CN112625118B - 一种金钱鱼促生殖细胞成熟基因igf3及其应用 - Google Patents
一种金钱鱼促生殖细胞成熟基因igf3及其应用 Download PDFInfo
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Abstract
本发明公开了一种金钱鱼促生殖细胞成熟基因IGF3，它的核苷酸序列如SEQ IDNO：3所示。还公开了编码所述基因IGF3的蛋白，它的氨基酸序列如SEQ IDNO：4所示。以及所述金钱鱼促生殖细胞成熟基因IGF3的cDNA，它的核苷酸序列如SEQ IDNO：1所示。和编码所述基因IGF3的cDNA的蛋白，它的氨基酸序列如SEQ IDNO：2所示。以及包括所述基因IGF3的表达载体、金钱鱼IGF3重组蛋白及其制备方法，和上述基因IGF3、编码所述基因IGF3的蛋白或所述的重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用，开发的IGF3重组蛋白有望应用于研发新型鱼类催熟催产制剂。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种金钱鱼促生殖细胞成熟基因IGF3及其应用。
技术背景
繁殖和生长是生物最基本的特征；它们之间有着密切的联系，但又是相互区别的。胰岛素样生长因子(IGFs)是调节生长和生殖轴的关键因素之一。IGFs由3种配体(IGF1、IGF2和最新发现的IGF3)、2种受体(IGF1R、IGF2R)和6种IGF结合蛋白(IGFBP)组成。在鱼类中，IGF1和IGF2主要是发挥促进生长的生理功能，但自从在鱼类卵巢中发现特异表达的IGF3以来，IGFs在鱼类性腺发育中的作用受到了广泛的关注。
IGF3是鱼类特有的IGF类型，最先是在斑马鱼和罗非鱼中克隆鉴定出来，并发现IGF3在性腺中特异性表达。随后，在欧洲鲈、虹鳟鱼、黄姑鱼、草鱼、青鳉、南方鲶以及石斑鱼等鱼类同样证实了IGF3的存在，并表明IGF3与性腺的发育相关，提示了IGF3在鱼类性腺发育中具有重要的生理功能。进一步研究发现，IGF3重组蛋白能在体外诱导斑马鱼卵母细胞成熟。最终证实了IGF3在诱导卵母细胞成熟过程中的重要生理功能，是一个诱导卵母细胞成熟的新型关键因子，由此开辟了研究鱼类卵子成熟的新通路。
随后，在探讨IGF3诱导卵母细胞成熟的机理相继展开。目前研究发现IGF3和LH具有显著的关联性。在斑马鱼的研究中发现，LH能正调控IGF3的表达，相关的研究在罗非鱼中同样发现LH激活的PKA通路同样能激活IGF3的启动子，并激活IGF3的表达。此外，借助基因敲除技术在斑马鱼中敲除LHβ基因后，IGF3的表达量显著下降，并且IGF3重组蛋白能够挽救LHβ基因敲除后导致的卵子不能成熟。同时，研究表明IGF3在斑马鱼卵巢发育过程中的表达模式和LH受体的表达模式相似，进而提示了IGF3是介导LH信号诱导鱼类卵母细胞成熟的重要衔接因子。
金钱鱼(Scatophagus argus)隶属鲈形目、金钱鱼科、金钱鱼属，俗称金鼓。作为南方名特优名贵海水经济鱼类，金钱鱼是名贵的水族观赏鱼类之一，同时金钱鱼味道鲜美，因此广受消费者青睐。2015年本申请所在团队突破金钱鱼全人工繁殖技术，但实际生产中发现金钱鱼和其他海水鱼类一样，传统的人工催熟效果不明显，极大限制了大规模化苗种的培育。因此，如何突破高效的人工催熟催产是海水鱼类面临的巨大挑战。IGF3作为基因表达的蛋白产物，将具有更便利的应用空间，具有传统的激素诱导卵母细胞成熟无可比拟的优势。
发明内容
本发明的目的在于提供一种金钱鱼促生殖细胞成熟基因IGF3，其编码的蛋白，cDNA、所述cDNA编码的蛋白以及包含所述基因IGF3的表达载体。
