CN107383181B - 一种对虾抗病Toll9蛋白及其编码cDNA和用途 - Google Patents
一种对虾抗病Toll9蛋白及其编码cDNA和用途 Download PDFInfo
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Abstract
本发明公开了一种对虾抗病Toll9蛋白，其氨基酸序列如SEQ ID NO.2所示。本发明还公开了一种编码上述对虾抗病Toll9蛋白的cDNA，其核苷酸序列为如SEQ ID NO.1所示。本发明还公开了含有该cDNA的表达载体，并提供了一种能与上述对虾抗病Toll9蛋白特异性结合的抗体。除此之外，本发明还提供了供上述对虾抗病Toll9蛋白的用途以及上述编码对虾抗病Toll9蛋白cDNA的用途。
Description
技术领域
本发明属于生物技术领域，具体涉及一种抗病Toll9蛋白及其编码cDNA，以及该对虾抗病Toll9蛋白及其编码cDNA在抵抗氨氮胁迫、降低细菌感染对虾死亡率及在对虾良种选育中的用途。
背景技术
我国是对虾养殖大国，2005年我国养殖的对虾产量为100多万吨，约占世界对虾总产量的50％。近年来，由于养殖生态环境恶化、非传染性疾病与传染性疾病频发等一连串问题，其中传染性疾病(如病毒性、细菌性及真菌性疾病等)的发生尤为严重，使对虾养殖业蒙受到了相当大的损失，特别是1993年白斑综合征的暴发致使对虾养殖产量锐减。2010年偷死病的暴发，导致对虾养殖池发病率和排塘率普遍超过50％，部分地区甚至高达80-90％，是对虾养殖业近10多年来遭受的最严重病害。每年因上述问题导致对虾养殖业的直接经济损失高达50亿元。由此可见，环境和病害问题已成为对虾养殖业的可持续健康发展的障碍。改善养殖环境，提高对虾抗病能力，培育抗病性强、免疫力高的对虾新品种是解决上述问题的有效途径。通过对虾免疫学研究，有望解释其抗病机理，开展有效的疾病防治措施，同时指导良种选育工作。
Toll受体及其介导的信号通路是无脊椎动物先天性免疫系统的重要组成部分。Toll受体蛋白通过识别微生物成分,将入侵的信号通过胞内级联免疫反应传递到细胞核内，促进抗菌肽等免疫相关分子的转录、表达与释放，从而激活机体免疫系统，提高抗病抗病能力。Toll受体在进化上较为保守，属于I型跨膜蛋白，由胞外区、跨膜区和胞内区三部分组成。其中胞外区富含亮氨酸重复，主要介导细胞外配体的识别；跨膜区富含半胱氨酸，而胞内区TIR区负责将信号传递到接头分子和细胞内通路相关因子，如MyD88，NF-κB，前炎性细胞活素等，从而发挥免疫作用。
Toll信号通路与病原侵染等各种刺激反应相关联，当对虾受到氨氮胁迫、硫化物胁迫等环境胁迫以及病害感染时Toll基因会高效表达(Duan et al.,2017a；Yudiati etal.,2016；)，下游的抗菌肽基因得以激活。有研究认为，对虾免疫力与Toll基因的表达相关(Duan et al.,2017b)。因此通过增强Toll受体蛋白及其通路关键因子的作用，可以提高宿主的免疫力，从而抵抗外部环境骤变和病害感染。
发明内容
本发明的第一个目的是提供一种对虾抗病Toll9蛋白。
本发明的第二个目的是提供编码上述对虾抗病Toll9蛋白的新cDNA。
本发明的第三个目的是提供含有上述cDNA的表达载体。
本发明的第四个目的是提供一种能与上述对虾抗病Toll9蛋白特异性结合的抗体。
本发明的第五个目的是提供上述对虾抗病Toll9蛋白的用途。
本发明的第六个目的是提供上述编码对虾抗病Toll9蛋白cDNA的用途。
本发明提供的对虾抗病Toll9蛋白，其氨基酸序列如SEQ ID NO.2所示。
为达到上述第一个目的，本发明采用的技术方案如下：
一种对虾抗病Toll9蛋白，其特征在于：其氨基酸序列如SEQ ID NO.2所示。
为达到上述第二个目的，本发明采用的技术方案如下：
一种编码上述对虾抗病Toll9蛋白的cDNA，其特征在于：其核苷酸序列为如SEQ IDNO.1所示。
本发明通过在斑节对虾转录组中筛选获得斑节对虾Toll9蛋白的部分序列，然后通过RACE技术克隆到Toll9全表达序列。对虾Toll9蛋白开放阅读框长3045bp，编码1014个氨基酸组成的蛋白，真核表达分子量约为115KDa。该基因可以阻断微生物感染，可应用于提高对虾免疫力，增强抗病能力。
为达到上述第三个目的，本发明采用的技术方案如下：
一种包含上述的cDNA的表达载体。
本发明的表达载体可以是本领域中已知的任何载体。
优选地，所述的表达载体为pRSET，pET或者pGEX-4T。
为达到上述的第四个目的，本发明采用的技术方案如下：
一种抗体，其特征在于：其与上述的对虾抗病Toll9蛋白特异性地结合。
这种抗体为重组Toll9蛋白经动物免疫后制取所得。
为达到上述的第五个目的，本发明采用的技术方案如下：
上述对虾抗病Toll9蛋白在制备对虾抗病抗病药物中的应用。
上述对虾抗病Toll9蛋白在制备对虾抗病抗病药物中的应用具体包括：所述对虾抗病Toll9蛋白的cDNA在制备免疫增强剂中的应用以及所述对虾抗病Toll9蛋白的cDNA在虾类抗病育种中的应用。即利用Toll9基因的多态性，开发与对虾抗病性状相关的单核苷酸多态性(SNP)标记位点，并利用该SNP位点作为辅助育种标记。
有益效果：
本发明公开了一种对虾抗病Toll9蛋白和其cDNA序列。经过实验证明，该基因在细菌感染后显著表达变化，该蛋白可以结合微生物成分，阻断微生物感染，同时可以激活抗菌肽表达，该基因敲降导致对虾抗病力下降，提示该基因参与抗病生物学过程，可应用于对虾增强抗病力药物开发、病害防治以及对虾免疫增强剂的生产等。而Toll9基因具有多态性，可开发与对虾抗病性状相关的单核苷酸多态性(SNP)标记位点，利用该SNP位点作为辅助育种标记，用于虾类良种选育。
附图说明
图1是Toll9蛋白的三维结构分析结果，含有胞外区、跨膜区和TIR结构域；
图2是斑节对虾Toll9蛋白基因在无乳链球菌、哈维氏弧菌刺激下的表达特征分析；
图3是斑节对虾pREST-Toll9-ED蛋白的表达及纯化情况，其中，图3A中的M为蛋白maker，1为经IPTG诱导，2为未经IPTG诱导；图3B中的1为纯化后的pREST-Toll9-ED原核蛋白，M为marker；
图4是斑节对虾pGEX-Toll9-ED蛋白的表达及纯化情况；
图5是斑节对虾pET-Toll9-ED蛋白的表达情况；
图6是斑节对虾Toll9蛋白结合细菌效果；
图7是斑节对虾Toll9蛋白结合病毒类似物效果；
图8是斑节对虾Toll9蛋白体外结合病原微生物成分的效果；
图9是斑节对虾真核Toll9蛋白的免疫印迹结果，其中M为marker；C为转染空质粒的对照组；G：转染Toll9真核质粒的组别；
图10是斑节对虾真核Toll9蛋白的细胞内定位；
图11是斑节对虾Toll9被微生物成分激活，参与抵抗微生物；
