CN113880932B - 锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用 - Google Patents
锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用 Download PDFInfo
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- CN113880932B CN113880932B CN202111138584.5A CN202111138584A CN113880932B CN 113880932 B CN113880932 B CN 113880932B CN 202111138584 A CN202111138584 A CN 202111138584A CN 113880932 B CN113880932 B CN 113880932B
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Abstract
本发明涉及基因工程技术领域，尤其涉及锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用。本发明针对维氏气单胞菌感染后发病锦鲤的脾脏进行研究得到一种表达水平明显上调的锦鲤Ladderlectin基因。本发明研究发现锦鲤Ladderlectin基因在维氏气单胞菌感染的发病锦鲤的各个组织中表达水平均发生显著变化，并且过表达了锦鲤Ladderlectin基因的锦鲤，其抗病原菌的能力得到显著提高，这在锦鲤养殖领域具有重要意义。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用。
背景技术
锦鲤(Cryprinus carpio)属于鲤形目鲤科鲤属，是风靡世界的高级观赏鱼，有“会游泳的艺术品”的美称，它具有优美的游姿，绚丽的花纹以及亲和的性格，深受人们的喜爱。锦鲤经过百余年的变异和选择，免疫力较食用鲤鱼低，并且易于受到应激而引发多种病原感染。锦鲤的疾病中大部分由细菌引起，如维氏气单胞菌(秦国民；姜娜et al.,2012)，嗜水气单胞菌(陈昶et al.,2009)和黄柱杆菌(陈明波,2015)，还有部分由寄生虫引起，以及少数病毒引起。在养殖过程中维持锦鲤的健康状态对于提高其观赏价值和延长寿命至关重要，所以应该对锦鲤采取“防重于治”的原则，减少养殖过程中锦鲤病原菌感染的出现。
Ladderlectin是最早从虹鳟血清中分离的一种凝集素，且可以在钙离子环境下集合多种病原菌，如维氏气单胞菌、嗜水气单胞菌、鲁氏耶尔森菌、和假单胞菌属(Young,K.M.et al.,2007),Bergljot Magnadottira等(2019)在大西洋鳙鲽血清中分离了一种新的ladderlectin，报道并在鳃、皮肤和肠道有较高的蛋白表达，表明其可能作为一种鱼类黏膜凝集素。
发明内容
为了解决现有技术存在的问题，本发明提供锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用。
第一方面，本发明提供锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用。
本发明进一步提供锦鲤Ladderlectin蛋白或其编码基因在抗病原菌锦鲤品种的培育中的应用。
进一步地，所述应用为通过提高所述锦鲤Ladderlectin蛋白的表达水平，提高锦鲤的抗病原菌感染能力。
进一步地，所述病原菌包括维氏气单胞菌、嗜水气单胞菌、温和气单胞菌或豚鼠气单胞菌中的一种或多种。
进一步地，所述锦鲤Ladderlectin蛋白包括如下任一项所述氨基酸序列：
i)如SEQ ID NO.1所示的氨基酸序列；
ii)由i)经过添加、替换、减少一个或多个氨基酸序列后具备相同或类似功能的氨基酸序列。
进一步地，所述锦鲤Ladderlectin蛋白由如下任一项所述的核苷酸序列编码得到：
i)如SEQ ID NO.2所示的核苷酸序列；
ii)由i)经过添加、替换、减少一个或多个核苷酸序列后具备相同或类似功能的核苷酸序列。
第二方面，本发明提供一种调控锦鲤抗病原菌感染能力的方法，包括：