本发明的目的还在于提供一种金钱鱼IGF3重组蛋白及其制备方法。
本发明的最后一个目的在于提供上述基因IGF3，其编码的蛋白或所述重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
本发明的上述第一个目的可以通过以下技术方案来实现：一种金钱鱼促生殖细胞成熟基因IGF3，它的核苷酸序列如SEQ IDNO：3所示。
编码所述基因IGF3的蛋白，它的氨基酸序列如SEQ IDNO：4所示。
所述金钱鱼促生殖细胞成熟基因IGF3的cDNA，它的核苷酸序列如SEQ IDNO：1所示。
本发明利用反转录PCR技术从金钱鱼中克隆得到新型胰岛素类生长因子(Insulin-ike growth factor 3，IGF3)的cDNA序列。
编码所述基因IGF3的cDNA的蛋白，它的氨基酸序列如SEQ IDNO：2所示。
金钱鱼IGF3基因的cDNA序列如SEQ ID NO：1所示，全长594bp，其编码蛋白共198个氨基酸，序列如SEQ ID NO：2所示，获得的金钱鱼促生殖细胞成熟基因IGF3，其不含有cDNA中的信息肽部分，扩增片段大小为507bp，序列如SEQ ID NO：3所示，其编码的蛋白共169个氨基酸，序列如SEQ ID NO：4所示。
本发明还提供了一种表达载体，包括所述基因IGF3。
本发明的上述第二个目的可以通过以下技术方案来实现：一种金钱鱼IGF3重组蛋白的制备方法，包括用所述的表达载体转化寄主细胞，培养转化体，获得重组的金钱鱼IGF3重组蛋白。
本发明还提供了一种金钱鱼IGF3重组蛋白，包括用所述的酯酶表达载体转化寄主细胞，培养转化体，从培养物中获得重组蛋白的步骤的方法制备。
本发明利用反转录PCR技术从金钱鱼中克隆到了促生殖细胞成熟基因IGF3，并构建了IGF3的原核表达载体，可大量表达获得金钱鱼促生殖细胞成熟基因IGF3所编码的重组蛋白。
本发明的上述最后一个目的可以通过以下技术方案来实现：所述基因IGF3、编码所述基因IGF3的蛋白或所述的重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
本发明具有如下优点：本发明利用反转录PCR技术从金钱鱼中克隆得到新型胰岛素类生长因子(insulin-like growth factor 3，IGF3)的cDNA序列，并根据实验结果说明了IGF3可能参与调控金钱鱼卵母细胞成熟的生理过程，为深入了解鱼类卵母细胞成熟的生殖内分泌调控机制提供一定的理论依据和重要线索。开发的金钱鱼IGF3重组蛋白有望应用于研发新型鱼类催熟催产制剂。
附图说明
图1是实施例1中PCR扩增金钱鱼IGF3基因电泳图；
图2是实施例1中金钱鱼IGF3重组蛋白表达与纯化，M：marker，1：过柱前，2：流穿，3-6：50mM浓度的咪唑洗脱液洗涤，7-10：250mM浓度的咪唑洗脱液洗脱；
图3是实施例1中金钱鱼IGF3重组蛋白SDS-PAGE检测，M：marker，1-2：洗涤液，3-4：洗脱液；
图4是实施例2中金钱鱼IGF3重组蛋白质谱结果，图中显示为下划线部分可信度在95％以上，显示为黑体字部分可认为完全不可信，完全忽略，IGF3重组蛋白质谱结果覆盖率为84.02％，确定重组蛋白为目的蛋白；
图5是实施例3中不同浓度金钱鱼IGF3重组蛋白对细胞周期基因在mRNA转录水平上的表达调节，A表示IGF3重组蛋白对cdc2表达的影响，B表示IGF3重组蛋白对cyclinB表达的影响。
具体实施实例
实施例1 IGF3重组蛋白的制备
1、金钱鱼RNA的提取
取健康金钱鱼全鱼，以冰浴麻醉约2min后，杀鱼取样，分离卵巢组织，采用Trizol试剂法抽提获得金钱鱼卵巢组织总RNA。
2、IGF3基因的克隆
第一链cDNA的合成：