图12是斑节对虾敲除后Toll9基因后抗菌能力下降，死亡率增加。
具体实施方式
下面通过以下具体实施方式对本发明作进一步阐述。
1.总RNA的提取和肝胰腺全长cDNA文库构建
1.1总RNA的提取
取鲜活健康斑节对虾(体重约150g)在实验室内暂养3d后(水温约24℃，气泵充气)，解剖虾体取出肝胰腺约100mg，放入1mL RLT Buffer(Qiagen,USA)中进行低温研磨，按照Qiagen Mini试剂盒使用说明提取总RNA，并使用DNase消化除去基因组。
1.2肝胰腺全长cDNA模板的制备
按照GeneRacer kit(Invitrogen，USA)制备全长cDNA模板。取3μg总RNA经过小牛碱性磷酸酶(CIP)反应去除RNA 5’磷酸基团，经过烟草焦磷酸酶(TAP)去除RNA 5’帽子结构，利用RNA连接酶连上GeneRacerTM RNA Oligo，再利用GeneRacerTM OligodT引物在逆转录酶Superscript III的作用下50℃反应60min，从而获得全长cDNA的模板。
2.Toll9蛋白基因cDNA全序列的克隆
2.1Toll9全长cDNA的获得
根据对已有的斑节对虾转录组数据库筛选获得的cDNA片段序列设计特异性引物。利用cDNA末端快速扩增(Rapid Amplification of cDNA ends,RACE)技术对目的基因的3ˊ和5ˊ末端进行PCR扩增。依据序列合成以下引物：
5’RACE引物：5’-CGTCATTGCGGCTGAGGCTTGC-3’
3’RACE引物：5’-TAGCCAAGCCTTCGGATAGTGA-3’
按照SMARTerTM Race的方法制备5’RACE所需的cDNA模板，利用UPM(为Long UPM和Short UPM的混合引物，浓度比值为5:1)引物和基因特异性引物5R-R获取5’端片段。PCR反应体系为：10×KOD-Plus Burffer 2.5μl；dNTP Mixture(各2mM)2.5μl；MgSO4(25mM)1.5μl；PmTLR22-5R-R(10μM)1μl；UPM primer(10μM)1μl；KOD-Plus(50U/μl)0.5μl；rTaq(5U/μl)0.15μl；cDNA 1μl；ddH2O 14.85μl；Total 25μl。反应程序为：94℃3min；94℃30s，72℃3min，5个循环；94℃30s，70℃30s，72℃3min，5个循环；94℃30s，68℃30s，72℃3min，25个循环。
3’RACE利用Adaptor primer和基因特异性引物3R-F获取PmToll9的3’端片段。PCR反应体系：10×KOD-Plus Burffer 2.5μl；dNTP Mixture(各2mM)2.5μl；MgSO4(25mM)1.5μl；PmTLR22-3R-F(10μM)1μl；Adaptor primer(10μM)1μl；KOD-Plus(50U/μl)0.5μl；rTaq(5U/μl)0.15μl；cDNA 1μl；ddH2O 14.85μl；Total 25μl。其PCR反应程序：94℃2min；94℃40s，55℃40s，72℃50s，循环32次；72℃10min；最后4℃恒温。
5’RACE和3’RACE PCR产物经1％琼脂糖凝胶电泳分离，分别亚克隆至pGEM-T载体，挑取阳性克隆，提取质粒DNA。将具有插入片段的质粒DNA用M13通用引物进行测序，所获片段拼接获得全长cDNA。
2.2Toll9的生物信息学分析
用BlastX(http://www.ncbi.nim.nih.gov/)进行同源性分析，结果如表1所示，该基因与克氏原螯虾Toll-like receptor、大水蚤Toll9、端足虫TLR13、鸡TLR21蛋白均有较高同源性，结合进化树分析，将其命名为Toll9基因。利用DNAstar等工具进行生物信息学分析，揭示开放读码框为3045个核苷酸，5’UTR为109bp，3’UTR为548bp，3’UTR存在mRNA快速降解信号ATTTA。推测编码一个1014个氨基酸的蛋白，分子量大小为115.8kDa、等电点为5.71。利用SMART在线生物信息学分析该蛋白含有三个保守结构域，包括一个富含LRR重复的胞外区(241-741aa)，一个跨膜结构域(814-833aa)，一个TIR结构域(861-1002aa)(图1)。利用SWISS-MODEL网页和Pymol软件进行三维结构模型构建，结果显示PmToll9胞外区有12个亮氨酸富集区(LRRs)，不含信号肽序列，其空间结构呈现为马蹄型结构，其凹面区形成一个光滑的弯曲状的β折叠结构。PmToll9的N端紧随的是LRRs区域，紧邻LRRs区域的是C端“帽子”结构，并与跨膜区相连(见图1)。
表1 Toll9基因的blast分析结果
| Description | <u>E value</u> | <u>Identity</u> | Accession |
| <u>Toll-like receptor[Procambarus clarkii]</u> | 0.0 | 49％ | <u>AJE28352.1</u> |
| <u>Toll-9[Daphnia magna]</u> | 1e-41 | 32％ | <u>KZS16474.1</u> |
| <u>toll-like receptor 13[Hyalella azteca]</u> | 2e-104 | 32％ | <u>XP_018021731.1</u> |
| <u>toll-like receptor 21[Gallus gallus]</u> | 8e-44 | 27％ | <u>AFD61602.1</u> |
3.荧光定量PCR检测Toll9mRNA在不同刺激物的表达
健康斑节对虾(7～12g)分别注射50μl无乳链球菌Streptococcus Agalactiae(革兰氏阳性菌)，和注射哈维氏弧菌Vibrio Harveyi(革兰氏阴性菌)，同时以注射PBS和不注射组作为对照。注射后3h，6h，12h，24h，48h，72h，96h取肝胰腺、肠、淋巴和鳃等组织，存放于RNAlater。每种刺激物每个时间点均取3尾虾。
取不同组织总RNA 1μg与反转录引物(Oligo-(dT)18接头引物)1μL(10pmol/L)混合，70℃加热5min后，立即放置冰上，然后加入5×buffer，2.5mmol/L dNTP混合液，Ribonuclease Inhibitor，M-MLV反转录酶(Promega，USA)，反应体系为25μL。反应过程为42℃60min，70℃15min，稀释1倍后作为模板使用。根据Toll9基因的全cDNA长设计荧光定量PCR引物：
Toll9-F1：5’-GCTGAACGATAACCCCTTGTG-3’
