CN108118061A - 斜带石斑鱼天然免疫受体tlr22基因及其真核表达载体与应用 - Google Patents
斜带石斑鱼天然免疫受体tlr22基因及其真核表达载体与应用 Download PDFInfo
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Abstract
本发明首次公开了一种斜带石斑鱼天然免疫受体TLR22基因及其真核表达载体与应用。TLR22基因的核苷酸序列如SEQ.ID.NO：1所示，TLR22基因的全长cDNA序列如SEQ.ID.NO：2所示，其序列上包含的一对引物序列如SEQ.ID.NO：4～5所示，TLR22蛋白的氨基酸序列如SEQ.ID.NO：3所示。本发明还提供一种含所述TLR22基因全长cDNA序列的真核表达载体pcDNA3.1‑EcTLR22。此外，本发明将TLR22基因或TLR22蛋白应用于调节炎症因子表达、在参与识别病毒RNA及调节免疫因子表达、细胞免疫应答方面的研究中。
Description
技术领域
本发明属于生物基因工程技术领域，更具体地，涉及一种斜带石斑鱼天然免疫受体TLR22基因及其真核表达载体与应用。
背景技术
模式识别受体（PRRs）能够识别多种微生物保守的分子结构（PAMPs）以及损伤的细胞释放的内源性的分子（DAMPs），用于激活先天性免疫应答。鱼类这一低等脊椎动物利用PRRs直接检测外源性微生物，使得先天性免疫***为机体提供了直接、快速的抗感染应答反应。
Toll受体家族（TLRs）是一类特异监控微生物分子结构的PRRs成员，是参与机体先天性免疫过程中的重要受体，是典型的I 型跨膜受体。它具有胞外不保守的富含亮氨酸重复区域（LRRs），以及胞内保守的TIR 结构域。TLRs 通过LRR 区识别多种微生物保守的分子组分——病原相关分子模式（PAMPs），之后通过TIR 区募集接头蛋白MyD88 和TRIF，介导MyD88 依赖型信号通路和TRIF 依赖型信号通路，活化转录因子，最终释放促炎因子和I型干扰素等免疫基因，从而调动获得性免疫应答，加强机体防御。鱼类特有的TLR 基因（如TLR21，TLR22，TLR23）是在自然界漫长的进化过程中保留下来的重要基因，更加具有研究价值。TLR22基因是鱼类特有的TLR 家族的成员，已经证明具有TLR 家族典型的分子结构，既能够募集MyD88接头蛋白也能结合TRIF接头蛋白，激活下游通路参与鱼类的免疫防御。
斜带石斑鱼（orange-spotted grouper,Epinepheluscoioides）是鲈形目（Perciformes），鮨科（Serranidae），石斑鱼亚科（Epinephelinae），石斑鱼属（Epinephelus）鱼类。斜带石斑鱼主要分布在包括我国华南沿海、台湾和香港在内的东南亚地区，其肉质鲜美，细嫩，营养价值极为丰富，具有较高的经济价值。但随着石斑鱼养殖业的发展，种质资源的衰退以及鱼类疾病（如病毒病、细菌病、寄生虫病等）的防控成为其发展的最大障碍。
因此，通过TLR22基因序列、cDNA序列以及蛋白氨基酸序列在研究斜带石斑鱼的先天性免疫防御机制中具有重要意义。加深人们对机体抵抗病原微生物感染过程的认识，以便在合适的时机采取有效的免疫保护措施，减少鱼药的滥用，保障鱼类的健康养殖。
发明内容
本发明的要解决的技术问题是弥补现有技术的空白，提供一种斜带石斑鱼天然免疫受体TLR22基因的核苷酸序列。
本发明要解决的另一技术问题是提供包含所述TLR22基因全长cDNA序列的真核表达载体。
本发明还一要解决的技术问题是提供所述TLR22基因或TLR22蛋白在调节炎症因子表达中的应用。
本发明还一要解决的技术问题是提供所述TLR22基因或TLR22蛋白在参与识别病毒RNA及调节免疫因子表达中的应用。
本发明还一要解决的技术问题是提供所述TLR22基因或TLR22蛋白在细胞免疫应答中的应用。
本发明的目的通过以下技术方案予以实现：
本发明提供了一种斜带石斑鱼天然免疫受体TLR22基因，所述基因的核苷酸序列如SEQ.ID.NO：1所示。
所述斜带石斑鱼天然免疫受体TLR22基因的全长cDNA序列如SEQ.ID.NO：2所示。
所述斜带石斑鱼天然免疫受体TLR22基因的cDNA序列全长3320bp，包括一个78bp的5’非编码区，一个359bp的3’非编码区，以及一个2880bp的开放阅读框。
所述斜带石斑鱼天然免疫受体TLR22基因的cDNA序列3’非编码区上有2个AATAA多腺苷酸化加尾信号。
所述斜带石斑鱼天然免疫受体TLR22基因编码的蛋白的氨基酸序列如SEQ.ID.NO：3所示。
所述斜带石斑鱼天然免疫受体TLR22基因的全长cDNA序列中包含1对引物序列，引物序列如SEQ.ID.NO：4～5所示。
本发明还提供包含所述TLR22基因全长cDNA序列的真核表达载体。
本发明还提供了所述TLR22基因或所述的TLR22蛋白在调节炎症因子表达中的应用。
本发明还提供了所述TLR22基因或所述的TLR22蛋白在参与识别病毒RNA及调节免疫因子表达中的应用。
本发明还提供了所述TLR22基因或所述的TLR22蛋白在细胞免疫应答中的应用。
本发明的有益效果：
本发明首次提供了所述斜带石斑鱼天然免疫受体TLR22基因的核苷酸序列基于所述序列，还提供了所述TLR22基因的真核表达载体，并且提供了所述TLR22基因及TLR22基因编码的蛋白在研究调节炎症因子的表达、识别病毒RNA及调节免疫因子表达、细胞免疫应答中的应用，具有突出的实质性进步。
附图说明
图1 TLR22基因ORF区域的PCR凝胶电泳图。
图2验证真核表达载体pcDNA3.1-EcTLR22的凝胶电泳图。
图3斜带石斑鱼TLR22的激活抑制NF-KB通路活化。
图4斜带石斑鱼TLR22的激活抑制AP-1通路活化。
图5斜带石斑鱼TLR22的激活促进ERK活化。
