CN108997491A - 斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 - Google Patents
斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 Download PDFInfo
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Abstract
本发明公开了斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白在识别细菌鞭毛、调控细胞内信号通路和调节炎症因子中的应用。有助于了解鱼体抵抗病原微生物,尤其弧菌入侵的生理过程,可在生产应用中为鱼类免疫,预防病害等研究提供科学的依据和实践的基础。在生产应用中可制备成具有抵御副溶血性弧菌增殖功效的药物,也可作为免疫增强剂应用。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白的新用途。
背景技术
先天免疫是机体抵御外来病原微生物入侵的第一道防线,是鱼类快速且直接的免疫防御。Toll样受体(Toll-like receptor,TLR)作为先天免疫***中主要的模式识别受体,参与多种病原相关分子模式(pathogen-associated molecular patterns,PAMPs)的识别,激活胞内MyD88(myeloid differentiation primary-response protein 88)依赖型信号途径或者MyD88非依赖型信号途径引起特异性免疫反应来抵御病原微生物的入侵。
在哺乳动物中,TLR5特异性识别并结合革兰氏菌鞭毛蛋白的保守区域,通过募集胞内的MyD88转导信号,活化NF-κB(nuclear factor-κB),启动促炎因子和I型干扰素等免疫基因的表达和介导机体炎症应答。目前在红鳍东方鲀、虹鳟、大西洋鲑、斑点叉尾鮰、牙鲆、大黄鱼和草鱼等硬骨鱼中鉴定出2种类型的TLR5:跨膜型TLR5(Membrane form ofTLR5,TLR5M)和可溶型TLR5(Soluble form of TLR5,TLR5S),其中TLR5S基因是鱼类所特有的TLR家族成员。在虹鳟TLR5研究中证明TLR5S能够结合细菌鞭毛蛋白,促进TLR5M介导的NF-κB通路活化。但对鱼类免疫中TLR5S能够识别何种细菌组分及识别后发挥怎样的作用机制调控胞内信号通路活化等功能尚不明确。
斜带石斑鱼(Epinephelus coioides)是一种暖水性近海底层名贵鱼类,肉质细嫩鲜美,富含营养物质,具有很高的经济价值,在分类上属于鲈形目(Perciformes),鮨科(Serranidae),石斑鱼亚科(Epinephelinae),石斑鱼属(Epinephelus)。伴随着斜带石斑鱼养殖业的发展,病毒、细菌和寄生虫鱼病日益猖獗,严重制约斜带石斑鱼产业的发展。细菌性鱼病由于其传播速度快,致死率高,危害性大,已在世界范围内对渔业养殖造成了巨大的经济损失。其中副溶血性弧菌是水生动物弧菌病的主要致病菌之一,是一种嗜盐性革兰氏阴性菌,给海水养殖业带来严重的经济损失。所选的副溶血性弧菌感染会引起较高的致死率和腹部充血的症状,在斑马鱼、黑青斑河豚和青岛文昌鱼注射副溶血性弧菌后均有观察到。副溶血性弧菌含有多种PAMPs,如LPS、RNA、鞭毛等,机体中的TLR可以通过识别副溶血性弧菌组分,激活胞内信号通路从而引发机免疫应答反应。胞内信号通路过度激活或失衡都会引发机体严重的炎症反应给机体带来损伤,为维护免疫稳态,机体演化出一套复杂的负调控体系对胞内信号进行严密的监控。
探究斜带石斑鱼天然免疫受体TLR5S的功能特征及其在针对细菌的先天免疫防御中发挥的作用,有助于了解鱼体抵抗病原微生物,尤其弧菌入侵的生理过程,可在生产应用中为鱼类免疫,预防病害等研究提供科学的依据和实践的基础。
发明内容
本发明的目的在于提供斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白的新用途。
本发明的上述目的是通过以下技术方案来实现的:斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白在识别细菌鞭毛、调控细胞内信号通路和调节炎症因子中的应用。
所述的细菌优选为副溶血性弧菌。
本发明中的斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白在识别到副溶血性弧菌的鞭毛后,可以调控细胞内信号通路,通过负调控细胞内信号通路的活化来调节下游炎症因子的表达,尤其是平衡炎症因子的表达。因此可以将其作为药物使用,用以抵制副溶血性弧菌的增殖,作为免疫调节剂来调节炎症反应。