通过调控锦鲤的基因组中锦鲤Ladderlectin蛋白的表达水平，调控锦鲤的抗病原菌感染能力；
所述锦鲤Ladderlectin蛋白包括如下任一项所述氨基酸序列：
i)如SEQ ID NO.1所示的氨基酸序列；
ii)由i)经过添加、替换、减少一个或多个氨基酸序列后具备相同或类似功能的氨基酸序列。
进一步地，通过提高所述锦鲤的基因组中Ladderlectin蛋白的编码基因的表达水平，提高所述锦鲤的抗病原菌感染的能力；
所述Ladderlectin蛋白的编码基因包括如下任一项所述的核苷酸序列：
i)如SEQ ID NO.2所示的核苷酸序列；
ii)由i)经过添加、替换、减少一个或多个核苷酸序列后具备相同或类似功能的核苷酸序列。
进一步地，将所述Ladderlectin蛋白的编码基因构建至载体上得到表达质粒；
将所述表达质粒转移至所述锦鲤体内进行表达。
进一步地，所述载体为pCDNA3.1、pCMV、pSI、pCGN、pCEP4、pcDNA4或pCI中的一种或多种。
本发明具备如下有益效果：
本发明针对维氏气单胞菌感染后的发病锦鲤进行研究得到一种感染病原菌后，表达水平发生显著变化的锦鲤Ladderlectin基因。锦鲤Ladderlectin基因和锦鲤的抗病原菌感染能力显著相关，当在锦鲤中过表达锦鲤Ladderlectin基因时，其抗病原菌的能力显著提高，存活率由62.5％显著提高至87.5％，这在锦鲤养殖领域具有重要意义。
附图说明
图1为本发明实施例1提供的锦鲤Ladderlectin基因在健康锦鲤不同组织中的表达水平对比示意图。
图2为本发明实施例2提供的维氏气单胞菌感染的锦鲤的脾脏组织中锦鲤Ladderlectin基因的表达水平变化示意图。
图3为本发明实施例2提供的维氏气单胞菌感染的锦鲤的头肾组织中锦鲤Ladderlectin基因的表达水平变化示意图。
图4为本发明实施例2提供的维氏气单胞菌感染的锦鲤的肝脏组织中锦鲤Ladderlectin基因的表达水平变化示意图。
图5为本发明实施例3提供的试验组和对照组的锦鲤脾脏的菌落数对比结果示意图。
图6为本发明实施例3提供的试验组和对照组的锦鲤存活率对比结果示意图。
图7为本发明实施例4提供的试验组和对照组的锦鲤脾脏的菌落数对比结果示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1锦鲤Ladderlectin基因的获得
为了研究病原菌感染锦鲤对鱼体免疫相关基因的激活上调情况，本实施例对维氏气单胞菌感染后的发病锦鲤的脾脏进行转录组测序，根据测序结果，从中找到一个表达水平显著上调(约9倍)的免疫相关的差异表达基因，并且进一步根据同源序列比对和蛋白结构模拟分析，推测该基因编码的蛋白质为Ladderlectin蛋白。
本实施例以锦鲤脾脏cDNA为模板，通过PCR扩增，获得锦鲤Ladderlectin基因的开发阅读框ORF，共489个核苷酸，编码得到一个由162个氨基酸组成的蛋白质，且不含信号肽。锦鲤Ladderlectin的氨基酸序列如SEQ ID NO.1所示，其ORF核苷酸序列如SEQ ID NO.2所示。
本实施例进一步研究了锦鲤Ladderlectin在健康锦鲤体内不同组织的相对表达量，用于研究Ladderlectin mRNA的组织分布，具体流程如下：
(1)随机选取健康锦鲤(体重约20g)的12个组织，分别为：鳃、眼、头肾、脾脏、肾、心脏、肌肉、皮肤、肝脏、血液、脑和肠。用Trizol法提取组织总RNA，用Takara反转录试剂盒反转录为cDNA。
(2)根据锦鲤Ladderlectin基因的cDNA序列设计用于荧光定量PCR的引物，引物序列如下：
qLadF：5’-CTTGCATCTATCCACAATCAACTC-3’；
qLadR：5’-GATCCGTCACTCCAAAACCAG-3’；
内参基因40S核糖体蛋白S11基因的引物序列如下：
qS11F：5’-CCGTGGGTGACATCGTTACA-3’；
qS11R：5’-TCAGGACATTGAACCTCACTGTCT-3’。
(3)采用ABI7500实时荧光定量PCR仪，检测上述12个组织中Ladderlectin的相对表达量。反应程序为：95℃，15s；95℃5s，59.6℃30s，72℃30s，40个循环。