图6过表达斜带石斑鱼TLR22对免疫因子IL-1β的影响。
图7过表达斜带石斑鱼TLR22对免疫因子IFNγ1的影响。
图8过表达斜带石斑鱼TLR22对免疫因子IFNγ2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购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TLR22基因的克隆
（1）斜带石斑鱼脾脏总RNA的提取
取健康斜带石斑鱼，于冰上麻醉3分钟，分离脾脏组织，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TLR22基因cDNA全序列的克隆
设计特异性引物ORF-F和ORF-R，如SEQ.ID.NO：4、SEQ.ID.NO：5所示，扩增得到的ORF片段，PCR扩增反应条件为：94 ℃，3min；94 ℃ 15s，58℃ 15s，72℃ 3min30s ，40个循环；72℃，10min；4℃ ∞。PCR反应后的凝胶电泳图见附图1。
实施例2真核表达载体pcDNA3.1-EcTLR22的构建及其验证
构建斜带石斑鱼pcDNA3.1-EcTLR22真核表达载体，选择NheI和KpnI双酶切位点构建引物。上游引物为 EcTLR22-NheI F：5’-ACCCAAGCTGGCTAGCATGGGTCCCGGTTTGAAAG-3’；下游引物为 EcTLR22-myc-KpnI R：5’-GTGGATCCGAGCTCGGTACCCAGATCTTCTTCAGAAATAAGTTTTTGTTCCGGTTTGTCCGTCACAGTCAG-3’。以斜带石斑鱼头肾反转录的cDNA为模板，扩增中间片段，KOD体系反应条件：94 ℃ 3min；94℃ 15s，56 ℃ 30s，68 ℃6min，40个循环，PCR产物于4℃保存。PCR反应后的凝胶电泳结果见附图2。真核表达载体pcDNA3.1分别用NheI和KpnI双酶切及纯化后，与带有酶切位点的TLR22连接，经转化、阳性克隆筛选、测序，提质粒得到含有目的基因的真核表达载体。经测序验证序列正确性、无突变无移码，证明pcDNA3.1-EcTLR22 载体构建成功。
实施例3激活TLR22后引起NF-KB信号通路变化
将HEK293T细胞转移至24孔细胞培养板中，密度为0.5-2×105个细胞，体积为500μl，当细胞汇合度达90～95%时方可进行转染。将真核表达载体pcDNA3.1-EcTLR22与pcDNA3.1-NF-kB-luc共转染HEK293T细胞，TK Renillia vectors作为内参同时转染。孵育细胞于 37℃ 5% CO2的培养箱中6h，之后换成DMEM完全培养基继续培养至24h。用5激动剂PGN(10 mg/mL)、CL-097 (1μg /mL)、poly AU (50 μg/mL)、HMW（5，25，50μg/mL）和LMW（5，25，50μg/mL）刺激细胞6 h后收获细胞，细胞用PBS洗涤2次，每孔加入50μL 1×Passive Lysis Buffer，室温温和震荡裂解细胞15min。取20μL细胞裂解液于96孔酶标板中，于Promega双荧光检测酶标仪中设定：加入50μL LAR II 溶液以检测萤火虫荧光素酶活性，加入50μL Stop &Glo溶液以淬灭萤火虫荧光素酶荧光、激活并检测海肾荧光素酶荧光。萤火虫荧光素酶荧光强度与海肾荧光素酶荧光强度比值表示各样品启动活性强度，进行数据分析。结果显示，在不同激动剂的刺激下，EcTLR22引起的NF-κB的活化情况不同。在CL-097和PGN的刺激下NF-κB被微弱活化；而poly AU的刺激使NF-κB的活化受到微弱抑制，但是与未刺激组相比都没有显著性差异。而在dsRNA类似物HMW和LMW刺激下（不论是低浓度还是高浓度），NF-κB的活化受到的显著性的抑制，并且HMW对于NF-κB的抑制更加显著，结果见附图3。表明长片段的dsRNA（HMW）是TLR22的配体，并且dsRNA刺激EcTLR22抑制NF-κB通路的活化。
实施例4激活TLR22后引起AP-1信号通路变化
转染前一天将HEK293T细胞转移至24孔细胞培养板中，密度为0.5～2×10 5个细胞，体积为500μL，当细胞汇合度达 90～95%时方可进行转染。分别将每孔500ng pcDNA3.1-EcTLR22或pcDNA3.1、200ng AP-1-luc和100ng TK Renillia vectors共转染到HEK293T细胞。24h后选取不同浓度（5，25，50μg/mL）的 HMW 刺激过表达了EcTLR22 基因的HEK293T细胞。细胞用 PBS 洗涤 2 次，每孔加入 50μl 1×Passive Lysis Buffer，室温温和震荡裂解细胞15min。取20μL细胞裂解液于96孔酶标板中，于Promega双荧光检测酶标仪中设定：加入 50μL LAR II 溶液以检测萤火虫荧光素酶活性，加入 50μL Stop &Glo溶液以淬灭萤火虫荧光素酶荧光、激活并检测海肾荧光素酶荧光。萤火虫荧光素酶荧光强度与海肾荧光素酶荧光强度比值表示各样品启动活性强度，进行数据分析。结果显示不同浓度的HMW刺激，均能显著抑制AP-1的活性，结果如附图4所示。
实施例5激活TLR22后引起ERK信号通路变化