因此,本发明中的斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白也可以在制备具有抑制副溶血性弧菌增殖功效的药物中应用;也可以作为免疫调节剂应用。
本发明所述斜带石斑鱼天然免疫受体TLR5S基因的核苷酸序列如SEQ.ID.NO:1所示。
本发明所述斜带石斑鱼天然免疫受体TLR5S基因编码蛋白的氨基酸序列如SEQ.ID.NO:2所示。
此外,本发明所述斜带石斑鱼天然免疫受体TLR5S基因的全长cDNA序列如SEQ.ID.NO:3所示。
本发明所述斜带石斑鱼TLR5S基因的全长cDNA序列全长2168bp,包括一个79bp的5`非编码区,一个154bp的3`非编码区,以及一个1935bp的开放阅读框。
本发明所述斜带石斑鱼天然免疫受体TLR5S基因编码的蛋白结构由17个LRR结构域构成。
本发明具有以下优点:本发明提供了斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白在识别细菌鞭毛、调控细胞内信号通路和调节炎症因子中的应用。有助于了解鱼体抵抗病原微生物,尤其弧菌入侵的生理过程,可在生产应用中为鱼类免疫,预防病害等研究提供科学的依据和实践的基础;以及进一步在制备具有抑制副溶血性弧菌增殖功效的药物中应用,也可以作为免疫调节剂应用。
附图说明
图1是实施例1中EcTLR5S基因ORF区域的PCR凝胶电泳图;
图2是实施例2中验证真核表达载体pcDNA4.0-TLR5S的凝胶电泳图;
图3是实施例3中真核表达载体的过表达EcTLR5S的效果;
图4是实施例4中EcTLR5S参与识别副溶血性弧菌鞭毛;
图5是实施例5中过表达EcTLR5S抑制副溶血性弧菌鞭毛引发的IκB-α磷酸化;
图6是实施例6中过表达EcTLR5S抑制副溶血性弧菌鞭毛引发的NF-κB入核,A图是副溶血性弧菌鞭毛刺激引发NF-κB入核模型,B图是过表达EcTLR5S对副溶血性弧菌鞭毛引发的NF-κB转录因子核位移过程的影响;
图7是实施例7中过表达EcTLR5S后抑制副溶血性弧菌鞭毛介导产生的炎症因子的转录;
图8是实施例8中敲降EcTLR5S后对副溶血性弧菌鞭毛介导产生的炎症因子的影响。
具体实施方式
下面结合具体实施例进一步说明本发明的应用方法。下述实施例和附图仅用于示例性说明,不能理解为对本发明的限制。除非特别说明,下述实施例中使用的试剂原料为常规市售或商业途径获得的生试剂原料,除非特别说明,下述实施例中使用的方法和设备为本领域常规使用的方法和设备。
实施例1斜带石斑鱼TLR5S基因的克隆
(1)斜带石斑鱼头肾总RNA的提取
取健康斜带石斑鱼,于冰上麻醉3分钟,分离头肾组织,采用Trizol试剂提取其总RNA。
(2)cDNA第一链的合成
取1μg斜带石斑鱼头肾总RNA样品进行DNA酶处理以去除基因组DNA的污染,与RNAOligodT混合,进行反转录,所得产物置于-20℃保存备用。
(3)斜带石斑鱼TLR5S基因cDNA全序列的克隆
设计特异性引物,上游引物为TLR5S-ORF-F:5`-ATGTGGACGCTGGGTCTTCA-3`;下游引物为TLR5S-ORF-R:5`-CTGCTGTGTGATGTCATCAG-3`,扩增得到的ORF片段大小为1935bp,电泳结果如图1所示,切胶回收目的条带并纯化产物。将纯化产物连接至pGEM-T载体,转化至DH5α大肠杆菌后挑选阳性克隆测序。
由测序结果可知,斜带石斑鱼天然免疫受体TLR5S基因的核苷酸序列如SEQ.ID.NO:1所示,斜带石斑鱼天然免疫受体TLR5S基因的全长cDNA序列如SEQ.ID.NO:3所示,基因的全长cDNA序列全长2168bp,包括一个79bp的5`非编码区,一个154bp的3`非编码区,以及一个1935bp的开放阅读框。分析核苷酸序列可知,斜带石斑鱼天然免疫受体TLR5S基因的编码蛋白的氨基酸序列如SEQ.ID.NO:2所示,所编码的蛋白结构由17个LRR结构域构成。
实施例2真核表达载体pcDNA4.0-TLR5S的构建及其验证
根据TLR5S基因全长cDNA序列,选择BamHI和KpnI双酶切位点设计引物,其中上游引物为EcTLR5S-BamHI-F:5`-CGCGGATCCGCCACCATGTGGACGCTGGGTCTTCA-3`;下游引物为EcTLR5S-KpnI-R:5`-CCGCTCGAGCTGCTGTGTGATGTCATCAG-3`。以含有TLR5S编码基因的pGEM-T质粒为模板,进行PCR扩增,得到特异性扩增产物大小在2000bp左右,将带有酶切位点的TLR5S连接至已用BamHI和KpnI双酶切及纯化后的pcDNA4.0真核表达载体中,得到重组表达载体,重组表达载体经PCR验证的凝胶电泳结果见图2,经测序验证序列正确性、无移码突变,证明pcDNA4.0-TLR5S载体构建完成。
