CN116144667B - 卵形鲳鲹胰岛素样生长因子结合蛋白1基因、蛋白及应用 - Google Patents
卵形鲳鲹胰岛素样生长因子结合蛋白1基因、蛋白及应用 Download PDFInfo
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Abstract
本发明涉及卵形鲳鲹胰岛素样生长因子结合蛋白1基因、蛋白及应用,属于分子生物学领域,所述卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)的基因的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示,本发明还提供了含有所述SEQ ID NO.1的真核表达载体、重组菌,所述卵形鲳鲹胰岛素样生长因子结合蛋白1。本发明所述卵形鲳鲹胰岛素样生长因子结合蛋白1真核表达质粒注射鱼体后能够促进炎症因子如IL‑8、IL‑10、IL‑1β和TNF‑α的表达,并且能够显著降低细菌侵染后鱼体内的细菌载量。
Description
技术领域
本发明涉及分子生物学领域,具体的说是一种卵形鲳鲹胰岛素样生长因子结合蛋白1(insulin-like growth factorbinding protein 1,IGFBP1)基因、蛋白及应用。
背景技术
卵形鲳鲹(Trachinotus ovatus)是我国南方名贵的海产鱼类之一,具有较高的经济价值。但是近年养殖过程中病害频发,造成巨大的经济损失。
IGFBP1属于IGFBP家族,是一类可以调节IGF配体和IGF受体相互结合的蛋白质,在调节IGFs通路中发挥重要作用。在哺乳动物中,IGFBP1除了可以促进肿瘤细胞凋亡外,还参与细胞增殖,调节免疫和宿主对病原体的免疫反应。然而鱼类中IGFBP1的研究还较少,仅在斑马鱼(Danio rerio)、草鱼(Ctenopharyngodon idellus)、建鲤(Cyprinus carpiovarJian)、牙鲆(Paralichthys olivaceus)、花鲈(Lateolabrax maculatus)、虹鳟(Oncorhynchus mykiss)几种鱼类中克隆出IGFBP1cDNA基因,但对其生物学功能研究开展的很少。卵形鲳鲹中目前尚无相关研究。
发明内容
本发明要解决的技术问题在于提供一种卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)的基因、蛋白及其在卵形鲳鲹抗细菌性疾病功能上的应用。
本发明是通过如下技术方案来实现的:
本发明提供一种卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)的基因,其cDNA核苷酸序列如SEQ ID NO.1所示:
atgcctggattacacgag aggcttacatttgtggcaggagcggctctggctgtcttagtcatggtgcggtcatccccagtggtgggaccg gagcctatccactgtgccccctgcactcaggagaaacggaacaactgtcctgccgtcccagcagagtgcaggcaggtgctgagggagcctggctgcggctgctgcatggcctgcgctctggagagaggggcatcctgtggagtccacacagcccactgtggtgagggtctccgctgcgctcccaggcctggtgaggccagacctctccacgctttgaccagggggcagggggtctgcactgaggacttgggccaagaggaaactgagggagtccccgaccacagctccttgcaccacttgctgggtctcaaccttccctttgaccaccaagacactgctgagggccacgagagcatcaaggccaaggccaacgctatccgcaacaagctggtacaacagggaccctgtcacattgaactgcacacagcactggacatgatagccagctctcagcagaaactaggagagaagttcacaactttctacctccccaactgtgacaagtacggcttctacaaggccaagcagtgtgagtcctctctggttggtccacccgctcgctgctggtgtgtctctccctggaatgggaagaagatcccaggatcgagtgacctgctccttgattcagagtgtcatcaagaagtcacacactaa
所述卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)的氨基酸序列如SEQ IDNO.2所示:
MPGLHERLTFVAGAALAVLVMVRSSPVVGPEPIHCAPCTQEKRNNCPAVPAECRQVLREPGCGCCMACALERGASCGVHTAHCGEGLRCAPRPGEARPLHALTRGQGVCTEDLGQEETEGVPDHSSLHHLLGLNLPFDHQDTAEGHESIKAKANAIRNKLVQQGPCHIELHTALDMIASSQQKLGEKFTTFYLPNCDKYGFYKAKQCESSLVGPPARCWCVSPWNGKKIPGSSDLLLDSECHQEVTH。
本发明还提供一种重组真核表达载体,所述重组载体中含有如SEQ ID NO.1所示的核苷酸序列。
本发明还提供一种重组菌,所述重组菌中含有所述重组载体。
本发明还提供所述卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)基因、蛋白、重组载体和重组菌在制备卵形鲳鲹抗细菌感染的制剂中的应用,所述细菌为哈维氏弧菌(Vibrio harveyi)。
本发明所述的卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)基因的克隆方法是:提取卵形鲳鲹肝脏组织总RNA,将RNA反转录为cDNA,再以cDNA为模板,以TroIGFBP1-F/TroIGFBP1-R为引物,通过PCR扩增。所述引物的序列为:TroIGFBP1-F:5’-gatatcgccaccATGCCTGGATTACACGAG-3’;TroIGFBP1-R:5’-gatatcGTGTGTGACTTCTTGATGACAC-3’。
PCR反应条件:95℃预变性5min,95℃30s,58℃30s,72℃1min,共35个循环,最后72℃延伸5min。
所述卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1基因的真核表达质粒pTroIGFBP1构建如下:IGFBP1克隆产物经纯化后与pEASYS-T1 Simple载体连接,转化至大肠杆菌DH5α感受态中,挑取阳性菌落进行PCR检测及测序验证;检测正确后提取质粒,用限制性内切酶EcoR V酶切回收符合目的基因大小的片段;提取改造后的真核表达载体pCN3质粒同时用限制性内切酶Sma I对pCN3载体进行酶切。利用T4 DNA连接酶将上述747bp回收片段与线性化的pCN3质粒连接,构建重组质粒;重组质粒经测序验证含有IGFBP1基因,将其命名为pTroIGFBP1。
本发明与现有技术相比的有益效果:
本发明的卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)真核表达质粒注射鱼体后能够促进炎症因子如IL-8、IL-10、IL-1β和TNF-α的表达,并且能够显著降低细菌侵染后鱼体内的细菌载量。
附图说明
图1为本发明实施提供的对卵形鲳鲹注射真核表达质粒pTroIGFBP15天后对免疫基因IL-8、IL-10、IL-1β和TNF-α的表达影响:A表示肝脏组织中免疫基因相对表达量;B表示脾脏组织中免疫基因相对表达量;C表示头肾组织中免疫基因相对表达量。
图2为本发明实施提供的卵形鲳鲹IGFBP1基因过量表达后体内抑制哈维氏弧菌的感染后的肝脏、脾脏和头肾组织中的细菌载量;A为肝脏、B为脾脏,C为头肾。
具体实施方式
下面通过实施例来对本发明的技术方案作进一步解释,但本发明的技术方案不受实施例以任何形式上的限制。
实施例1:卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)基因的克隆与真核表达质粒的构建。
(1)卵形鲳鲹肝脏组织总RNA使用Promega公司的Super Total RNAExtractionKit按照操作步骤提取。
(2)cDNA第一链的合成:以上述提取的总RNA为模板,用Promega公司的逆转录试剂盒RT Master Mix Kit进行cDNA第一链的合成。保存于-20℃备用。
(3)卵形鲳鲹胰岛素样生长因子结合蛋白1(IGFBP1)基因的克隆
1)根据实验室卵形鲳鲹转录组库中胰岛素样生长因子结合蛋白1(IGFBP1)的编码区序列设计引物:TroIGFBP1-F:5’-gatatcgccaccATGCCTGGATTACACGA G-3’;TroIGFBP1-R:5’-gatatcGTGTGTGACTTCTTGATGACAC-3’用于TroIGF BP1基因的扩增。
2)按照北京全式金TransTaq高保真DNA聚合酶说明书配置PCR混合液,PCR条件为:94℃5min预变性;然后94℃30s,60℃30s,72℃60s,30个循环;最后72℃延伸7min。
3)PCR反应结束后,使用1.2%的琼脂糖凝胶检测扩增产物,然后使用南京诺唯赞生物科技股份有限公司的Gel DNA Extraction Mini Kit对PCR产物进行回收。
4)连接T载体连接:按照北京全式金-T1simple Cloning Vector使用说明书操作,置于室温连接10min。将连接产物转化至大肠杆菌DH5α感受态,在含Amp抗生素的LB培养基对克隆进行筛选。从平板挑选单克隆进行PCR检测,及测序,获得阳性克隆,命名为pTroIGFBP1。