取适量新鲜斜带石斑鱼脾组织立即浸没于 RPMI 1640 完全培养液中，并置于冰上，在细胞房超净工作台内，将脾脏组织用无菌剪刀剪碎，并用无菌磨砂玻片的磨砂面进行研磨直至浆状，并剔除***。将研磨分散出的含有脾脏细胞的培养基过滤70μm的细胞筛网，去除杂质，滤液收集至50mL离心管中，于4℃ 500×g离心10 min，弃上清，加入4mL RPMI1640组织培养液重悬细胞，于4℃ 500×g离心10min，弃上清。加入 4mL RPMI 1640 组织培养液重悬细胞，将细胞悬液缓缓加入2mL淋巴细胞分离液（LMS）液面之上，于4℃ 500×g，离心30min。此时离心管中由上至下细胞分为四层：第一层为血浆层；第二层为乳白色淋巴细胞层；第三层为分离液层；第四层为红细胞层。用巴氏管缓慢收集第二层淋巴细胞悬液至新的 15mL离心管，加入10mL RPMI 1640 组织培养液，混匀后于 4℃ 500×g离心5min，弃上清，重复一次。以适量的组织培养液重悬细胞，转移至细胞培养瓶后于 27℃，5% CO2培养箱中培养备用。
将EcTLR22-myc-pcDNA3.1或pcDNA3.1真核表达载体转染入斜带石斑鱼脾脏淋巴细胞中过表达，24小时后用50μg/mL HMW刺激6小时后收集细胞。加入RIPA 裂解液，轻轻混匀，冰浴裂解30min，4℃，12000g 离心 10min，收集上清，100℃加热变性 5min 后置于 -20℃保存。将蛋白样品用 10％聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离，然后经 2mA/cm2恒流将蛋白质转移至硝酸纤维素薄膜上。转膜完成后，将硝酸纤维素膜经5%脱脂牛奶/PBST(PBS0.1%Tween20)室温封闭2h，加一抗稀释液4℃过夜，然后用1‰Tween PBST洗涤五次，每次5min。加入二抗稀释液，室温下避光孵育1h，1‰ Tween 的 PBST洗膜五次，每次5min。用ECL化学发光工作液显色，用化学发光成像分析***拍照分析。对照组的淋巴细胞在 HMW 刺激下，ERK的磷酸化水平在刺激20min 升高，之后直到刺激 120min 趋于稳定；而在过表达了EcTLR22 的淋巴细胞中，ERK 的磷酸化在刺激 40min 时表达量骤然升高，之后恢复到与对照组同一水平，结果如附图5所示。因此，在斜带石斑鱼中，EcTLR22 能够促进HMW引起的ERK通路的活化。
实施例6激活TLR22后引起免疫因子表达量变化
用斜带石斑鱼IL-1β、IFNγ1和IFNγ2抗体研究在dsRNA刺激下EcTLR22对IFNr的影响。用50ug/mL激动剂dsRNA（HMW）刺激过表达TLR22的斜带石斑鱼脾脏淋巴细胞后0min、20min、40min、60min、120min、180min时收细胞提总蛋白，用western检测p-ERK表达量。如附图6所示，在过表达了EcTLR22 的脾脏淋巴细胞中，HMW刺激60min和刺激120min，IL-1β表现出显著上调表达。如附图7所示，HMW 刺激20min 即检测到IFNγ1蛋白快速的上调表达，在刺激40min 到 180min 时间段，可以观察到过表达细胞系与对照组有相似的变化趋势。如附图8所示，EcTLR22引起的IFNγ2的表达却在HMW刺激的早期（20min～40min）出现了小幅度的下调，但是在刺激后期（60min～180min）又迅速发生上调的表达变化。
序列表
<110> 中山大学
<120> 斜带石斑鱼天然免疫受体TLR22基因及其真核表达载体与应用
<140> 2017105577432
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2883
<212> DNA
<213> 斜带石斑鱼TLR22基因核苷酸序列(Epinepheluscoioides)
<400> 1
atgggtcccg gtttgaaaga ggaacaaaca aaaagaggat caaaatgttt taaactaaat 60
tttgtttttt tcttgctgaa tatcagcagt ttttttgttc cagtcacagg gtatgtactg 120
aggtcttgcc atatcagtta cagcactgcc atatgttcca agaggagact cactgctgtc 180
cctcaagata ttccctcaac agtgaaaggt ttggacttgt atggaaacaa aatttcaaaa 240
atccaagtat cagatttcaa aaagctgtca aatttgacag agttaaatct gaaatggaat 300
ataatctcag agatagatac aggctccttt accaatttga attccctcaa gatgttaaat 360
ctaaatgata acaaacttac tggacttgga gacaatgttt ttgatggttt gggcagccta 420
acacatctca gaatgaacag aaatcagatc aaaacagtgg catccacctc ttttaagtcc 480
ttgacgaact tgatggtttt ggacatctcc gtcaacaaac tgtaccgcat aacaaatgtt 540
catactatat tacagcacct gccaaatcta ctggagctgt cagtcgcaaa caacaactta 600
acctcttttc agtcgtggga actgacaaac agctcactaa aacttggcca acttgatttg 660
tctcaaaatc ccattggaga ctttaggatc actgcagatg tttttcctaa tctcacctgg 720
ttaaacactg ggggctcttc taagaggcat cacatgaaat gggacgttcg taacaagact 780
tttcttcgca gagtgtctac ttttgatatt agtgggcttc aaataccttc agaagacatg 840
aaaacaatac tagggacctt caactcctca ctgacgacag tgaagatgaa tgatatgaaa 900
