CN109593766A - 草鱼铁调素基因及其编码的重组蛋白和应用 - Google Patents
草鱼铁调素基因及其编码的重组蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种草鱼铁调素基因，具有如序列表SEQ ID NO：1所示的核苷酸序列。本发明还公开了表达草鱼铁调素基因的重组蛋白，具有如序列表SEQ ID NO：2所示的氨基酸序列。所述重组蛋白与饲料混匀后投喂草鱼，可显著降低草鱼感染柱状黄杆菌的死亡率，提高草鱼的免疫保护率，并且可以调节草鱼铁代谢和免疫指标，增强草鱼抵抗细菌感染的能力。
Description
技术领域
本发明属于基因工程领域，具体涉及一种草鱼铁调素基因，以及该基因编码的重组蛋白及其应用。
背景技术
随着限抗甚至无抗时代的到来，开发新型生物药物替代现有化学类抗生素的绿色防控技术已势在必行。由柱状黄杆菌(Flavobacterium columnare)感染所引起的鱼类柱形病对多种重要经济鱼类造成严重危害，在临床处置中尚缺乏有效措施。柱状黄杆菌分类地位上属于拟杆菌门、黄杆菌纲、黄杆菌目、黄杆菌科、黄杆菌属，它是一种在世界范围内广泛分布的对多种经济鱼类引发高死亡率的重要致病菌。由柱状黄杆菌引起的细菌性烂鳃病是草鱼养殖过程中发生最频繁的疾病，也是危害严重、较难治愈的疾病，该病死亡率较高，呈现发病快、死亡迅速、继发性感染能力强等特点。
在过去十年间，许多科研小组对细菌性烂鳃病进行了研究，目的是探索烂鳃病的病理起因，以找出宿主和病原体之间复杂的相互作用。目前研究认为，铁对于所有动物和几乎所有病原微生物都是必需的营养元素。在感染条件下，宿主和病原微生物对铁的利用存在激烈的竞争，病原菌能否从宿主中获得铁是决定病原菌在宿主体内生存或死亡的关键。因此，铁调控是控制细菌感染和提高宿主免疫力的关键环节。铁调素hepcidin是一种由肝脏特异表达的富含半胱氨酸的新型抗菌肽，属于防御素蛋白家族，是机体天然免疫的重要效应分子。目前研究发现铁调素hepcidin是调节铁代谢的重要枢纽，hepcidin发挥铁代谢的分子基础是：在病原体或炎症刺激下，在肝脏中被大量诱导表达的hepcidin与细胞膜(肝脏、肠和巨噬细胞)上唯一的铁输出蛋白-膜铁转运蛋白(ferroportin,Fpn)结合，引起Fpn的内化和降解，进而控制铁释放和巨噬细胞对铁的再循环，降低铁在血液循环中的含量，从而抑制病原菌的生长。
发明内容
本发明的目的之一是提供一种草鱼铁调素基因。从草鱼肝脏中提取总mRNA后反转录成cDNA，设计扩增引物，用RT-PCR技术扩增出目的基因，该基因具有如序列表SEQ IDNO：1所示的核苷酸序列。
本发明的目的之二是提供表达草鱼铁调素基因的重组蛋白。扩增出目的基因后，将pGEX-KG质粒与目的基因双酶切后连接，构建出重组质粒将其转入大肠杆菌BL21(DE3)表达菌株中，将重组菌株经诱导、纯化后得到重组蛋白，该重组蛋白具有如序列表SEQ IDNO：2所示的氨基酸序列。
本发明目的之三是提供草鱼铁调素重组蛋白的用途。其一处理法是将草鱼铁调素重组融合蛋白做成饲料添加剂做预防用，投喂10d后，实验动物再用柱状黄杆菌感染草鱼进行攻毒实验，发现与对照组(投喂普通饲料)相比，铁调素预防组可以明显减低草鱼的死亡率，说明重组蛋白对柱状黄杆菌所致疾病具有保护作用。
其二处理法是将草鱼铁调素重组融合蛋白做成饲料添加剂做治疗用，实验动物先用柱状黄杆菌感染草鱼进行攻毒实验，再投喂草鱼铁调素重组融合蛋白7天，发现与对照组(投喂普通饲料)相比，铁调素治疗组可以明显减低草鱼的死亡率，说明铁调素重组蛋白对柱状黄杆菌所致疾病具有治疗作用。