实施例3重组质粒pcDNA4.0-TLR5S的过表达效果
将适量的293T细胞接种到12孔板中,待细胞长至70%~80%时转染,转染前换为无血清的Opti-MEM培养液。每孔分别转染1500ng重组质粒pcDNA4.0-TLR5S,或pcDNA4.0空白载体对照。转染6小时后换为含有5%的胎牛血清的DMEM培养液。转染36小时后收集细胞,提取总蛋白,进行western blot检测EcTLR5S融合蛋白表达,实验结果如图3所示,其中β-actin作为内参蛋白。结果表明,重组质粒构建成功。
实施例4 EcTLR5S参与识别副溶血性弧菌鞭毛
将适量的GS细胞接种到96孔板中,待细胞汇合度达到90%以上时,使用一些病原相关分子模式,如:LPS(脂多糖)、PGN(肽聚糖)、Poly I:C(聚肌胞苷酸)、灭活的副溶血性弧菌、副溶血性弧菌鞭毛蛋白分别刺激细胞,刺激6小时后收获细胞,提取总RNA,反转录,进行荧光定量PCR检测EcTLR5S的mRNA表达变化,其中EF1作为内参基因。根据检测得到的CT值进行结果的分析,利用2-△△ct的方法计算出以上EcTLR5S的相对表达量,实验结果如附图4所示。检测中用到的特异性引物序列如下表1:
表1荧光定量PCR检测EcTLR5S中用到的引物
从图4中可以看出,副溶血性弧菌鞭毛蛋白的刺激能引起EcTLR5S mRNA表达量的显著性上调,这一结果提示EcTLR5S参与免疫防御过程,有能力对副溶血性弧菌鞭毛蛋白进行免疫识别。
这一结果提示EcTLR5S能参与对副溶血性弧菌鞭毛蛋白的免疫识别过程。
实施例5过表达EcTLR5S抑制副溶血性弧菌鞭毛引发的IκB-α磷酸化
将适量的GS细胞接种到12孔板中,待细胞长至70%~80%时转染,转染前将含10%胎牛血清的L15培养基换为无血清的Opti-MEM培养液。每孔分别转染1500ngpcDNA4.0-TLR5S表达载体,或pcDNA4.0空白载体对照。转染6小时后换为含有5%的胎牛血清的L15培养液。转染36小时后使用1μg/mL副溶血性弧菌鞭毛刺激细胞,分别在刺激0、20、40、60、120、180min时收集细胞,提取总蛋白,进行western blot检测NF-κB通路中IκB-α磷酸化水平变化。实验结果如图5,其中β-actin作为内参蛋白。
IκB-α磷酸化是NF-κB通路激活的重要标志之一,图5所示,过表达EcTLR5S能抑制NF-κB通路中重要信号分子IκB-α磷酸化,表明EcTLR5S基因或EcTLR5S基因编码蛋白可识别副溶血性弧菌鞭毛并抑制其所诱导的NF-κB通路激活。
实施例6过表达EcTLR5S抑制副溶血性弧菌鞭毛引发的NF-κB转录因子入核
首先构建副溶血性弧菌鞭毛刺激引发NF-κB入核模型,将适量的GS细胞接种到8孔腔室载玻片中,待细胞长至40%~60%时转染,转染前将含10%胎牛血清的L15培养基换为无血清的Opti-MEM培养液。每孔分别转染200ngpcDNA4.0-EcNF-κB-p65表达载体,转染6小时后换为含有5%的胎牛血清的L15培养液。转染36小时后使用1μg/mL副溶血性弧菌鞭毛刺激细胞,分别刺激0、30、60、100、140min后制片。用4%多聚甲醛固定细胞,DAPI染细胞核,每孔滴加抗淬灭剂后封片,通过双分子激光共聚焦显微镜观察结果,结果如图6中A图所示。
随后检测过表达EcTLR5S对副溶血性弧菌鞭毛引发的NF-κB转录因子核位移过程的影响,将适量的GS细胞接种到8孔腔室载玻片中,待细胞长至40%~60%时转染,转染前将含10%胎牛血清的L15培养基换为无血清的Opti-MEM培养液。每孔转染200ng pcDNA4.0-EcNF-κB-p65和200ng pcDNA4.0-TLR5S表达载体,转染6小时后换为含有5%的胎牛血清的L15培养液。转染36小时后使用1μg/mL副溶血性弧菌鞭毛刺激细胞,分别刺激0、30、60、100、140min后制片。用4%多聚甲醛固定细胞,DAPI染细胞核,每孔滴加抗淬灭剂后封片,通过双分子激光共聚焦显微镜观察结果,结果如图6中B图所示。
NF-κB亚基p65活化入核是NF-κB通路激活的重要标志之一,从图6A中可以看出,副溶血弧菌鞭毛刺激能引发NF-κB亚基p65活化入核。图6B可以看出,过表达EcTLR5S后显著抑制了副溶血性弧菌鞭毛所引发的NF-κB亚基p65活化入核,表明EcTLR5S基因或EcTLR5S基因编码蛋白可识别副溶血性弧菌鞭毛并抑制其所诱导的NF-κB通路激活。