cgcacatacc tgagagcctt aattaacatc tcctgcagca tcccaacagt gtcctcactt 960
caactcagaa gtaacaacct cttttttatg agctcaaatt tgttatcgtt gtgttttaag 1020
gtaacagagt tagatttagc agataactca atgaaaaata tcaaagacga tgccttcaaa 1080
catcttcaag gtataaaggt cttggctctg aatcacaaca ggctttcatc tgttccagct 1140
gccataagga atctaccagc tcttgaagag ctagatctca gcaaaaataa tatcaccaca 1200
cttggttgtc aagatttcgc caatctgaca aagctcagac agctcagcct tcatcagaat 1260
tcaatctcag ctctacaggg gtgtgttttc aaggatttaa tacacctgga agttttaaag 1320
ctacagaata acagcatttc taaactgaat gatgctttca taaaatactt gccaaatctg 1380
agacaactgc atctgaattc aaataaactc acagcaatta aacatgggga atttaggggc 1440
ttgcggtccc tccagaactt gtcattacat gacaatcaga taacagtact tgaaaaagag 1500
tgttttactg gactgaggaa tcttatggct atccagctgc aaatgaatca tattaaaaga 1560
cataaagatt acaaagatag tttcagagat ctgataaatt taaaacgatt ggatttgagt 1620
cacaatagca ttaaatatat tgagagctca gctttgcctg atccaccatt ttctcatctg 1680
tccaatttgg agagactggc ttttactgga aatcaccgca gagtgaagtc ccagctgcct 1740
tgcaacttcc tgcaaggctt gactaatctt ttagatttca ctggcaggaa tcttcagctt 1800
ttatccttgc acaaagacac atttaatcac acgccaaaac tacaaaaact tgatataagc 1860
tcaaatgaac ttatggatct ttcttcagaa ttgttttacc caattagaaa tctcaaaagc 1920
ctctatatat ctagaataaa ccttgggtct ctagacttct ttaaagatgc caacctaact 1980
aagctggagt tcctgcaggc aagaaggaat cagttttcag ttttcagtga ggaagtactg 2040
gagtccatgc cagctctggc ttacttggat cttcaaggca atagtttcac ctgtgactgt 2100
gataacgcct ggttcctcaa gtggacagaa aacaaccagc aaacacaagt ttctgatgcc 2160
tacaactttg tgtgcaacta tcctccacac cttaaaggta tgaaactgtt gaagctcgat 2220
gtccggtcct gcacagtgga cactgaattc atctgcttta tctccaccac atgtacgatc 2280
ctcgtgttta tggtggcatc cttcacctac catttcctga ggtggtactt ggcctatgcc 2340
tactacatct tcttggcttt tctctttgac acaaagcata aaaacaagca agctccaaat 2400
cagtatgacg ccttcatctc ctacaacacc cacgatgagc catgggtcat cagacagctg 2460
ttgccaaaac tggaagggga gcaaggctgg agattgtgtt tgcaccatcg agatttcgag 2520
ccaggtaaac ccatcataga caacattaca gatgccatct atggaagcag gaagaccatc 2580
tgtgtgatca gtcacagata ccttgagagt gagtggtgct ccagagagat ccaggtggcc 2640
agcttccgcc tgtttgatga gcagaaggac gtgctcatcc tggtgtttct ggaggacatt 2700
cccaccactc aactgtctcc ttactaccgc atgaggaagc ttctgaagag gcagacctat 2760
ttgagctggc ctcgagcctc agagcaccct gaactattct gggagaaact ccgccaagct 2820
atgaagacca gagaggatct tggggaggat gaattgaacc tgactgtgac ggacaaaccg 2880
tga 2883
<210> 2
<211> 3321
<212> DNA
<213> 斜带石斑鱼TLR22基因全长cDNA序列(Epinepheluscoioides)
<400> 2
caggtggact atacgttgag gaagttcaat tccattttgg ttggacatct cttgttcttt 60