得到如图1所示的结果，结果显示锦鲤Ladderlectin基因在上述12个组织中均有表达。其中，锦鲤Ladderlectin基因在皮肤中的表达量最高，其后依次是肌肉、肾脏、鳃、头肾和心脏。
实施例2锦鲤Ladderlectin基因在维氏气单胞菌感染后的表达变化
维氏气单胞菌(Aeromonas veronii，A.v)购自中国普通微生物菌种保藏管理中心(CGMCC，菌株编号：1.927)。将该菌种从超低温冰箱取出后，在LB平板划线，并于28℃培养箱培养，复苏菌种，挑取单菌落于LB液体培养基中扩大培养，离心收集菌体，用PBS重悬，调至5×106CFU/mL。
随机选取健康锦鲤(约20g)60尾，平均分为攻毒组和对照组，每组3个平行缸。攻毒组每尾锦鲤腹腔注射100μL维氏气单胞菌悬液，对照组每尾锦鲤腹腔注射100μL PBS。于感染前(0h)和感染后6h、12h、24h、48h、96h以及7d，分别从攻毒组和对照组随机选取6尾锦鲤，采集脾脏和头肾组织，置于液氮中速冻后存放于-80℃用于RNA提取，为减少个体误差，每2尾鱼进行混样。采用实时荧光定量PCR方法检测锦鲤Ladderlectin基因的相对表达量。
得到如图2-图4所示的结果，结果显示相对于对照组而言，攻毒组锦鲤在维氏气单胞菌感染后第6h，锦鲤ladderlectin基因在头肾组织表达出现显著上调，而后又显著下降的情况；而在脾脏和肝脏组织中，该基因自细菌感染后先发生下调反应，分别在感染后48h和96h，ladderlectin基因表达迅速升高，显著高于对照组(P＜0.05)。由此可以看出，在锦鲤感染维氏气单胞菌后头肾组织ladderlectin基因上调反应最迅速，且表达升高的程度最大，随后该基因在脾脏和肝脏也出现上调变化，表明该基因可能参与到锦鲤抗维氏气单胞菌感染的过程中。
实施例3锦鲤Ladderlectin基因在抗病原菌感染中的应用
1、构建锦鲤Ladderlectin基因真核表达质粒
(1)根据测序结果中锦鲤Ladderlectin基因的mRNA序列设计分别携带HindIII和XhoI酶切位点的扩增引物F:5’-CCAAGCTTGGGATGGGGATTTCTGCAGCATTCCTC-3’和R：5’-CCCTCGAGTCAGTGGTGGTGGTGGTGGTGGAAAT-3’。以锦鲤脾脏cDNA为模板，以F和R为引物进行PCR扩增，得到含有Ladderlectin基因ORF的cDNA片段。
PCR扩增反应体系(25μL)含有2.5μL 10×Ex buffer，2μLdNTP，F和R引物各0.3μmol，1U Ex taq酶(Takara)，500ng cDNA模板；PCR反应程序为：95℃5min；92℃15s，53℃25s，72℃30s(10个循环)；95℃15s，60℃25s，72℃30s(25个循环)；72℃7min。扩增产物经1％琼脂糖凝胶电泳分析后，用DNA凝胶回收试剂盒(Takara)进行回收纯化。
(2)将真核表达载体pCDNA3.1(Invitrogen)用限制性内切酶Hind III和XhoI酶切后胶回收，将其与上述纯化PCR产物用T4DNA连接酶4℃连接过夜，连接产物转化至大肠杆菌DH5α，在含氨苄霉素(50μg/mL)的LB琼脂平板上培养18小时，筛选阳性克隆进行测序，将正确重组了Ladderlectin片段的阳性菌提取质粒，命名为pCDNA3.1-Ladderle ctin。
2、质粒CDNA3.1-Ladderlectin对锦鲤病原菌的清除效果
(1)质粒注射：将pCDNA3.1-Ladderlectin在PBS缓冲液中稀释至200μg/mL，即得pCDNA3.1-Ladderlectin注射液。将空白质粒pCDNA3.1在PBS缓冲液中稀释至200μg/mL，即得对照质粒注射液。将8尾锦鲤(20g左右)随机分为2组，每组4尾，两组分别为对照组和试验组。将试验组的每尾鱼分别肌肉注射100μL质粒注射液(注射于背鳍根部前方肌肉丰富处)，将对照组的每尾鱼分别注射100μL对照质粒注射液。
(2)病原菌悬液制备：LB培养基培养维氏气单胞菌(CGMCC，1.927)至OD600为0.6-0.8，离心(8000g，2min)，倾倒上清，将菌体用PBS缓冲液悬浮，调至终浓度为1×107CFU/mL。