实施例7过表达EcTLR5S后抑制副溶血性弧菌鞭毛介导产生的炎症因子的转录
将适量的GS细胞接种到96孔板中过夜培养,待细胞长至70%~80%时转染,转染前将含10%胎牛血清的L15培养基换成无血清的Opti-MEM培养液。每孔转染300ngpcDNA4.0-TLR5S表达载体,或pcDNA4.0空白载体对照。转染6小时后换为含有5%的胎牛血清的L15培养液。转染36小时后使用1μg/mL副溶血性弧菌鞭毛刺激细胞,刺激6小时后,收获细胞,提取总RNA,反转录,进行荧光定量PCR检测免疫因子IFN-γ2、IL-6、以及TNF-α的mRNA水平,其中EF1作为内参基因。根据检测得到的CT值进行结果的分析,利用2-△△ct的方法计算出以上免疫因子的相对表达量,实验结果如附图7所示。检测中用到的特异性引物序列如下表2:
表2荧光定量PCR检测免疫因子IFN-γ2、IL-6、以及TNF-α中采用的引物序列
从图7中可以看出,过表达EcTLR5S后显著性抑制副溶血性弧菌鞭毛刺激诱发的下游炎症因子mRNA的表达。
实施例8敲降EcTLR5S后对副溶血性弧菌鞭毛介导产生的炎症因子的影响
将适量的GS细胞接种到96孔板中过夜培养,待细胞长至70%~80%时转染,转染前将含10%胎牛血清的L15培养基换成无血清的Opti-MEM培养液。所用到的siRNA序列为,EcTLR5S-siRNA1(序列:sense:5`-CCAGCCUUCAAUGUUCUUUTT-3`;antisense:5`-AAAGAACAUUGAAGGCUGGTT-3`)EcTLR5S-siRNA2(序列:sense:5`-CCUUCUGUUGAGUGACAAUTT-3`;antisense:5`-AUUGUCACUCAACAGAAGGTT-3`)及Negative Control序列:(序列:sense:5`-UUCUCCGAACGUGUCACGUTT-3`;antisense:5’-AUGGAGACAGC UGAUAGGCTT-3’),稀释浓度为20nM。转染时,将EcTLR5S-siRNA1和EcTLR5S-siRNA2以1:1比例混合制成EcTLR5S-siRNA-mix。转染液为:A液(Opti-MEM培养液5μL,加入0.3μL siRNA,混匀),B液(Opti-MEM培养液5μL,加入0.4μL lipo3000,混匀,室温放置5min);将A液和B液轻轻混匀即为转染液,室温放置15min,加入相应的转染孔中,孵育6小时;最后换为含有5%的胎牛血清的L15培养液。转染36小时后,加入副溶血性弧菌鞭毛蛋白进行刺激,刺激所用的鞭毛的浓度为1μg/mL,等体积的PBS作为对照。刺激6h后,收获细胞,提取总RNA,逆转录,进行荧光定量PCR检测检测各免疫因子表达量,实验结果如图8所示。
从图8中可以看出,敲降EcTLR5S后可以显著性增加副溶血性弧菌鞭毛刺激介导下游炎症因子mRNA的表达。结果表明,EcTLR5S基因或EcTLR5S基因编码蛋白可识别副溶血性弧菌鞭毛并调节炎症因子表达。
本发明不局限于上述特定的实施方案范围内,上述实施方案仅仅是为了能够对本发明的使用过程进行详细地说明,而且有相等功能的生产方法和技术细节也属于本发明内容的一部分。事实上,本领域技术人员根据前文的描述,就能够根据各自需要找到不同的调整方案,这些调整都应在本文所附的权利要求书的范围内。
序列表
<110> 中山大学
<120> 斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白的新用途
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1935
<212> DNA
<213> 斜带石斑鱼(Epinephelus coioides)
<400> 1
atgtggacgc tgggtcttca ggtggctgtc atctgtgttt tcctacaggt gccaggttgt 60
ttcccatcat gcctcatcgt gaactctgta gccaactgtg cctacaagaa cctccgctca 120
gttcctcccc tccctcctca catcacccac ctgtacctgg aggggaaccg catcagtgag 180
atcaactcca actccctgtc gggcctgaag gagctgcagg agctggacct tggagggcag 240
tttgtgcgtc tcatgatcag aaacaatgcc ttcagggagc aaagacacct gaggaggctg 300