使用TaKaRa公司的PrimeScriptTM RT reagent Kit with gDNA Eraser(PerfectReal Time)试剂盒，先除去金钱鱼卵巢组织总RNA中的基因组DNA，然后将总RNA反转录得到第一链的单链cDNA，于-20℃冰箱保存备用。
以cDNA为模板的IGF3基因克隆：
根据生物信息学中相关的序列比对和结构分析结果，在基因IGF3的cDNA序列(如SEQ ID NO：1所示，其编码的蛋白质序列如SEQ ID NO：2所示)中去除编码29个氨基酸的信号肽的序列，以剩下的序列在两端设计特异性引物，如下：
IGF3-RT-F：5’-tgggatccggtaccaagcttGCCTGGCCGCTCTCCTCTGA-3’(SEQ ID NO：5)
IGF3-RT-R：5’-tggtggtggtggtgctcgagTTATGATTCAAATCCCATCATTC-3’(SEQ IDNO：6)
以第一链cDNA为模板，采用引物IGF3-RT-F和IGF3-RT-R进行PCR扩增，获得不含信息肽的IGF3基因，扩增片段大小为507bp，如图1所示，扩增序列为如SEQ IDNO：3所示，其编码的蛋白质序列如SEQ ID NO：4所示。电泳分离DNA片段，切胶回收目的产物。将纯化后的目的产物连接至pMBP载体，转化大肠杆菌DH5α，挑选阳性克隆测序，测序结果经BLAST比对分析，比对结果表明目的产物确为基因IGF3。
3、基因IGF3原核表达载体的构建
挑选阳性克隆的大肠杆菌DH5α，使用质粒小抽试剂盒抽提质粒，然后转化至原核表达工程菌rosetta2，将获得的阳性单克隆菌落进行菌液PCR验证，PCR扩增的片段大小与目的片段大小相似，约507bp，并将该阳性单菌落进行测序验证，测序结果与目片段的序列一致，说明成功构建了IGF3的原核重组表达载体。
4、基因IGF3的原核表达及纯化
重组蛋白的诱导表达：
将含有重组表达载体(pMBP-IGF3)的工程菌rosetta2先经过LB培养基的扩大培养，当菌液OD600值介于0.6～0.8之间时，加入IPTG(0.5mM，37℃，4h)进行诱导培养，诱导培养后收集菌液，离心获得菌体，加入含0.5M氯化钠的10mM Tris-HCl(pH 8.0)溶液重悬菌体，菌体重悬液超声波破碎，然后入蛋白Loading buffer，混匀，沸水浴中煮5分钟，进行SDS-PAGE电泳检测。
重组蛋白的纯化、脱盐、酶切及冻干：
将诱导表达后的菌体超声破碎，离心，弃沉淀，将上清经孔径0.45微米的滤膜过滤备用。
用去离子水洗镍柱，至pH 7.0，往柱子内加入约50mL含0.5M氯化钠的10mM Tris-HCl(pH 8.0)溶液平衡镍柱，稀释后的样品液过镍柱，收集穿过液，再重复过镍柱两次。重组蛋白挂柱后，先用含0.5M氯化钠的10mM Tris-HCl(pH 8.0)漂洗液漂洗，再分别以50mM、250mM浓度的咪唑洗脱液洗脱，对不同浓度咪唑洗脱液洗脱下来的物质进行SDS-PAGE检测(图2)，获得纯化的重组蛋白。
将纯化后的重组蛋白进行脱盐，先用0.02M的PB缓冲液进行清洗和平衡柱子。加入样品，收集穿过液。待样品完全进入下端柱子后，再加入PB缓冲液洗脱，收集洗脱液，即为脱盐后的重组蛋白。
往脱盐后的重组蛋白加tev蛋白酶酶切，质量比10∶1(MBP-IGF3：MBP-tev)，4℃酶切过夜，然后电泳检测酶切效率。酶切效率超过90％以上后，过amylose resin，收集流穿重组蛋白并进行SDS-PAGE检测，IGF3重组蛋白的分子量约为19kDa(图3)。