Toll9-R1：5’-GATAAAGCCTGGTGACATTACTG-3’
扩增Toll9基因同时选用EF-1α作为内参基因，引物序列：
EF-F2：5’-AAGCCAGGTATGGTTGTCAACTTT-3’
EF-R2：5’-CGTGGTGCATCTCCACAGACT-3’
荧光定量PCR反应体系为20μL，包含10μL的2×SYBR Green Real-time PCRMaster Mix(TaKaRa，Japan)，1μL cDNA模板，2μL引物和7μL的双蒸水，以蒸馏水代替模板作为阴性对照,每个样品设置3个重复，反应参数为95℃预变性10s，然后95℃变性15s，55℃退火30s，72℃延伸30s，共40个循环。实验数据采用相对CT法进行数据分析。
结果显示革兰氏阳性菌无乳链球菌能激活PmToll9表达，且肝胰腺的PmToll9反应最为灵敏(图2A)，在肝胰腺(图2A)、肠(图2B)、淋巴(图2C)、鳃组织(图2D)中分别在12h、24h、48h、24h达到表达高峰，为对照组的7.65倍、4.15倍、4.16倍、3.45倍。而革兰氏阴性菌哈维氏弧菌感染对PmToll9的表达具有一定的抑制作用，肝胰腺在24h(图2A)，肠道在72h(图2B)，淋巴中在72h(图2C)，鳃中在12h(图2D)分别被显著抑制98％，97％，99％，98％。说明Toll9基因可能参与的斑节对虾对细菌的免疫防御过程。
4.Toll9融合蛋白的制备
根据拼接好的Toll9蛋白基因cDNA胞外区序列，设计合成一对引物，上游引物(rToll9-F)CGGGATCCGTGTCTGGGCTCGAGGTCC引入BamHI酶切位点，下游引物(rToll9-R)CCGGAATTCGTCCTCCTCCATCGCGTC引入EcoRI酶切位点。使用肝胰腺cDNA为模板进行PCR：94℃2min，94℃30s，55℃，30s，72℃，1min，共30循环，72℃,10min。PCR产物经BamHI和EcoRI双酶切后亚克隆到pRSET-A质粒，质粒经测序确认无误后，转化到大肠杆菌BL21细胞，挑取单克隆培养至对数期，加入终浓度为0.4mM的IPTG在37℃诱导表达3-6h。附图3A显示pRSET-Toll9-ED诱导表达为一个约55kD的融合蛋白，与预测的分子量一致。
利用融合表达蛋白的6个组氨酸标签，使用Ni-NTA+树脂和His BindPurification Kit(Novagen,USA)对pRSET-Toll9-ECD进行变性条件下亲和层析纯化。纯化流程见Novagen试剂盒说明书。附图3B显示His纯化效果较好，经纯化的pRSET-Toll9-ED融合蛋白约55KD。经8M，6M，4M，2M，1M，0M尿素梯度稀释复性后，保存在-20度冰箱备用。
相似的，通过上游引物5’-CGGGATCCGTGTCTGGGCTCGAGGTCC-3’和下游引物5’-CCGGAATTCGTCCTCCTCCATCGCGTC-3’分别引入BamHI和EcoRI酶切位点，酶切后与pGEX-4T质粒连接，获得重组克隆，经测序正确后诱导表达获得约70kD的重组蛋白(图4)，经GST.bind纯化试剂盒(Merck，USA)对所获重组蛋白进行纯化获得较为单一的条带(图4)。
同样，通过引物5’-CCGGGATCCGTGTCTGGGCTCGAGGTCC-3’和下游引物5’-CCGGAATTCGTCCTCCTCCATCGCGTC-3’分别引入BamHI和EcoRI酶切位点，酶切后与pET28质粒连接，获得重组克隆，经测序正确后诱导表达获得约60kD的重组蛋白(图5)。
5.Toll9基因编码蛋白的多克隆抗体制备
将重组到pRSET的纯化蛋白(约100-200μg)与3ml完全弗氏佐剂充分混匀孵化，在小鼠皮下多点注射，共注射8只小鼠，每只小鼠注射约50μg蛋白。第一次注射3周后加强免疫3次。加强注射剂量为10-20μg蛋白，使用不完全弗氏佐剂进行乳化。每次注射间隔10天。第4次注射后将小鼠拔除眼球取血，将鼠血于37℃静置1h后，4000rpm离心10min，吸取上层多抗血清，分装保存于-80℃。图6显示所制备的多克隆抗体可特异性识别Toll9蛋白。
6、rToll9蛋白对细菌的结合特性
将过夜培养的创伤弧菌(Vibrio vulnificus)，嗜水气单胞菌(Aeromonashydrophila)，大肠杆菌(Escherichia coli)，美人鱼发光杆菌(Photobacteriumdamselae)，以及金黄色葡萄球菌(Staphylococcus aureus)细菌接入9ml培养基中,将细菌接入培养基中，37℃培养至OD600＝0.5～0.7，加入1ml 37％的甲醛继续培养1h；4℃2000rmp离心10min；去上清，用5ml PBS洗涤两次；4℃，2000rpm离心10min收集菌体用2ml PBS重悬；加入重组表达并纯化的pRSET-Toll9蛋白，室温轻摇30min后离心收集菌体，用2ml PBS洗涤3次后，加入300ul洗脱液(50mM柠檬酸钠，pH＝3.5)洗脱结合的蛋白；4℃2000rpm离心10min，取出上清立即用1M Tris中和。取100ul冼脱液,用anti-His单克隆抗体进行Westernblot。结果显示，PmToll9对革兰氏阴性菌有明显的结合能力，特别对创伤弧菌表现出较强的结合能力，而对嗜水气单胞菌和金黄色葡萄球菌结合能力稍弱(图7)。说明PmToll9可以通过结合细菌抵抗细菌感染。
7、rPmToll9对病原的结合特性
为验证PmToll9是否对细菌或病毒具有结合能力，利用ELISA技术检测了rPmToll9对不同病原组分(病毒中间产物双链RNA poly(I:C)-L和poly(I:C)-H，革兰氏阴性组分脂多糖LPS，病毒和细菌DNA类似物ODN2006以及Resoquimod)的结合活性。将含有不同病原成分的50m M碳酸钠-碳酸氢钠缓冲液(pH 9.8)，按每孔100μL加入到96孔酶标板上，4℃包被过夜；次日用PBST溶液洗涤3次，每次5min，然后按每孔200μL加入3％BSA溶液，在37℃条件下封闭1h；移去BSA溶液，PBST洗涤3次，每次5min，然后在每孔中加入100μL的pRST-Toll9重组蛋白溶液，在18℃条件下孵育2h，以BSA蛋白作为对照；移去蛋白溶液，PBST洗涤三次，每次5min，然后加入100μL小鼠抗His的单克隆抗体(1:2000)，于37℃条件下孵育1h；移去抗体，PBST洗涤三次，每次5min，然后加入100μL AP标记的兔抗小鼠的二抗(1:2000)，于37℃条件下再孵育1h；移去二抗，TBST洗涤三次，每次5min，然后通过p NPP试剂显色，并酶标仪读数记录。每个孔设三个重复用于统计分析。结合活性记录为405nm处的P/N，当P/N>2.1时被认为是阳性结果。结果表明rPmToll9对5种检测的PAMPs都有较高的结合活性(图11)，其中对病毒和细菌中间产物poly(I:C)-L、poly(I:C)-H以及细菌脂多糖LPS的结合活性呈现浓度的梯度依赖性，但对ODN2006以及Resoquimod的浓度依赖性效应不显著(图8)。说明PmToll9能结合细菌和病毒的主要成分，进而启动细胞内的免疫防御反应抵御细菌和病毒感染。