tgtaacaaag attttaaaat gggtcccggt ttgaaagagg aacaaacaaa aagaggatca 120
aaatgtttta aactaaattt tgtttttttc ttgctgaata tcagcagttt ttttgttcca 180
gtcacagggt atgtactgag gtcttgccat atcagttaca gcactgccat atgttccaag 240
aggagactca ctgctgtccc tcaagatatt ccctcaacag tgaaaggttt ggacttgtat 300
ggaaacaaaa tttcaaaaat ccaagtatca gatttcaaaa agctgtcaaa tttgacagag 360
ttaaatctga aatggaatat aatctcagag atagatacag gctcctttac caatttgaat 420
tccctcaaga tgttaaatct aaatgataac aaacttactg gacttggaga caatgttttt 480
gatggtttgg gcagcctaac acatctcaga atgaacagaa atcagatcaa aacagtggca 540
tccacctctt ttaagtcctt gacgaacttg atggttttgg acatctccgt caacaaactg 600
taccgcataa caaatgttca tactatatta cagcacctgc caaatctact ggagctgtca 660
gtcgcaaaca acaacttaac ctcttttcag tcgtgggaac tgacaaacag ctcactaaaa 720
cttggccaac ttgatttgtc tcaaaatccc attggagact ttaggatcac tgcagatgtt 780
tttcctaatc tcacctggtt aaacactggg ggctcttcta agaggcatca catgaaatgg 840
gacgttcgta acaagacttt tcttcgcaga gtgtctactt ttgatattag tgggcttcaa 900
ataccttcag aagacatgaa aacaatacta gggaccttca actcctcact gacgacagtg 960
aagatgaatg atatgaaacg cacatacctg agagccttaa ttaacatctc ctgcagcatc 1020
ccaacagtgt cctcacttca actcagaagt aacaacctct tttttatgag ctcaaatttg 1080
ttatcgttgt gttttaaggt aacagagtta gatttagcag ataactcaat gaaaaatatc 1140
aaagacgatg ccttcaaaca tcttcaaggt ataaaggtct tggctctgaa tcacaacagg 1200
ctttcatctg ttccagctgc cataaggaat ctaccagctc ttgaagagct agatctcagc 1260
aaaaataata tcaccacact tggttgtcaa gatttcgcca atctgacaaa gctcagacag 1320
ctcagccttc atcagaattc aatctcagct ctacaggggt gtgttttcaa ggatttaata 1380
cacctggaag ttttaaagct acagaataac agcatttcta aactgaatga tgctttcata 1440
aaatacttgc caaatctgag acaactgcat ctgaattcaa ataaactcac agcaattaaa 1500
catggggaat ttaggggctt gcggtccctc cagaacttgt cattacatga caatcagata 1560
acagtacttg aaaaagagtg ttttactgga ctgaggaatc ttatggctat ccagctgcaa 1620
atgaatcata ttaaaagaca taaagattac aaagatagtt tcagagatct gataaattta 1680
aaacgattgg atttgagtca caatagcatt aaatatattg agagctcagc tttgcctgat 1740
ccaccatttt ctcatctgtc caatttggag agactggctt ttactggaaa tcaccgcaga 1800
gtgaagtccc agctgccttg caacttcctg caaggcttga ctaatctttt agatttcact 1860
ggcaggaatc ttcagctttt atccttgcac aaagacacat ttaatcacac gccaaaacta 1920
caaaaacttg atataagctc aaatgaactt atggatcttt cttcagaatt gttttaccca 1980
attagaaatc tcaaaagcct ctatatatct agaataaacc ttgggtctct agacttcttt 2040
aaagatgcca acctaactaa gctggagttc ctgcaggcaa gaaggaatca gttttcagtt 2100
ttcagtgagg aagtactgga gtccatgcca gctctggctt acttggatct tcaaggcaat 2160