其三处理法是将铁调素重组融合蛋白注射到草鱼体内，用空载体作为对照，而后感染柱状黄杆菌，检测铁调素重组融合蛋白对鱼的保护作用，结果显示，与对照组相比，铁调素处理组同样可以降低草鱼感染柱状黄杆菌的死亡率，另外，感染后对铁调节相关基因hepcidin和Ferroportin具有明显调节作用，而且免疫相关基因TNF-α、IgD、IgM、IL-8、MHCⅡ的表达明显高于对照组。
附图说明
图1为草鱼铁调素基因扩增电泳图，1：克隆引物从草鱼肝脏cDNA中扩增的目的基因；M：DNA分子量标准。
图2利用同源重组方法扩增草鱼铁调素重组载体PCR鉴定电泳图，M：DNA分子量标准；1-5：表达引物从cDNA中扩增目的基因。
图3为pGEX-KG定性分析的SDS-PAGE电泳结果，M：蛋白质分子质量标准；2：未诱导的pGEX-KG-hep；3：IPTG诱导的pGEX-KG-hep表达产物。
图4为草鱼铁调素处理后对铁代谢相关基因hepcidin、ferroportin(Fpn)表达水平的调节，处理组为带GST标签的空载体组(GST-Tag)和铁调素处理组(hepcidin-GST)。
图5为草鱼铁调素处理后14天对草鱼免疫基因IgD、IgM、IL-8、MHCII和IL-1β表达水平的调节，分为对照组和草鱼铁调素处理组。
具体实施方式
实施例1：草鱼铁调素基因的克隆及序列分析
1)Trizol法提取总RNA
使用麻醉剂MS-222处理草鱼后，解剖取其肝脏组织50-100mg，放入冰浴的玻璃匀浆器内，加入1.0mL Trizol，置于冰上研磨，将研磨液转移至1.5mL EP管中，室温静置5min，已裂解好的研磨液中加入氯仿、异丙醇抽提，再加入1mL 75％乙醇洗涤沉淀，溶于无RNA酶的双蒸水中备用。
2)cDNA第一条链的合成及PCR扩增
以草鱼肝脏总RNA为模板，反转录合成第一条链，反转录体系为20μL，根据草鱼基因组数据上草鱼铁调素序列信息，比对鱼类铁调素核苷酸序列的保守性，用Primer 5.0软件在cDNA序列开放阅读框的上下游区域，设计一对特异性引物引物(铁调素F，铁调素R)：引物如下：
铁调素-F：5’TTCGGTACCATGAAGTGCGCACACGTGGC 3’
铁调素-R：5’GGCAAGCTTGAATTTACAGCAATATCCAC 3
引物由武汉擎科测序服务公司合成合成。
即：
PCR扩增反应条件为：94℃预变性3min；94℃变性30秒，55℃退火30秒，72℃延伸30秒，进行35个循环反应；循环结束后在72℃再延伸10min。
扩增结束后，取5μL PCR扩增产物，用10×核酸上样缓冲液点样，1.2％琼脂糖凝胶，1×TAE，120V，电泳20min观察结果，以DL2000Marker作为参照，结果显示在250bp左右有一条明亮的条带，其大小符合预期(见图1)。
3)目的基因的克隆、筛选与测序
将PCR扩增为阳性的单克隆，取1mL菌液送武汉擎科测序服务公司，用双脱氧链终止法测序。用BLAST软件对所测定序列进行分析，该基因有完整的开放阅读框(ORF)(282bp)，与已发表的鱼类铁调素序列进行同源性比较，获得草鱼铁调素ORF序列，如序列表SEQ ID NO：1所示的序列。
序列号：SEQ ID NO：1
序列长度：282bp
序列类型：cDNA
来源：草鱼铁调素
序列特征：有正确的开放阅读框(ORF)：282bp；决定位置：起始、终止密码子存在位置：ATG，1位；TAA，280位。
实施例2：草鱼铁调素原核表达载体构建及在大肠杆菌中的诱导表达
1)草鱼铁调素PCR产物与pGEX-KG双酶切