8、真核细胞表达
设计引物进行真核表达。引物pCMV-Toll9-F引入Pst I酶切位点(5′-GCTTCTGCAGGAATTCATGAAAATGACGGACAACGG-3′)，pCMV-Toll9-R引入Xba I酶切位点(5′-GTTTCTGCTCTCTAGATAACTTCGTCGACGCTGCGA-3′)，以肝胰腺cDNA为模板，进行PCR，产物和空质粒pCMV经PstI和Xba I双酶切后，连接、转化，抽提质粒，经测序正确后大量抽提备用。按照转染试剂
3000的操作说明书转染Hela细胞，转染6h后将转染液换为完全培养基继续培养24h后收集细胞，用预冷1×PBS漂洗一次，加入4×Loading buffer重悬细胞并行SDS-PAGE，转膜后用myc单克隆抗体进行Western blot杂交、显色。获得真核表达的PmToll9大小约为115kD(图9)，与预测分子量大小一致。
将PmToll9基因的开放读码框的核苷酸序列与pGAPZA和pGAPαB表达载体制备成真核表达质粒，并转入克隆菌株DH5α。将表达质粒用限制性内切酶BspI消化，浓缩线性化DNA片段后，电转化毕赤酵母，在Zeocin抗性平板上筛选出重组克隆，并在3ml YPD(100μg/ml)Zeocin培养，48h后分别取菌体和上清于12％的聚丙烯酰胺凝胶电泳。筛选出游特异蛋白表达的克隆。
9、真核细胞内定位
Toll9-GFP-F引物引入Bgl II酶切位点(5′-GAAGATCTCATGAAAATGACGGACAACGG-3′)，Toll9-GFP-R引物引入EcoRI酶切位点(5′-AATTCCCCATAACTTCGTCGACGCTGC-3′)，经PCR扩增后与pEGFP-N2质粒分别进行Bgl II和EcoRI双酶切，用T4连接酶连接过夜，次日转化DH5a感受态，挑取阳性克隆测序正确后抽提质粒备用。
转染前24h将活力较强的Hela细胞铺于24孔板，待细胞生长密度达到60％-70％即可准备进行转染。按照转染试剂
3000的操作说明书预先向1.5ml EP管中按比例分别加入PmToll9-GFP重组质粒和pEGFP-N1空载；将孔板中旧培养基吸掉，加入无血清新鲜培养基，再将预混合的转染液均匀滴加到24孔板中；转染6h后将转染液换为完全培养基继续培养24h；去掉培养基，并用预冷1×PBS漂洗一次预冷1×PBS漂洗一次；加入4％多聚甲醛(PFA)，4℃放置15min；1×PBS洗2次，每次10min；加入500μl 1μM的DAPI染液，室温染细胞核10min；1×PBS洗3次，每次5min；加入200μl抗荧光衰减剂于每孔中，锡箔纸封闭在Nikon显微镜下观察荧光蛋白的表达与定位。结果表明PmToll9-GFP的绿色荧光分布于除细胞核外的其他区域，在细胞质中观察到较强的绿色荧光，在细胞膜上也有一定的分布。
10.PmToll9激活宿主免疫系统
PmToll9全长真核重组质粒以斑节对虾cDNA为模板，片段在5’端引入Xho I酶切位点，3’端引入Hind III酶切位点(特异性引物需要补充)，经Xho I和Hind III双酶切后插入果蝇表达载体pAc5.1/V5-His B。报告质粒pGL3-PEN453、pGL3-PEN536和pGL3-Crustin-like的构建以斑节对虾基因组DNA为模板；pGL3-Drosomycin、pGL3-Drosocin和pGL3-Defensin以果蝇S2细胞基因组DNA扩增得到，经过酶切、连接、转化、测序等步骤，将获得的阳性克隆菌大量扩增并提纯备用。
表2抗菌肽质粒引物设计
S2细胞生长密度达到40％-80％以上，进行转染操作。转染按Qiagen公司Effectene Transfection Reagent说明书进行：0.2μg质粒加Buffer EC至60ul，混合1.6uLEnhancer；室温孵育2-5min；1ml PBS漂洗，加入350ul新鲜培养基；转染混合液滴加到24孔板中，轻轻震荡混匀；28℃培养24h后，将8种不同种类的免疫刺激剂分别以适宜浓度加入到其中一组实验组中；孵育6-8h后，更换为新鲜培养基继续培养12-18h；收集细胞，用1×positive lysis buffer裂解，离心取部分上清液进行荧光素酶表达测定。
表3 TLR配体种类
结果显示PmToll9可以明显激活果蝇抗真菌肽Drosomycin启动子上调2.2倍，而果蝇防御素Defensin和Drosocin启动子上调幅度较低，分别是2.0倍和1.1倍(图10)。同时，三种来源于斑节对虾的抗菌肽基因Penaeidin 453、Penaeidin536和Crustin-like启动子可以被PmToll9激活，相比对照组分别上调表达水平2.1倍、4.4倍和5.1倍(P<0.01)(图10)。说明PmToll9可以明显激活果蝇和对虾的抗菌肽，启动宿主免疫系统进行免疫防御，增强对虾抗病力。
11、PmToll9对细菌、病毒成分具有免疫响应
利用上述方法构建的双荧光素酶报告系统，探究细菌和病毒成分是否对PmToll9，分别添加了8种免疫刺激剂后搜集细胞，提取RNA并检测PmToll9信号通路的免疫应答情况。结果显示病毒模拟物poly(I:C)-LMW、poly(I:C)-HMW和革兰氏阳性菌成分peptidoglycan(PGN)免疫刺激后，报告基因的荧光值显著上调1.9倍；而经LPS刺激的实验组报告基因的荧光值表达明显下降1.9倍。CL097、ODN2006、R848、Rec-FLA、虽然能引起报告基因上调1.1～1.3倍，但尚未达到显著水平(图11)。说明PmToll9对病毒和革兰氏阳性菌的成分具有免疫应答，参与对病毒和革兰氏阳性菌的免疫防御。
12.Toll9抵御细菌感染的作用
按照Promega公司的T7RiboMAXTM Express RNAi System试剂盒说明书进行dsRNA合成。先用dsToll-up(5’-CCTGAGGTAGTCTTCCCAGATGC-3’)和dsToll-T7-dn(5’-TAATACGACTCACTATAGGGAGAGAGGTGGATAAGTTATGTGGTTGCTTTG-3’)引物对，以及dsToll-dn(5’-GAGGTGGATAAGTTATGTGGTTGCTTTG-3’)dsToll-T7-up(5’-TAATACGACTCACTATAGGGAGACCTGAGGTAGTCTTCCCAGATGC-3’)引物对分别以cDNA为模板，扩增并纯化获得正义模板以及反义模板，接着进行体外转录反应，具体为RiboMAX Express T7 2×Buffer 10μL，加入正义模板4μL，反义模板4μL，混匀后37℃温浴4h；温浴结束后将反应液于70℃水浴10min，之后缓慢降至室温(～20min)；然后加1μL RNase A Solution和1μL RQ1RNase-FreeDNase，37℃孵育30min；最后用3M醋酸钠沉淀后，70％乙醇洗涤，加无RNase的水溶解沉淀后备用。