gtgctcggtg gcaacgtcca ccttcagctg gagccgcagg cttttgtggg actgtccagt 360
ttgcagaatc tctacttata tcactgttcg ctccaacagt ctatactgga ggaggactac 420
ctggagccac tcacctcttt agagactctt gacctctttg gtaacaagat aaagagactc 480
cagccttcaa tgttctttgc aaacatgact aatttgaaaa ttctgaatct caaactgaat 540
gaaattgaca aaatatgtga gtctgatctg gtaggttttc agggaaagca ctttgaggtt 600
ctgaatttag actctgttta ccttaagacc atgtttaaaa aacgcgaatg gcagaaatgt 660
gggaatcctt tcagggggat gtcctttcag acactcgacc tgtccaacaa cgggttaagc 720
gtgagtaaat cgaaacagtt gttcacagcc attaggggga ccaagatttc caatctcaaa 780
ctgtcactgg gactcatggg taaaggattt tcattcaata tttaccctga tccagacagc 840
agcatgtttg aaagcctgaa tgacagttca gtccacactc tggatctgtc taaaaacaag 900
atatttgcac tccaagaggg ggttttcagt gcactaaaag aagttgcaat cattgatgtt 960
tcccaaaaca atgtgaatca gatacacaga aatgcctttg aaggccttca gggacattta 1020
caaatgctca acctgtcaca caacctgctg ggggaaatcc attctgacac ttttgcttct 1080
ctgacaaacc tgcaggtgtt ggacttgtct tacaatcaca ttggtgttct gggtcatgac 1140
tcatttagtg aacttcccaa attaaaagta ttaaatctaa caggaaactc tctgcgagac 1200
attggcttcc ctgccttact tccgagttta gattaccttc tgttgagtga caataaactg 1260
atagactcgc cggggagaag tatccatagg tttgctgggg atattttgca tctggacatt 1320
cgggataaca gattaacaaa cctggggggt gtttataccc ttgtgactct gctggaccgc 1380
cttcagcatc tctcttatgg aggaaaccca atcaagtggt gcaatctcgg cagagaagtc 1440
aggttcattg attcgaataa tgtcacaacc ctggatcttc acggcagctc cctgcagtct 1500
gtctggtctc agtggacatg tctggatctg tttgacaatt tcggacatgt gattactctg 1560
aacttgagcc acaacgcgct gcagtctctt cctaggggca ttttcaaggg tctcacctca 1620
gttgtggaga tggacctctc atccaacagc ttgacttatc tccagcctga tgtattgccc 1680
aaaagtctca aagtactcga cctctccaac aacttcatag cctcccctga ccctgatgct 1740
tttcgctctc tcagctccct tgacctgaac atgaaccgat ttcactgtga tgcaaacctg 1800
aagagttttc tgaattgggt aaccaacacc accgtgacac tcctgacacc tgtcaaggag 1860
ctcaaatgtg aatttccatc tgatttctat aatgttcctc tgttacgatt ctctgatgac 1920
atcacacagc agtaa 1935
<210> 3
<211> 644
<212> PRT
<213> 斜带石斑鱼(Epinephelus coioides)