agtttcacct gtgactgtga taacgcctgg ttcctcaagt ggacagaaaa caaccagcaa 2220
acacaagttt ctgatgccta caactttgtg tgcaactatc ctccacacct taaaggtatg 2280
aaactgttga agctcgatgt ccggtcctgc acagtggaca ctgaattcat ctgctttatc 2340
tccaccacat gtacgatcct cgtgtttatg gtggcatcct tcacctacca tttcctgagg 2400
tggtacttgg cctatgccta ctacatcttc ttggcttttc tctttgacac aaagcataaa 2460
aacaagcaag ctccaaatca gtatgacgcc ttcatctcct acaacaccca cgatgagcca 2520
tgggtcatca gacagctgtt gccaaaactg gaaggggagc aaggctggag attgtgtttg 2580
caccatcgag atttcgagcc aggtaaaccc atcatagaca acattacaga tgccatctat 2640
ggaagcagga agaccatctg tgtgatcagt cacagatacc ttgagagtga gtggtgctcc 2700
agagagatcc aggtggccag cttccgcctg tttgatgagc agaaggacgt gctcatcctg 2760
gtgtttctgg aggacattcc caccactcaa ctgtctcctt actaccgcat gaggaagctt 2820
ctgaagaggc agacctattt gagctggcct cgagcctcag agcaccctga actattctgg 2880
gagaaactcc gccaagctat gaagaccaga gaggatcttg gggaggatga attgaacctg 2940
actgtgacgg acaaaccgtg atggaaaagg aaatacaacc ttctgtgaaa ggttcagtcc 3000
cattgtggtc aactggtttg agctttctca agcaacaacc tccagggctg aaaaatgaag 3060
ccaacatgga agtgtcaaaa actgcagttc cttgagtgac cacttggggc tagctacaga 3120
ggtgacttaa tctttatcta ccctcatgtt taattataca atatgatatg ataccatacg 3180
atatgatacg atacaatacg atatacttta ttgtcccata gggaagggac tcatatgcgc 3240
tgcatacaat aaaaatgcag tacagtaagt acagtaaaca taacaacaaa taacacatac 3300
cccctcccca gtgaattgcc c 3321
<210> 3
<211> 960
<212> PRT
<213> 斜带石斑鱼TLR22氨基酸序列(Epinepheluscoioides)
<400> 3
Met Gly Pro Gly Leu Lys Glu Glu Gln Thr Lys Arg Gly Ser Lys Cys
1 5 10 15
Phe Lys Leu Asn Phe Val Phe Phe Leu Leu Asn Ile Ser Ser Phe Phe
20 25 30
Val Pro Val Thr Gly Tyr Val Leu Arg Ser Cys His Ile Ser Tyr Ser
35 40 45
Thr Ala Ile Cys Ser Lys Arg Arg Leu Thr Ala Val Pro Gln Asp Ile
50 55 60
Pro Ser Thr Val Lys Gly Leu Asp Leu Tyr Gly Asn Lys Ile Ser Lys
65 70 75 80
Ile Gln Val Ser Asp Phe Lys Lys Leu Ser Asn Leu Thr Glu Leu Asn
85 90 95
Leu Lys Trp Asn Ile Ile Ser Glu Ile Asp Thr Gly Ser Phe Thr Asn
100 105 110
Leu Asn Ser Leu Lys Met Leu Asn Leu Asn Asp Asn Lys Leu Thr Gly
115 120 125
Leu Gly Asp Asn Val Phe Asp Gly Leu Gly Ser Leu Thr His Leu Arg
130 135 140
Met Asn Arg Asn Gln Ile Lys Thr Val Ala Ser Thr Ser Phe Lys Ser
145 150 155 160
Leu Thr Asn Leu Met Val Leu Asp Ile Ser Val Asn Lys Leu Tyr Arg
165 170 175
Ile Thr Asn Val His Thr Ile Leu Gln His Leu Pro Asn Leu Leu Glu
180 185 190
Leu Ser Val Ala Asn Asn Asn Leu Thr Ser Phe Gln Ser Trp Glu Leu
195 200 205
Thr Asn Ser Ser Leu Lys Leu Gly Gln Leu Asp Leu Ser Gln Asn Pro