将Toll9 dsRNA接种斑节对虾，每组18-20尾。对照组一注射磷酸缓冲液(PBS)。24h后注射PBS；对照组二注射磷酸缓冲液(PBS)24h后接种106cfu/mL浓度哈维氏弧菌25ul。实验组一注射dsRNA 25ug，24h后注射PBS，实验组二注射dsRNA24h后接种106cfu/mL浓度哈维氏弧菌25ul，5d后记录存活率。结果显示，对照组一均无死亡对虾，对照组二存活率为64.3％，实验组二在注射dsRNA基因敲降Toll9后，死亡率增加，最后累计存活率为28.6％(图12)。说明体内Toll9基因被敲除后，虾体免疫力显著下降，细菌感染加剧。
13.Toll9蛋白基因在对虾抗病育种中的应用
利用Toll9基因的多态性，开发与对虾抗病性状相关的单核苷酸多态性(SNP)标记位点，并利用该SNP位点作为辅助育种标记，具体方法如下：用剪刀剪取对虾肌肉组织25mg，利用Omega Bio-Tek Inc.E.Z.N.A.Mollusk DNA试剂盒提取养殖家系亲本DNA。利用Toll9基因上的SNP相关引物和HRM方法对样品进行SNP分型。PCR反应在7500Fast Real-time PCRSystem(Applied Biosystems Inc.)上进行，反应条件为95℃预变性5分钟，然后95℃变性30秒，55℃退火延伸1分钟，进行40个循环。PCR反应结束后，样品从65℃到95℃缓慢升温，进行熔解曲线分析。熔解曲线结果用ABI公司提供的High Resolution Melting Analysis2.0分析软件进行分型。筛选获得对虾抗病家系亲本，培育具有抗病性状的对虾新品系，提高对虾养殖的成活率，增加经济效益。
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。
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<110> 中国水产科学研究院南海水产研究所
<120> 一种对虾抗病Toll9蛋白及其编码cDNA和用途
<160> 2
<170> BiSSAP 1.3.6
<210> 1
<211> 3702
<212> DNA
<213> 人工序列
<220>
<223> 对虾抗病毒Toll9蛋白的全长cDNA序列
<400> 1
attcgtcatt gcggctgagg cttgcagaca ccctgttgag gttcctgccg aagaccatgg 60
ggaagcagcg attacagaag agccgcgagg tctggactta accttggaca tgaaaatgac 120
ggacaacgga aacggtagat atgacagcgc tacaatctca ctttccaaac agccgggaga 180
taatggctac acggcggagg aaagcgttac gaaggaactg gtcagaagtg gagaaagact 240
gacctctagc tccatgactg acgaagtcct aacaacgaaa gttaagactc acgtgagaaa 300
agaagtcagt aagaataaag tccctctcct ttcctcgact gagagtctcc agaaatcaga 360
agaatcttcg ttggccccag aagacgtcat gtttcctata acaggaaaga gaataaagga 420
ggaaaagggt gatagggtta aaggcgacac cctttcgtca cattcgaaga acaggtgcga 480
gttcaggtcg acgccacctg aggtagtctt cccagatgcg ttcgaagaat tttctaaatc 540
gatgaaagca agcctcagcc gcaatgacga atctgaagac gtgttcctcg acagcctcct 600
tcctgacgga tgccactaca tggagaggca gaagaagaaa gtcatgtgca ccggatccaa 660
catgacgtcc atccccgagt tcgaccacgc gaggaacatc gaaacactgc acttcagcgg 720
gacgtccatc gtacaggtga ccaacctcga ccccctcccg agatccctca aggggctcta 780
cttctccaac ggcatgctca aggtctttga cggcaggaac ctcaacaggg tgtctgggct 840
cgaggtcctc tccctcgacg ccaacttcat caacagctgg agcttcgtca ccaccttcta 900
ctccatgggc ggcttcgctg agcagaacac catcaaattg ctcgacattc aaagcaacca 960
cataacttat ccacctcagc cggtcgggga caacgagaca gtattgcctt tcctggagac 1020
tttcgtgctg aacgataacc ccttgtgtta cttgccagac acgctcttca agcctcttag 1080
gaacagtaat gtcaccaggc tttatctgaa gaactgcaac atcaacgaat tttacggatc 1140
tcccttatcc tacttaccaa acttggaaat cttggacctc acgaacaaca gggccattaa 1200
cgaaaccgag ctgagggacc ttctgatgcc tcttggccgc ctgaaggaac tgtacctcgg 1260
taacaacaac tacccgaccg tccctaccag agcactctcc ttggtgaacg gcacgctaca 1320
gaagctggac ctccattcct cgaccttcac atgcctcgac aactcttcat ttccagtgat 1380
gccgatcctg acgcacctca acctcatgta ctgcaggatc aacgcgatcc gggaatacac 1440