(3)真核表达质粒pTroIGFBP1的构建:
1)质粒提取:提前准备好上述测序正确的含完整ORF序列的pTroIGFBP1质粒和真核表达载体pCN3质粒的菌液,使用Omega公司的Plasmid Mini Kit I质粒提取试剂盒进行质粒提取。
2)酶切:将上述提取的质粒(pCN3和pTroIGFBP1),分别用SmaⅠ和EcoRⅤ于37℃水浴30min进行酶切,随后向pCN3载体的酶切体系中加入3μLFastAP继续孵育10min。
3)胶回收:使用南京诺唯赞生物科技股份有限公司的Gel DNAExtraction Mini Kit试剂盒对上述酶切产物进行回收。
4)连接:用T4 DNA连接酶将上述TroIGFBP1和pCN3线性化片段进行16℃过夜连接。
5)转化:将保存于-80℃的DH5α感受态细胞拿出并放置在冰上至溶融状态,加入上述连接产物,冰浴30min。42℃热激90s,冰浴5min。加入800μL的LB液体培养基,37℃,180rpm振荡培养1h。取100μL涂布于含Amp抗生素LB固体平板,37℃倒置培养过夜。
6)阳性克隆检测:挑单菌落至10μL无菌ddH2O中,吹打混匀。取1μL含菌的ddH2O作为模板,用引物TroIGFBP1-F/CN-R、TroIGFBP1-R/CN-F进行PCR扩增。1.5%琼脂糖凝胶电泳检测PCR产物,将检测阳性的菌株培养保种,测序,将测序正确的菌株放入-80℃保存。
上面所述引物为:
TroIGFBP1-F:5’-gatatcgccaccATGCCTGGATTACACGAG-3’
TroIGFBP1-R:5’-gatatcGTGTGTGACTTCTTGATGACAC-3’
CN-F:5’-CTTGCGTTTCTGATAGGCACCTA-3’
CN-R:5’-TGCGGGCCTCTTCGCTATT-3’
实施例2:真核表达质粒pTroIGFBP1对卵形鲳鲹IL-8、IL-10、TGF-β和TNF-α表达的影响。
(1)质粒的注射:提取的pTroIGFBP1、pCN3无内毒素质粒用PBS稀释至150μg/mL。将15尾卵形鲳鲹随机分为三组,每组5尾。三组分别肌肉注射上述稀释好的pTroIGFBP1、pCN3和PBS各100μL。
(2)基因表达检测
质粒注射后第5天,取三组鱼的肝脏、脾脏和头肾组织,提取RNA并反转成cDNA用于qRT-PCR检测IL-8、IL-10、IL-1β和TNF-α的表达。
结果表明,pTroIGFBP1注射组鱼的肝脏、脾脏和头肾的炎症因子IL-8、IL-10、IL-1β和TNF-α的表达量显著高于对照组;但pCN3组各组织的IL-8、IL-10、IL-1β和TNF-α的表达量与对照组无显著差异。
实施例3:卵形鲳鲹TroIGFBP1真核表达质粒的应用。
(1)质粒的注射:
提取的pTroIGFBP1、pCN3无内毒素质粒用PBS稀释至150μg/mL。将45尾卵形鲳鲹随机分为三组,每组15尾。三组分别肌肉注射上述稀释好的pTroIGFBP1、pCN3和PBS各100μL。
(2)菌悬液的制备:
在LB培养基中培养哈维氏弧菌至OD600为0.8,然后5000g离心10min。收集菌体,将其悬浮于PBS中至终浓度为5×106cfu/mL。
(3)攻毒:
卵形鲳鲹注射质粒后第5天,将三组的每条鱼注射100μL上述细菌悬液。分别在感染6h、9h、12h后,取鱼肝脏、脾脏和头肾组织在1mLPBS中匀浆,将100μL匀浆液涂布于LB平板。将平板置于30℃培养24h,计算菌落数。结果可见,注射pTroIGFBP1的肝脏、脾脏和头肾的细菌数显著低于对照组,但pCN3组与PBS组的组织中细菌数并无显著差异(图2)。
这些结果表明,在卵形鲳鲹中过量表达pTroIGFBP1能够显著增强其抵抗细菌的侵染能力。
Claims (8)
1.一种卵形鲳鲹胰岛素样生长因子结合蛋白1的基因,其特征在于,所述基因的cDNA核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述基因编码的卵形鲳鲹胰岛素样生长因子结合蛋白1,其特征在于,所述结合蛋白1的氨基酸序列如SEQ ID NO.2所示。
3.一种重组真核表达载体,其特征在于,所述载体中含有权利要求1中所述的SEQ IDNO.1所示的核苷酸序列。
4.一种重组菌,其特征在于,所述重组菌中含有权利要求3所述重组真核表达载体。
5.权利要求1所述卵形鲳鲹胰岛素样生长因子结合蛋白1基因在制备卵形鲳鲹抗细菌感染的制剂中的应用,所述细菌为哈维氏弧菌。
6.权利要求2所述卵形鲳鲹胰岛素样生长因子结合蛋白1在制备卵形鲳鲹抗细菌感染的制剂中的应用,所述细菌为哈维氏弧菌。
7.权利要求3所述重组真核表达载体在制备卵形鲳鲹抗细菌感染的制剂中的应用,所述细菌为哈维氏弧菌。
8.权利要求4所述重组菌在制备卵形鲳鲹抗细菌感染的制剂中的应用,所述细菌为哈维氏弧菌。
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