<400> 3
Met Trp Thr Leu Gly Leu Gln Val Ala Val Ile Cys Val Phe Leu Gln
1 5 10 15
Val Pro Gly Cys Phe Pro Ser Cys Leu Ile Val Asn Ser Val Ala Asn
20 25 30
Cys Ala Tyr Lys Asn Leu Arg Ser Val Pro Pro Leu Pro Pro His Ile
35 40 45
Thr His Leu Tyr Leu Glu Gly Asn Arg Ile Ser Glu Ile Asn Ser Asn
50 55 60
Ser Leu Ser Gly Leu Lys Glu Leu Gln Glu Leu Asp Leu Gly Gly Gln
65 70 75 80
Phe Val Arg Leu Met Ile Arg Asn Asn Ala Phe Arg Glu Gln Arg His
85 90 95
Leu Arg Arg Leu Val Leu Gly Gly Asn Val His Leu Gln Leu Glu Pro
100 105 110
Gln Ala Phe Val Gly Leu Ser Ser Leu Gln Asn Leu Tyr Leu Tyr His
115 120 125
Cys Ser Leu Gln Gln Ser Ile Leu Glu Glu Asp Tyr Leu Glu Pro Leu
130 135 140
Thr Ser Leu Glu Thr Leu Asp Leu Phe Gly Asn Lys Ile Lys Arg Leu
145 150 155 160
Gln Pro Ser Met Phe Phe Ala Asn Met Thr Asn Leu Lys Ile Leu Asn
165 170 175
Leu Lys Leu Asn Glu Ile Asp Lys Ile Cys Glu Ser Asp Leu Val Gly
180 185 190
Phe Gln Gly Lys His Phe Glu Val Leu Asn Leu Asp Ser Val Tyr Leu
195 200 205
Lys Thr Met Phe Lys Lys Arg Glu Trp Gln Lys Cys Gly Asn Pro Phe
210 215 220
Arg Gly Met Ser Phe Gln Thr Leu Asp Leu Ser Asn Asn Gly Leu Ser
225 230 235 240
Val Ser Lys Ser Lys Gln Leu Phe Thr Ala Ile Arg Gly Thr Lys Ile
245 250 255
Ser Asn Leu Lys Leu Ser Leu Gly Leu Met Gly Lys Gly Phe Ser Phe
260 265 270
Asn Ile Tyr Pro Asp Pro Asp Ser Ser Met Phe Glu Ser Leu Asn Asp
275 280 285
Ser Ser Val His Thr Leu Asp Leu Ser Lys Asn Lys Ile Phe Ala Leu
290 295 300
Gln Glu Gly Val Phe Ser Ala Leu Lys Glu Val Ala Ile Ile Asp Val
305 310 315 320
Ser Gln Asn Asn Val Asn Gln Ile His Arg Asn Ala Phe Glu Gly Leu
325 330 335
Gln Gly His Leu Gln Met Leu Asn Leu Ser His Asn Leu Leu Gly Glu
340 345 350
Ile His Ser Asp Thr Phe Ala Ser Leu Thr Asn Leu Gln Val Leu Asp
355 360 365
Leu Ser Tyr Asn His Ile Gly Val Leu Gly His Asp Ser Phe Ser Glu
370 375 380
Leu Pro Lys Leu Lys Val Leu Asn Leu Thr Gly Asn Ser Leu Arg Asp
385 390 395 400
Ile Gly Phe Pro Ala Leu Leu Pro Ser Leu Asp Tyr Leu Leu Leu Ser
405 410 415