将草鱼铁调素PCR产物和原核表达载体质粒pGEX-KG质粒进行双酶切，反应体系为50μL，即：
37℃酶切反应3h，用1.2％琼脂糖凝胶电泳，按照OMEGA公司DNA凝胶回收试剂盒操作将目的基因与表达载体分别回收。-20℃保存。
2)克隆质粒的构建与筛选
取双酶切后的目的基因4μL与表达载体pGEX-KG 1μL混合后，加入5μL Sultion I，混匀后16℃连接4h。连接产物转化至大肠杆菌DH5α中。转化步骤为：取10μL连接产物，无菌条件下加入大肠杆菌DH5α感受态细胞中，用移液器吹打混匀，冰浴放置30分钟。42℃水浴，热激90秒后立即冰浴5分钟使之冷却。取500μL LB液体培养基加入，150r/min37℃温和振荡1h，使细菌恢复抗药性。取150μL菌液涂布于含AMP(100μg/mL)的LB琼脂平板上。倒置平皿于37℃恒温培养箱培养12-16h，挑取白色菌落接种于含100μg/mL氨苄的LB液体培养基中，37℃振荡培养12-16h后鉴定。
3)重组克隆质粒的PCR鉴定及测序
PCR扩增反应条件和琼脂糖电泳鉴定同实施例1，将PCR扩增为阳性的单克隆，见图2，M为DL2000分子量标准(购自大连宝生物)，1-5为PCR扩增的单克隆，可以发现，在250bp左右有一条预期的PCR条带。取1mL阳性菌液送武汉擎科测序服务公司，用双脱氧链终止法测序。用BLAST软件对所测定序列进行分析，并与草鱼铁调素序列进行同源性比较。将测序正确的质粒命名为pGEX-KG-铁调素。
3)原核重组表达质粒的诱导表达
首先将原核重组表达质粒pGEX-KG-铁调素按上述方法转化至大肠杆菌感受态细胞表达菌株BL21(DE3)，挑取PCR鉴定为阳性重组表达菌进行诱导表达。同时转化pGEX-KG空载体质粒至表达菌株BL21(DE3)。活化带有pGEX-KG-Hepcidin质粒的菌液(12h)，转接2h，加IPTG终浓度1.0mM，16℃诱导12h。活化带有pGEX-KG空载体的菌液(12h)，转接2h，加IPTG终浓度1.0mM，16℃诱导12h，取诱导表达菌液，12000g离心1min，弃去上清，用PBS洗涤沉淀后重悬。取部分重悬菌液加入1/5体积的5×SDS蛋白上样缓冲液并混匀；同时取重悬菌液冰浴超声破碎，高速离心分离上清和沉淀，并加入蛋白上样缓冲液并混匀。样品沸水浴15min，使其充分变性。
配制SDS-PAGE(聚丙烯酰胺凝胶电泳)12％的分离胶和5％的浓缩胶。先将12％的分离胶注入玻璃板间，待分离胶凝固后，再加入注入配好的5％浓缩胶，并将样品槽模板插入浓缩胶内。待浓缩胶凝固后，加入配备好的甘氨酸电泳缓冲液。向每个样品槽中加入5-10μL处理过的蛋白样品，以蛋白标准marker作为参照。先用低电压80V运行，当样品进入到分离胶后调整电压到120V继续电泳3-4h。用配制好的考马斯亮蓝染色液染色1h后用脱色液脱色，利用凝胶成像仪观察并照相保存，结果如图3所示，与未诱导相比，在16℃浓度为1mM的IPTG诱导12h后在35KD处，有一条明显的条带，说明草鱼铁调素重组融合蛋白得到重组表达。
实施例3：新型免疫增强剂草鱼铁调素纯化重组蛋白的制备
1)草鱼铁调素在生物发酵罐中的大量诱导表达
重组草鱼铁调素菌株在100L发酵罐中进行连续培养，工作体积60L。重组草鱼铁调素菌株的发酵为高细胞高密度补料发酵，发酵过程分为菌株培养阶段和诱导表达阶段。
培养过程：
(1)挑保种单克隆菌液，按1:1000接种于25mL的加入氨苄青霉素抗性LB培养基中，置于50mL的摇瓶内，37℃,150rpm摇床过夜活化培养12h。
(2)将活化菌液按1:100比例接种600mL加入氨苄青霉素抗性LB培养基中，置于1L的摇瓶内，37℃，150rpm摇床过夜培养12h，菌体浓度可达到2～5个OD 600单位。