210 215 220
Ile Gly Asp Phe Arg Ile Thr Ala Asp Val Phe Pro Asn Leu Thr Trp
225 230 235 240
Leu Asn Thr Gly Gly Ser Ser Lys Arg His His Met Lys Trp Asp Val
245 250 255
Arg Asn Lys Thr Phe Leu Arg Arg Val Ser Thr Phe Asp Ile Ser Gly
260 265 270
Leu Gln Ile Pro Ser Glu Asp Met Lys Thr Ile Leu Gly Thr Phe Asn
275 280 285
Ser Ser Leu Thr Thr Val Lys Met Asn Asp Met Lys Arg Thr Tyr Leu
290 295 300
Arg Ala Leu Ile Asn Ile Ser Cys Ser Ile Pro Thr Val Ser Ser Leu
305 310 315 320
Gln Leu Arg Ser Asn Asn Leu Phe Phe Met Ser Ser Asn Leu Leu Ser
325 330 335
Leu Cys Phe Lys Val Thr Glu Leu Asp Leu Ala Asp Asn Ser Met Lys
340 345 350
Asn Ile Lys Asp Asp Ala Phe Lys His Leu Gln Gly Ile Lys Val Leu
355 360 365
Ala Leu Asn His Asn Arg Leu Ser Ser Val Pro Ala Ala Ile Arg Asn
370 375 380
Leu Pro Ala Leu Glu Glu Leu Asp Leu Ser Lys Asn Asn Ile Thr Thr
385 390 395 400
Leu Gly Cys Gln Asp Phe Ala Asn Leu Thr Lys Leu Arg Gln Leu Ser
405 410 415
Leu His Gln Asn Ser Ile Ser Ala Leu Gln Gly Cys Val Phe Lys Asp
420 425 430
Leu Ile His Leu Glu Val Leu Lys Leu Gln Asn Asn Ser Ile Ser Lys
435 440 445
Leu Asn Asp Ala Phe Ile Lys Tyr Leu Pro Asn Leu Arg Gln Leu His
450 455 460
Leu Asn Ser Asn Lys Leu Thr Ala Ile Lys His Gly Glu Phe Arg Gly
465 470 475 480
Leu Arg Ser Leu Gln Asn Leu Ser Leu His Asp Asn Gln Ile Thr Val
485 490 495
Leu Glu Lys Glu Cys Phe Thr Gly Leu Arg Asn Leu Met Ala Ile Gln
500 505 510
Leu Gln Met Asn His Ile Lys Arg His Lys Asp Tyr Lys Asp Ser Phe
515 520 525
Arg Asp Leu Ile Asn Leu Lys Arg Leu Asp Leu Ser His Asn Ser Ile
530 535 540
Lys Tyr Ile Glu Ser Ser Ala Leu Pro Asp Pro Pro Phe Ser His Leu
545 550 555 560
Ser Asn Leu Glu Arg Leu Ala Phe Thr Gly Asn His Arg Arg Val Lys
565 570 575
Ser Gln Leu Pro Cys Asn Phe Leu Gln Gly Leu Thr Asn Leu Leu Asp
580 585 590
Phe Thr Gly Arg Asn Leu Gln Leu Leu Ser Leu His Lys Asp Thr Phe
595 600 605
Asn His Thr Pro Lys Leu Gln Lys Leu Asp Ile Ser Ser Asn Glu Leu
610 615 620
Met Asp Leu Ser Ser Glu Leu Phe Tyr Pro Ile Arg Asn Leu Lys Ser
625 630 635 640
Leu Tyr Ile Ser Arg Ile Asn Leu Gly Ser Leu Asp Phe Phe Lys Asp
645 650 655
Ala Asn Leu Thr Lys Leu Glu Phe Leu Gln Ala Arg Arg Asn Gln Phe
660 665 670
Ser Val Phe Ser Glu Glu Val Leu Glu Ser Met Pro Ala Leu Ala Tyr
675 680 685
Leu Asp Leu Gln Gly Asn Ser Phe Thr Cys Asp Cys Asp Asn Ala Trp
690 695 700
Phe Leu Lys Trp Thr Glu Asn Asn Gln Gln Thr Gln Val Ser Asp Ala
705 710 715 720
Tyr Asn Phe Val Cys Asn Tyr Pro Pro His Leu Lys Gly Met Lys Leu
725 730 735