将酶切后的重组蛋白进行冻存：将含有目的蛋白的收集液置于合适大小的干净管子中，用封口膜封好，在膜上扎几个针眼，以便冻存时水分的散失。管子竖直放于-20度冰箱约2小时以上，待全部冻成冰状，迅速放于-80度冰箱。第二天，将-80度保存的管子置于调试完好的冻存机挂瓶内，开始冻存。冻存时间视水分散失快慢而定，一般8小时以上或者过夜。将冻存完成的、含有目的蛋白的管子取下迅速放于-80度冰箱保存，以便下一步的实验。
实施例2 IGF3重组蛋白的质谱检验
首先是蛋白质的还原烷基化，加入终浓度10mM二硫苏糖醇(DTT)还原IGF3重组蛋白，接着加入终浓度55mM碘乙酰胺(IAM)，最后加入1μg的Trypsin酶，过夜酶解8h～16h。酶解产生的多肽用C18柱子除盐，已经除盐的多肽抽干后用15μL Loading Buffer(0.1％甲酸，3％乙腈)溶解多肽。多肽上LC-MS/MS(ekspertTMnanoLC；AB Sciex TripleTOF 5600-plus)仪器进行分析，然后对结果进行评估。LC-MS/MS下机后，将原始下机数据直接提交到与AB SCIEX Triple TOFTM 5600plus质谱仪连接的Proteinpilot软件中进行数据库检索。
质谱结果(如图4所示)序列显示为下划线部分可信度在95％以上，显示为黑体字部分可认为完全不可信，完全忽略。但是如果序列处于-COOH端，即使是黑体字部分也不见得是错误的，原因在于-COOH极易被LC-MS/MS过滤掉。
图4显示IGF3重组蛋白的质谱结果可信度在95％以上部分覆盖度为84.02％，确定该蛋白为目的蛋白。
实施例3 IGF3重组蛋白调节生殖细胞成熟调控基因的表达
从湛江市场购买获得金钱鱼为研究对象，以特异表达基因IGF3的卵巢组织为材料，用IGF3重组蛋白离体孵育卵巢碎片，研究IGF3重组蛋白对相关生殖细胞成熟调控基因cdc2和cyclinB在mRNA转录水平表达的调节情况。
分别以浓度0、1、100、10000nM的IGF3重组蛋白(实施例1中制备)孵育卵巢碎片3h，然后使用实时荧光定量技术分别检测生殖细胞成熟调控基因cdc2和cyclinB在mRNA转录水平上的表达情况，检测结果如图5所示。金钱鱼cdc2和cyclinB基因序列由转录组数据库中获得，进而设计引物进行实时荧光定量实验。
从图5的检测结果可获知，不同浓度的IGF3重组蛋白对cdc2和cyclinB均起促进作用，说明IGF3重组蛋白对金钱鱼卵母细胞具有显著的促成熟作用。
以上列举具体实施例对本发明进行补充说明，需要指出的是，以上实施例只用于对本发明作进一步说明，不代表本发明的保护范围，其他人根据本发明的提示做出的非本质的修改和调整，仍属于本发明的保护范围。
序列表
<110> 广东海洋大学
中山大学
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Claims (7)
1.一种金钱鱼促生殖细胞成熟基因IGF3，其特征是：它的核苷酸序列如SEQ ID NO:3所示。
2.权利要求1所述基因IGF3编码的蛋白，其特征是：它的氨基酸序列如SEQ ID NO:4所示。
3.一种金钱鱼促生殖细胞成熟基因IGF3的cDNA，其特征是：它的核苷酸序列如SEQ IDNO:1所示。
4.权利要求3所述cDNA编码的蛋白，其特征是：它的氨基酸序列如SEQ ID NO:2所示。
5.一种表达载体，其特征是包括权利要求1所述基因IGF3。
6.一种金钱鱼IGF3重组蛋白的制备方法，其特征是：包括用权利要求5所述的表达载体转化寄主细胞，培养转化体，获得重组的金钱鱼IGF3重组蛋白。
7.权利要求1所述基因IGF3或权利要求1所述基因IGF3编码的蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
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