(3)向100L发酵罐中加入60L LB培养基，启动发酵罐自动高温灭菌程序20min，待温度降至37摄氏度时，手动加入600mL上述活化菌液,启动发酵罐发酵程序，控制温度37℃，150rpm培养5h，待菌液浓度达到2-5个OD 600单位后，调节发酵温度降至25℃，手动加入1mM的IPTG诱导3h。
(4)诱导结束后20000r/min离心机离心去除上清液，保存固体菌液于-80℃冰箱。
2)草鱼铁调素重组蛋白的纯化
用Buffer A(PBS缓冲液)重悬菌体，进行高压破碎3min，将破碎菌液在4℃12000r/min离心40min，收集上清；使用100mL PBS缓冲液清洗层析柱，将上清液过柱2次后，使用100mLPBS缓冲液清洗一次，将杂蛋白冲洗干净，再用Buffer B(PBS缓冲液，谷胱甘肽10mM)将目的蛋白洗脱出来，并进行收集；最后使用Buffer A4℃过夜透析，将透析液进行冷冻干燥，即得纯化蛋白。
实施例4：草鱼铁调素重组蛋白作为饲料添加剂的抗菌试验
柱状黄杆菌是从中国患病的黄颡鱼中分离到的一株基因运动Ⅰ型菌株，将细菌进行培养后制成5×109CFU/ml的菌悬液。
从武汉大北农水产科技有限公司获得基础日粮，将纯化后的草鱼铁调素重组蛋白冻干粉添加至粉碎后的基础日粮中，制成含十万分之6草鱼铁调素重组蛋白的颗粒饲料，
取草鱼，随机分成对照组、预防组和治疗组，每组55尾。攻毒前，对照组和治疗组投喂基础日粮，预防组投喂含十万分之6重组蛋白的饲料，每天早晚各投喂一次，平均1g/天*条。饲喂10天后，将三组草鱼用柱状黄杆菌进行攻毒实验，以5×109CFU/ml为攻毒浓度，每尾鱼注射300μl柱状黄杆菌悬液。攻毒后，对照组和预防组投喂基础日粮，治疗组组投喂含十万分之6重组蛋白的饲料，观察各处理组死亡率，结果如表1所示。
表1草鱼感染柱状黄杆菌后的死亡率
| 攻毒后时间 | 对照组 | 治疗组 | 预防组 |
| 24h | 45％ | 21.5％ | 22.5％ |
| 30h | 55.5％ | 36.5％ | 37％ |
| 36h | 56％ | 43.5％ | 44％ |
以上结果表明，草鱼铁调素重组蛋白能明显降低草鱼感染柱状黄杆菌后的死亡率，说明对感染柱状黄杆菌后的草鱼具有保护作用。
实施例5：注射草鱼铁调素对草鱼感染柱状黄杆菌的免疫保护率实验
将柱状黄杆菌制成5×106CFU/ml的菌悬液，用于草鱼注射攻毒。
将实施例3制备的草鱼铁调素纯化重组蛋白用注射用水溶解，制成浓度为400μg/mL的注射剂；同时将表达pGEX-KG空载体质粒的重组蛋白(GST蛋白)用注射用水溶解，制成浓度为400μg/mL的注射剂100μL，作为对照。
将草鱼随机分为对照组和预防组，每组20尾鱼，治疗组注射草鱼铁调素纯化重组蛋白，对照组注射GST蛋白，注射剂量均为100μl。24h后再给草鱼注射柱状黄杆菌菌悬液，剂量为300μL。
将注射完的草鱼转移回养殖鱼缸中，感染后不喂食，观察鱼的状况14d，记录死亡情况，结果见表2。同时每个处理组取三条草鱼，解剖后取肝胰脏等组织器官样品，提取肝胰脏总RNA，采用荧光定量RT-PCR技术检测铁代谢相关基因hepcidin、ferroportin(Fpn)表达水平，以及IL-1β、IL-8、IgD、IgM、MHCⅡ等免疫相关基因的表达水平，结果见图4和图5。
结果显示，与对照组相比，草鱼铁调素注射组可以显著降低草鱼感染柱状黄杆菌的死亡率，而且草鱼铁调素注射组铁代谢基因hepcidin、ferroportin(Fpn)和免疫相关基因IL-1β、IL-8、IgM、MHCⅡ的mRNA的表达水平均明显高于对照组，说明草鱼铁调素可以明显增强草鱼的免疫抵抗力。