attccaggga ttcccgatgc tgcaggaact gaacttggac gggaacagcc tcaccacagt 1500
ccccccggag gtgctgcttc cctcgttgca gaccctgacg ctgagcgaca acccgcgcgc 1560
caacggcaac gacggcgacc agcgcttcac catggacgac gtcagcttcc ggaacatggt 1620
caacctcaag acgatcacct taaatcaggt cataatggaa aagatagaga gctcttactt 1680
caatgacctg tacaatctcg aggaactttt tctaacggga tgtggcataa aaaccatcga 1740
aaacttcagc ttcatgaacc tgaccaagct gcagcggctg cacctcagcg acaactccat 1800
cagctccctc tataacgaca gcttggttgg cctcgtgagt ctgacctacc ttgacctctc 1860
caacaacaag cttgaagtga ccaatcgcat ggcgcgctct gggctctcgt ccctcgcgtc 1920
tcgagccgag tcgctctcgc ccgatagtgt ttcggacact cgagaaatag acatgttttt 1980
agagagcatc agagcccctt cgcctctcat tccttggctg tccaagatac gaaggaacgc 2040
gagggccgtg ggaacccgag aatgggagga ggagaatctg cagaagaaaa ttatagcaac 2100
tttgcccttc agcgacctcg taaacctgag gacgctcaac ctctccgaaa acaagatcat 2160
tcaaatctcg cccgaattat tccacaacct cacgaacctc ctgtttctcg acatcaacaa 2220
caacaggctg atgacctggg acgagccggt gttcgggtcc atccccaacc taacggagct 2280
ccacctgagg atgaacttgc tcgatggcat caccgacgcg atggaggagg acttccggaa 2340
agagagcctc aaactcgtcg acctccaaaa caacagcttc aagtgcgact gtagcctgag 2400
caagttcaat aggtcgctca acacatcgaa cttcctgaac tggccttatc agtgcacgga 2460
gggggagaca gacgtggaca tggaggaata cattgcgagg gcgccgtgta actttgctac 2520
ccagccgcag ggccacgttc gggtgcggac catcgtcatc gccttccttc tctcgggctt 2580
gctcctggtg gcctccgtca tggtctacag aaagcgatgg tatgtgcgct acttcgtgta 2640
caccgtgagg atgagagcaa aggtcacgca tgatgaagcc gacaagtacc tgtacgacac 2700
cttcgtctgt tactcgcaga cggaccgcca gtgggtgttc gagcacctgg tggccaagct 2760
ggaggacggg gggaggtacc gagtgtgtat ccacgagcga gacttcacag ttggccaaga 2820
gataacagaa aacattatca gtagtgtcga acggtcacgc aaggtcgtag tggttctgtc 2880
gccgtccttc atcaggagca gctggtgcat gttcgaactc cagatggtca gcaacaagat 2940
cctcgacgaa aggaaatcca aactgatcat gctgcttctg gagcgcatcc cggaggagga 3000
gcagcccaag aaactgaagt acttgctcaa gacgagaacc tacatcgagt gggttccgga 3060
cctcgagagt caaaagctct tctgggcaag gttgctgaga gccatagcca agccttcgga 3120
tagtgaggcc atcgcagcgt cgacgaagtt atagctgagg ctctgagtga agtcctgact 3180
cctgtttaca tatagtgttt gcttttaaag gtgactaaac tgtgttattg gtaaaacatt 3240
ccgttttgga acctgaacag gctgcctctg tggaatgtct gaactattcc aagagagctt 3300
atagacatcg tgtatgatat tcctattaat ctttggttct cttgtgagtt ttgttgtata 3360
aacagtgaag tcttgtatga taatgaatcc atattatttg ttcttgttgc taatcacaga 3420
ggtctgtagg aaaatgtgat actgaataca gttacaataa cacacacaca tacacacaca 3480
cacacacacg cacacacaca cacacacaca cacacacaca cacacacaca catatatata 3540
cagacacact cacacaatct gtgtatatat aatacacaca catatgcgcg ctttctgtga 3600
caaaacgtac gtgacattac acatatatat ttattatata ctgtatgggg ttgtgtacat 3660
gagagtaaca cggggggaca aaaaaaaaaa aaaaaaaaaa aa 3702
<210> 2
<211> 1014
<212> PRT
<213> 人工序列
<220>
<223> 对虾抗逆Toll9蛋白的氨基酸序列
<400> 2
Met Lys Met Thr Asp Asn Gly Asn Gly Arg Tyr Asp Ser Ala Thr Ile
1 5 10 15
Ser Leu Ser Lys Gln Pro Gly Asp Asn Gly Tyr Thr Ala Glu Glu Ser
20 25 30