Asp Asn Lys Leu Ile Asp Ser Pro Gly Arg Ser Ile His Arg Phe Ala
420 425 430
Gly Asp Ile Leu His Leu Asp Ile Arg Asp Asn Arg Leu Thr Asn Leu
435 440 445
Gly Gly Val Tyr Thr Leu Val Thr Leu Leu Asp Arg Leu Gln His Leu
450 455 460
Ser Tyr Gly Gly Asn Pro Ile Lys Trp Cys Asn Leu Gly Arg Glu Val
465 470 475 480
Arg Phe Ile Asp Ser Asn Asn Val Thr Thr Leu Asp Leu His Gly Ser
485 490 495
Ser Leu Gln Ser Val Trp Ser Gln Trp Thr Cys Leu Asp Leu Phe Asp
500 505 510
Asn Phe Gly His Val Ile Thr Leu Asn Leu Ser His Asn Ala Leu Gln
515 520 525
Ser Leu Pro Arg Gly Ile Phe Lys Gly Leu Thr Ser Val Val Glu Met
530 535 540
Asp Leu Ser Ser Asn Ser Leu Thr Tyr Leu Gln Pro Asp Val Leu Pro
545 550 555 560
Lys Ser Leu Lys Val Leu Asp Leu Ser Asn Asn Phe Ile Ala Ser Pro
565 570 575
Asp Pro Asp Ala Phe Arg Ser Leu Ser Ser Leu Asp Leu Asn Met Asn
580 585 590
Arg Phe His Cys Asp Ala Asn Leu Lys Ser Phe Leu Asn Trp Val Thr
595 600 605
Asn Thr Thr Val Thr Leu Leu Thr Pro Val Lys Glu Leu Lys Cys Glu
610 615 620
Phe Pro Ser Asp Phe Tyr Asn Val Pro Leu Leu Arg Phe Ser Asp Asp
625 630 635 640
Ile Thr Gln Gln
<210> 3
<211> 2168
<212> DNA
<213> 斜带石斑鱼(Epinephelus coioides)
<400> 3
acgcgggggg ggagaacaga gctctgatcc tggagtgttg tttgacctca tatcattgag 60
ggtttccctg gtgagcaaga tgtggacgct gggtcttcag gtggctgtca tctgtgtttt 120
cctacaggtg ccaggttgtt tcccatcatg cctcatcgtg aactctgtag ccaactgtgc 180
ctacaagaac ctccgctcag ttcctcccct ccctcctcac atcacccacc tgtacctgga 240
ggggaaccgc atcagtgaga tcaactccaa ctccctgtcg ggcctgaagg agctgcagga 300
gctggacctt ggagggcagt ttgtgcgtct catgatcaga aacaatgcct tcagggagca 360
aagacacctg aggaggctgg tgctcggtgg caacgtccac cttcagctgg agccgcaggc 420
ttttgtggga ctgtccagtt tgcagaatct ctacttatat cactgttcgc tccaacagtc 480
tatactggag gaggactacc tggagccact cacctcttta gagactcttg acctctttgg 540
taacaagata aagagactcc agccttcaat gttctttgca aacatgacta atttgaaaat 600
tctgaatctc aaactgaatg aaattgacaa aatatgtgag tctgatctgg taggttttca 660
gggaaagcac tttgaggttc tgaatttaga ctctgtttac cttaagacca tgtttaaaaa 720