表2不同处理组对草鱼感染柱状黄杆菌的死亡率
| 攻毒后时间 | 对照组 | 预防组 |
| 2d | 28％ | 20％ |
| 3d | 44％ | 28％ |
| 4d | 52％ | 32％ |
| 14天 | 52％ | 32％ |
序列表
<110> 武汉大北农水产科技有限公司
<120> 草鱼铁调素基因及其编码的重组蛋白和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 282
<212> DNA
<213> 草鱼铁调素(Ctenopharyngodon idella hepcidin)
<400> 1
atgaagtgcg cacacgtggc tctcgctgct gcagtcgtcc tcgcatgcgt ctgcatcctt 60
cagaccgcag ccgttccgtt cgtacagcag gagcaggatg agcatcaaat ggagattgaa 120
acaccacagc agaacgaaca ctcgacagaa acaacagaaa cacagggaca aacaaatccc 180
ctggcatttt tcaggacaaa acgtcaaagc catctttccc tgtgcagata ctgctgcaac 240
tgctgtcgta acaaaggctg tggatattgc tgtaaattca tg 282
<210> 2
<211> 94
<212> PRT
<213> 草鱼铁调素(Ctenopharyngodon idella hepcidin)
<400> 2
Met Lys Cys Ala His Val Ala Leu Ala Ala Ala Val Val Leu Ala Cys
1 5 10 15
Val Cys Ile Leu Gln Thr Ala Ala Val Pro Phe Val Gln Gln Glu Gln
20 25 30
Asp Glu His Gln Met Glu Ile Glu Thr Pro Gln Gln Asn Glu His Ser
35 40 45
Thr Glu Thr Thr Glu Thr Gln Gly Gln Thr Asn Pro Leu Ala Phe Phe
50 55 60
Arg Thr Lys Arg Gln Ser His Leu Ser Leu Cys Arg Tyr Cys Cys Asn
65 70 75 80
Cys Cys Arg Asn Lys Gly Cys Gly Tyr Cys Cys Lys Phe Met
85 90
Claims (6)
1.草鱼铁调素基因，具有如序列表SEQ ID NO：1所示的核苷酸序列。
2.表达权利要求1所述草鱼铁调素基因的重组蛋白，具有如序列表SEQ ID NO：2所示的氨基酸序列。
3.权利要求2所述的重组蛋白在制备防治草鱼感染柱状黄杆菌所致疾病的药物中的应用。
4.权利要求2所述的重组蛋白用作草鱼饲料添加剂的用途，所述饲料添加剂用于防治草鱼感染柱状黄杆菌所致疾病。
5.一种具有防治感染柱状黄杆菌所致疾病功能的草鱼饲料，其特征在于：所述饲料中含有权利要求2所述的重组蛋白。
6.如权利要求5所述的具有防治感染柱状黄杆菌所致疾病功能的草鱼饲料，其特征在于：所述重组蛋白占饲料总重量的万分之一到万分之十。
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