Val Thr Lys Glu Leu Val Arg Ser Gly Glu Arg Leu Thr Ser Ser Ser
35 40 45
Met Thr Asp Glu Val Leu Thr Thr Xaa Val Lys Thr His Val Arg Lys
50 55 60
Glu Val Ser Lys Asn Lys Val Pro Leu Leu Ser Ser Thr Glu Ser Leu
65 70 75 80
Gln Lys Ser Glu Glu Ser Ser Leu Ala Pro Glu Asp Ala Met Phe Pro
85 90 95
Ile Thr Gly Lys Arg Ile Lys Glu Glu Lys Gly Asp Arg Val Lys Gly
100 105 110
Asp Thr Leu Ser Ser His Ser Lys Asn Arg Cys Glu Phe Arg Ser Thr
115 120 125
Pro Pro Glu Val Val Phe Pro Asp Ala Phe Glu Glu Phe Ser Lys Ser
130 135 140
Met Lys Ala Ser Leu Ser Arg Asn Asp Glu Xaa Glu Asp Val Phe Leu
145 150 155 160
Asp Ser Leu Leu Pro Asp Gly Cys His Tyr Met Glu Arg Gln Lys Lys
165 170 175
Lys Val Met Cys Thr Gly Ser Asn Met Thr Ser Ile Pro Glu Phe Asp
180 185 190
His Ala Arg Asn Ile Glu Thr Leu His Phe Ser Gly Thr Ser Ile Val
195 200 205
Gln Val Thr Asn Leu Asp Pro Leu Pro Arg Ser Leu Lys Gly Leu Tyr
210 215 220
Phe Ser Asn Gly Met Leu Lys Val Phe Asp Gly Arg Asn Leu Asn Arg
225 230 235 240
Val Ser Gly Leu Glu Val Leu Ser Leu Asp Ala Asn Phe Ile Asn Ser
245 250 255
Trp Ser Phe Val Thr Thr Phe Tyr Ser Met Gly Gly Phe Ala Glu Gln
260 265 270
Asn Thr Ile Lys Leu Leu Asp Ile Gln Ser Asn His Ile Thr Tyr Pro
275 280 285
Pro Gln Pro Val Gly Asp Asn Glu Thr Val Leu Pro Phe Leu Glu Thr
290 295 300
Phe Val Leu Asn Asp Asn Pro Leu Cys Tyr Leu Pro Asp Thr Leu Phe
305 310 315 320
Lys Pro Leu Arg Asn Ser Asn Val Thr Arg Leu Tyr Leu Lys Asn Cys
325 330 335
Asn Ile Asn Glu Phe Tyr Gly Ser Pro Leu Ser Tyr Leu Pro Asn Leu
340 345 350
Glu Ile Leu Asp Leu Thr Asn Asn Arg Ala Ile Asn Glu Thr Glu Leu
355 360 365
Arg Asp Leu Leu Met Pro Leu Gly Arg Leu Lys Glu Leu Tyr Leu Gly
370 375 380
Asn Asn Asn Tyr Pro Thr Val Pro Thr Arg Ala Leu Ser Leu Val Asn
385 390 395 400
Gly Thr Leu Gln Lys Leu Asp Leu His Ser Ser Thr Phe Thr Cys Leu
405 410 415
Asp Asn Ser Ser Phe Pro Val Met Pro Ile Leu Thr His Leu Asn Leu
420 425 430
Met Tyr Cys Arg Ile Asn Ala Ile Arg Glu Tyr Thr Phe Gln Gly Phe
435 440 445
Pro Met Leu Gln Glu Leu Asn Leu Asp Gly Asn Ser Leu Thr Thr Val
450 455 460
Pro Pro Glu Val Leu Leu Pro Ser Leu Gln Thr Leu Thr Leu Ser Asp
465 470 475 480
Asn Pro Arg Ala Asn Gly Asn Asp Gly Asp Gln Arg Phe Thr Met Asp
485 490 495
Asp Val Ser Phe Arg Asn Met Val Asn Leu Lys Thr Ile Thr Leu Asn
500 505 510
Gln Val Ile Met Glu Lys Ile Glu Ser Ser Tyr Phe Asn Asp Leu Tyr
515 520 525
Asn Leu Glu Glu Leu Phe Leu Thr Gly Cys Gly Ile Lys Thr Ile Glu
530 535 540
Asn Phe Ser Phe Met Asn Leu Thr Lys Leu Gln Arg Leu His Leu Ser
545 550 555 560
Asp Asn Ser Ile Ser Ser Leu Tyr Asn Asp Ser Leu Val Gly Leu Val
565 570 575
Ser Leu Thr Tyr Leu Asp Leu Ser Asn Asn Lys Leu Glu Val Thr Asn
580 585 590
Arg Met Ala Arg Ser Gly Leu Ser Ser Leu Ala Ser Arg Ala Glu Ser
595 600 605
Leu Ser Pro Asp Ser Val Ser Asp Thr Arg Glu Ile Asp Met Phe Leu
610 615 620
Glu Ser Ile Arg Ala Pro Ser Pro Leu Ile Pro Trp Leu Ser Lys Ile
625 630 635 640