(3)攻毒感染：上述步骤(1)的注射流程后第72h，将对照组和试验组的每尾鱼注射100μL上述步骤(2)制备得到的菌悬液。在感染后第24h，用MS222麻醉锦鲤，解剖，取出脾脏组织，称重。按照10μL/mg加入灭菌PBS缓冲液，用无菌研磨棒研磨，取100μL脾脏匀浆液在LB平板上涂布，每个样品涂2个平板取平均值，待平板在28℃恒温箱培养24h后进行菌落计数，并进行统计分析。结果如图5和表1所示，试验组锦鲤脾脏的菌落数(6个/mg)显著(P＜0.05)低于对照组锦鲤脾脏的菌落数(44个/mg)。
表1锦鲤(20g)脾脏菌落计数(个/mg)
3、质粒CDNA3.1-Ladderlectin对锦鲤抵抗病原菌感染的保护效果
(1)质粒注射：将步骤(1)得到的真核表达质粒pCDNA3.1-Ladderlectin在PBS缓冲液中稀释至200μg/mL，即得pCDNA3.1-Ladderlectin注射液。将空白质粒pCDNA3.1在PBS缓冲液中稀释至200μg/mL，即得对照质粒注射液。将16尾锦鲤(20g左右)随机分为2组，每组8尾，两组分别为对照组和试验组。将试验组的每尾鱼分别肌肉注射100μL质粒注射液(注射于锦鲤背鳍基部前方肌肉丰富处)，将对照组的每尾鱼分别注射100μL对照质粒注射液。
(2)病原菌悬液制备：LB培养基培养维氏气单胞菌(CGMCC，1.927)至OD600为0.6-0.8，离心(8000g，2min)，倾倒上清，将菌体用PBS缓冲液悬浮，调至终浓度为5×107CFU/mL。
(3)在上述步骤(1)注射后第72h，将对照组和试验组的每尾鱼注射100μL上述步骤(2)制备得到的菌悬液。观察和统计7d内各组锦鲤的死亡情况，发现死鱼及时捞出，用Graphpad绘制生存曲线。
得到如图6所示的结果，结果表明，试验组7天的存活率为(87.5％)，明显高于对照组10天的存活率(62.5％)。这表明Ladderlectin能够增强锦鲤抵抗病原菌的侵染。
实验例4锦鲤Ladderlectin基因在抗嗜水气单胞菌感染中的应用
(1)质粒注射：将实施例3涉及的质粒pCDNA3.1-Ladderlectin在PBS缓冲液中稀释至200μg/mL，即得pCDNA3.1-Ladderlectin注射液。将空白质粒pCDNA3.1在PBS缓冲液中稀释至200μg/mL，即得对照质粒注射液。将6尾锦鲤(30g左右)随机分为2组，每组3尾，两组分别为对照组和试验组。将试验组的每尾鱼分别肌肉注射100μL质粒注射液(注射于背鳍根部前方肌肉丰富处)，将对照组的每尾鱼分别注射100μL对照质粒注射液。
(2)病原菌悬液制备：LB培养基培养嗜水气单胞菌(Aeromonashydrophila,A.h)NX830(保藏于中国农业部水生动物病原库，保藏编号：BYK20130805)至OD600为0.6-0.8，离心(8000g，2min)，倾倒上清，将菌体用PBS缓冲液悬浮，调至终浓度为1×108CFU/mL。
(3)攻毒感染：上述步骤(1)的注射流程后第72h，将对照组和试验组的每尾鱼注射100μL上述步骤(2)制备得到的菌悬液。在感染后第96h，用MS222麻醉锦鲤，解剖，取出脾脏组织，称重。按照10μL/mg加入灭菌PBS缓冲液，用无菌研磨棒研磨，取50μL脾脏匀浆液在LB平板上涂布，每个样品涂2个平板取平均值，待平板在28℃恒温箱培养24h后进行菌落计数，并进行统计分析。结果如图7和表2所示，试验组锦鲤脾脏的菌落数(7个/mg)显著(P＜0.05)低于对照组锦鲤脾脏的菌落数(81个/mg)。
表2锦鲤(30g)脾脏菌落计数(个/mg)
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 北京市水产科学研究所（国家淡水渔业工程技术研究中心）
<120> 锦鲤Ladderlectin蛋白或其编码基因在调控锦鲤抗病原菌感染中的应用
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Ala Ser Ile His Asn Gln Leu Thr His Asn Phe Leu Lys Thr Phe Val
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tttaaatatt tcagcgagtt aaaatcatgg gctgaagcag agaaacaatg tgttgaactt 180
