile Cys Glu Asp Ser Asp Glu Leu Leu Arg Tyr Ile
210 215 220
Glu Gly Gln Lys Gln Gln Leu Gln Gln Gln Asn Arg Pro Gly Asn Gln
225 230 235 240
Gly Gln Gly Asn Arg Gly Gln Gly Asn Arg Gly Gln Gly Asn Asn Asp
245 250 255
Asn Ala Asn Gln Arg Ala Arg Ala His Phe Gly
260 265
Claims (4)
1.一种三疣梭子蟹C型凝集素PtCLec2基因,其特征在于:三疣梭子蟹C型凝集素PtCLec2基因为序列表SEQ ID No.1中的碱基序列所示。
2.一种权利要求1所述的三疣梭子蟹C型凝集素PtCLec2基因编码蛋白,其特征在于:所述PtCLec2基因编码蛋白为序列表SEQ ID No.2中的氨基酸序列所示。
3.一种按权利要求2所述的三疣梭子蟹C型凝集素PtCLec2基因编码蛋白的应用,其特征在于:所述三疣梭子蟹C型凝集素PtCLec2基因的重组表达产物用于制备革兰氏阴性菌或革兰氏阳性菌的抗菌药物或凝集制剂;
所述革兰氏阴性菌为溶藻弧菌或铜绿假单胞菌;革兰氏阳性菌为金黄色葡萄球菌或藤黄微球菌。
4.按权利要求2所述的三疣梭子蟹C型凝集素PtCLec2基因编码蛋白的应用,其特征在于:所述三疣梭子蟹C型凝集素PtCLec2基因的重组表达产物用于制备菌清除制剂;
所述菌为溶藻弧菌。
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