Arg Arg Asn Ala Arg Ala Val Gly Thr Arg Glu Trp Glu Glu Glu Asn
645 650 655
Leu Gln Lys Lys Ile Ile Ala Thr Leu Pro Phe Ser Asp Leu Val Asn
660 665 670
Leu Arg Thr Leu Asn Leu Ser Glu Asn Lys Ile Ile Gln Ile Ser Pro
675 680 685
Glu Leu Phe His Asn Leu Thr Asn Leu Leu Phe Leu Asp Ile Asn Asn
690 695 700
Asn Arg Leu Met Thr Trp Asp Glu Pro Val Phe Gly Ser Ile Pro Asn
705 710 715 720
Leu Thr Glu Leu His Leu Arg Met Asn Leu Leu Asp Gly Ile Thr Asp
725 730 735
Ala Met Glu Glu Asp Phe Arg Lys Glu Ser Leu Lys Leu Val Asp Leu
740 745 750
Gln Asn Asn Ser Phe Lys Cys Asp Cys Ser Leu Ser Lys Phe Asn Arg
755 760 765
Ser Leu Asn Thr Ser Asn Phe Leu Asn Trp Pro Tyr Gln Cys Thr Glu
770 775 780
Gly Glu Thr Asp Val Asp Met Glu Glu Tyr Ile Ala Arg Ala Pro Cys
785 790 795 800
Asn Phe Ala Thr Gln Pro Gln Gly His Val Arg Val Arg Thr Ile Val
805 810 815
Ile Ala Phe Leu Leu Ser Gly Leu Leu Leu Val Ala Ser Val Met Val
820 825 830
Tyr Arg Lys Arg Trp Tyr Val Arg Tyr Phe Val Tyr Thr Val Arg Met
835 840 845
Arg Ala Lys Val Thr His Asp Glu Ala Asp Lys Tyr Leu Tyr Asp Thr
850 855 860
Phe Val Cys Tyr Ser Gln Thr Asp Arg Gln Trp Val Phe Glu His Leu
865 870 875 880
Val Ala Lys Leu Glu Asp Gly Gly Arg Tyr Arg Val Cys Ile His Glu
885 890 895
Arg Asp Phe Thr Val Gly Gln Glu Ile Thr Glu Asn Ile Ile Ser Ser
900 905 910
Val Glu Arg Ser Arg Lys Val Val Val Val Leu Ser Pro Ser Phe Ile
915 920 925
Arg Ser Ser Trp Cys Met Phe Glu Leu Gln Met Val Ser Asn Lys Ile
930 935 940
Leu Asp Glu Arg Lys Ser Lys Leu Ile Met Leu Leu Leu Glu Arg Ile
945 950 955 960
Pro Glu Glu Glu Gln Pro Lys Lys Leu Lys Tyr Leu Leu Lys Thr Arg
965 970 975
Thr Tyr Ile Glu Trp Val Pro Asp Leu Glu Ser Gln Lys Leu Phe Trp
980 985 990
Ala Arg Leu Leu Arg Ala Ile Ala Lys Pro Ser Asp Ser Glu Ala Ile
995 1000 1005
Ala Ala Ser Thr Lys Leu
1010
Claims (7)
1.一种对虾抗病Toll9蛋白，其特征在于：其氨基酸序列如SEQ ID NO.2所示。
2.一种编码如权利要求1所述的对虾抗病Toll9蛋白的cDNA，其特征在于：其核苷酸序列为如SEQ ID NO.1所示。
3.一种表达载体，其特征在于：所述的表达载体包含权利要求2所述的编码对虾抗病Toll9蛋白的cDNA。
4.根据权利要求3所述的表达载体，其特征在于：所述的表达载体为pRSET，pET或者pGEX-4T。
5.一种多克隆抗体，其特征在于：其与权利要求1所述的对虾抗病Toll9蛋白特异性地结合。
6.权利要求1所述的对虾抗病Toll9蛋白在制备对虾抗病药物中的应用。
7.根据权利要求6所述对虾抗病Toll9蛋白在制备对虾抗病药物中的应用，其特征在于：所述的应用包括：所述对虾抗病Toll9蛋白的cDNA在制备免疫增强剂中的应用。
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| CN102318771A (zh) * | 2011-09-20 | 2012-01-18 | 广东粤海饲料集团有限公司 | 一种中草药对虾饲料抗病添加剂 |
| WO2013063388A1 (en) * | 2011-10-28 | 2013-05-02 | The Regents Of The University Of California | Diatom-based vaccines |
| CN103497241A (zh) * | 2013-09-10 | 2014-01-08 | 中国水产科学研究院南海水产研究所 | 一种对虾抗病毒Argonaute蛋白及其编码cDNA和用途 |
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| CN102318771A (zh) * | 2011-09-20 | 2012-01-18 | 广东粤海饲料集团有限公司 | 一种中草药对虾饲料抗病添加剂 |
| WO2013063388A1 (en) * | 2011-10-28 | 2013-05-02 | The Regents Of The University Of California | Diatom-based vaccines |
| CN103497241A (zh) * | 2013-09-10 | 2014-01-08 | 中国水产科学研究院南海水产研究所 | 一种对虾抗病毒Argonaute蛋白及其编码cDNA和用途 |
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