acgcgaatgg cagaaatgtg ggaatccttt cagggggatg tcctttcaga cactcgacct 780
gtccaacaac gggttaagcg tgagtaaatc gaaacagttg ttcacagcca ttagggggac 840
caagatttcc aatctcaaac tgtcactggg actcatgggt aaaggatttt cattcaatat 900
ttaccctgat ccagacagca gcatgtttga aagcctgaat gacagttcag tccacactct 960
ggatctgtct aaaaacaaga tatttgcact ccaagagggg gttttcagtg cactaaaaga 1020
agttgcaatc attgatgttt cccaaaacaa tgtgaatcag atacacagaa atgcctttga 1080
aggccttcag ggacatttac aaatgctcaa cctgtcacac aacctgctgg gggaaatcca 1140
ttctgacact tttgcttctc tgacaaacct gcaggtgttg gacttgtctt acaatcacat 1200
tggtgttctg ggtcatgact catttagtga acttcccaaa ttaaaagtat taaatctaac 1260
aggaaactct ctgcgagaca ttggcttccc tgccttactt ccgagtttag attaccttct 1320
gttgagtgac aataaactga tagactcgcc ggggagaagt atccataggt ttgctgggga 1380
tattttgcat ctggacattc gggataacag attaacaaac ctggggggtg tttataccct 1440
tgtgactctg ctggaccgcc ttcagcatct ctcttatgga ggaaacccaa tcaagtggtg 1500
caatctcggc agagaagtca ggttcattga ttcgaataat gtcacaaccc tggatcttca 1560
cggcagctcc ctgcagtctg tctggtctca gtggacatgt ctggatctgt ttgacaattt 1620
cggacatgtg attactctga acttgagcca caacgcgctg cagtctcttc ctaggggcat 1680
tttcaagggt ctcacctcag ttgtggagat ggacctctca tccaacagct tgacttatct 1740
ccagcctgat gtattgccca aaagtctcaa agtactcgac ctctccaaca acttcatagc 1800
ctcccctgac cctgatgctt ttcgctctct cagctccctt gacctgaaca tgaaccgatt 1860
tcactgtgat gcaaacctga agagttttct gaattgggta accaacacca ccgtgacact 1920
cctgacacct gtcaaggagc tcaaatgtga atttccatct gatttctata atgttcctct 1980
gttacgattc tctgatgaca tcacacagca gtaaagtagc aactaaaagc caacacataa 2040
cggataatgt acagcgagtg ggccattatt gagaaataaa cctgcagagg ggtcttgaat 2100
caccatttct gtacataatc ccacttgtta catggccaac taatcaagtc aataatttga 2160
cacaaaat 2168
Claims (4)
1.斜带石斑鱼天然免疫受体TLR5S基因及其编码蛋白在识别细菌鞭毛、调控细胞内信号通路和调节炎症因子中的应用。
2.根据权利要求1所述的应用,其特征是:所述的细菌为副溶血性弧菌。
3.根据权利要求1或2所述的应用,其特征是:所述斜带石斑鱼天然免疫受体TLR5S基因的核苷酸序列如SEQ.ID.NO:1所示。
4.根据权利要求3所述的应用,其特征是:所述斜带石斑鱼天然免疫受体TLR5S基因编码蛋白的氨基酸序列如SEQ.ID.NO:2所示。
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