ggaggaaacc ttgcatctat ccacaatcaa ctcactcaca atttcctaaa aacctttgtg 240
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Claims (8)
1.锦鲤Ladderlectin蛋白或其编码基因在制备调控锦鲤抗病原菌感染的药物中的应用；
所述锦鲤Ladderlectin蛋白的氨基酸序列如SEQ ID NO.1所示；
所述病原菌为维氏气单胞菌或嗜水气单胞菌。
2.锦鲤Ladderlectin蛋白或其编码基因在抗病原菌锦鲤品种的培育中的应用；
所述锦鲤Ladderlectin蛋白的氨基酸序列如SEQ ID NO.1所示；
所述病原菌为维氏气单胞菌或嗜水气单胞菌。
3.根据权利要求1所述的应用，其特征在于，所述应用为通过提高所述锦鲤Ladderlectin蛋白的表达水平，提高锦鲤的抗病原菌感染能力。
4.根据权利要求1所述的应用，其特征在于，所述锦鲤Ladderlectin蛋白由如SEQ IDNO.2所示的核苷酸序列编码得到。
5.根据权利要求2所述的应用，其特征在于，所述应用为通过提高所述锦鲤Ladderlectin蛋白的表达水平，提高锦鲤的抗病原菌感染能力。
6.根据权利要求2所述的应用，其特征在于，所述锦鲤Ladderlectin蛋白由如SEQ IDNO.2所示的核苷酸序列编码得到。
7.根据权利要求6所述的应用，其特征在于，将所述Ladderlectin蛋白的编码基因构建至载体上得到表达质粒；
将所述表达质粒转移至所述锦鲤体内进行表达。
8.根据权利要求7所述的应用，其特征在于，所述载体为pCDNA3.1、pCMV、pSI、pCGN、pCEP4、pcDNA4或pCI中的一种或多种。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066321A2 (en) * | 1998-06-12 | 1999-12-23 | University Of Guelph | Pattern recognition proteins with lectin homology from several animal species and method to use them for measure or modulate innate resistance against bacteria and other pathogenic agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066321A2 (en) * | 1998-06-12 | 1999-12-23 | University Of Guelph | Pattern recognition proteins with lectin homology from several animal species and method to use them for measure or modulate innate resistance against bacteria and other pathogenic agents |
Non-Patent Citations (3)
| Title |
|---|
| Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss);Karrie M. Young 等;Fish & Shellfish Immunology;第23卷;305-315 * |
| Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss);S. Russell 等;Fish & Shellfish Immunology;第26卷;154-163 * |
| ladderlectin-like [Cyprinus carpio];无;NCBI Reference Sequence: XP_018960242.1;1 * |
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