Leu Lys Leu Asp Val Arg Ser Cys Thr Val Asp Thr Glu Phe Ile Cys
740 745 750
Phe Ile Ser Thr Thr Cys Thr Ile Leu Val Phe Met Val Ala Ser Phe
755 760 765
Thr Tyr His Phe Leu Arg Trp Tyr Leu Ala Tyr Ala Tyr Tyr Ile Phe
770 775 780
Leu Ala Phe Leu Phe Asp Thr Lys His Lys Asn Lys Gln Ala Pro Asn
785 790 795 800
Gln Tyr Asp Ala Phe Ile Ser Tyr Asn Thr His Asp Glu Pro Trp Val
805 810 815
Ile Arg Gln Leu Leu Pro Lys Leu Glu Gly Glu Gln Gly Trp Arg Leu
820 825 830
Cys Leu His His Arg Asp Phe Glu Pro Gly Lys Pro Ile Ile Asp Asn
835 840 845
Ile Thr Asp Ala Ile Tyr Gly Ser Arg Lys Thr Ile Cys Val Ile Ser
850 855 860
His Arg Tyr Leu Glu Ser Glu Trp Cys Ser Arg Glu Ile Gln Val Ala
865 870 875 880
Ser Phe Arg Leu Phe Asp Glu Gln Lys Asp Val Leu Ile Leu Val Phe
885 890 895
Leu Glu Asp Ile Pro Thr Thr Gln Leu Ser Pro Tyr Tyr Arg Met Arg
900 905 910
Lys Leu Leu Lys Arg Gln Thr Tyr Leu Ser Trp Pro Arg Ala Ser Glu
915 920 925
His Pro Glu Leu Phe Trp Glu Lys Leu Arg Gln Ala Met Lys Thr Arg
930 935 940
Glu Asp Leu Gly Glu Asp Glu Leu Asn Leu Thr Val Thr Asp Lys Pro
945 950 955 960
<210> 4
<211> 20
<212> DNA
<213> 斜带石斑鱼TLR22基因上游引物序列(Epinepheluscoioides)
<400> 4
ctrgvgasvg tmaactcctc 20
<210> 5
<211> 18
<212> DNA
<213> 斜带石斑鱼TLR22基因上游引物序列(Epinepheluscoioides)
<400> 5
ggtrggrats tcctccag 18
Claims (10)
1.一种斜带石斑鱼天然免疫受体TLR22基因，其特征在于，所述基因的核苷酸序列如SEQ.ID.NO：1所示。
2.根据权利要求1所述的斜带石斑鱼天然免疫受体TLR22基因，其特征在于，所述基因的全长cDNA序列如SEQ.ID.NO：2所示。
3.根据权利要求2所述斜带石斑鱼天然免疫受体TLR22基因的全长cDNA序列，其特征在于，所述cDNA序列全长3320bp，包括一个78bp的5’非编码区，一个359bp的3’非编码区，以及一个2880bp的开放阅读框。
4.根据权利要求2所述,斜带石斑鱼天然免疫受体TLR22基因的全长cDNA序列，其特征在于，所述cDNA序列3’非编码区上有2个AATAA多腺苷酸化加尾信号。
5.根据权利要求1所述的斜带石斑鱼天然免疫受体TLR22基因编码的蛋白，其特征在于，所述蛋白的氨基酸序列如SEQ.ID.NO：3所示。
6.根据权利要求1所述的斜带石斑鱼天然免疫受体TLR22基因，其特征在于，所述基因的全长cDNA序列中包含1对引物序列，引物序列如SEQ.ID.NO：4～5所示。
7.包含权利要求2所述TLR22基因全长cDNA序列的真核表达载体。
8.权利要求1或2所述的基因或权利要求3所述的蛋白在研究调节炎症因子表达方面的应用。
9.权利要求1或2所述的基因或权利要求3所述的蛋白在研究识别病毒RNA及调节免疫因子表达方面的应用。
10.权利要求1或2所述的基因或权利要求3所述的蛋白在研究细胞免疫应答方面的应用。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997491A (zh) * | 2018-06-26 | 2018-12-14 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 |
| CN109943651A (zh) * | 2019-02-28 | 2019-06-28 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5m基因及其编码蛋白的新用途 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997491A (zh) * | 2018-06-26 | 2018-12-14 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5s基因及其编码蛋白的新用途 |
| CN109943651A (zh) * | 2019-02-28 | 2019-06-28 | 中山大学 | 斜带石斑鱼天然免疫受体tlr5m基因及其编码蛋白的新用途 |
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