CN114573673A - 双叉犀金龟表皮蛋白，编码核苷酸序列及其应用 - Google Patents
双叉犀金龟表皮蛋白，编码核苷酸序列及其应用 Download PDFInfo
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Abstract
本发明提供了一种双叉犀金龟表皮蛋白，编码核苷酸序列及其应用，涉及生物技术领域。本发明以鞘翅目昆虫双叉犀金龟为研究对象，从其CPR家族的RR‑2亚族发现了一种新的RR‑2亚家族蛋白基因，命名为Td12611，并成功获得该基因的重组蛋白，具有在常温下发生液液相分离的特性，能够结合壳聚糖与几丁质，丰富了昆虫RR‑2亚家族表皮蛋白的种类，为研究RR‑2亚家族表皮蛋白提供了新的基因资源。Td12611重组蛋白可以作为增强剂，提升了单独壳聚糖膜的机械强度，具有较大潜在价值。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种双叉犀金龟表皮蛋白，编码核苷酸序列及其应用。
背景技术
仿生材料是材料学的重要研究方向，昆虫表皮凭借高强度、轻量型的特性，一直是仿生材料研究的热点。昆虫表皮是由表皮蛋白和几丁质等主要基质形成的高强度保护层。几丁质指的是N-乙酰氨基葡糖聚合物，是昆虫表皮的重要组分。脱乙酰几丁质又称壳聚糖，由于其分子结构中大量游离氨基的存在，溶解性能大大改观，具有一些独特的物化性质及生理功能，在农业、医药、食品、化妆品、环保诸方面具有广阔的应用前景。
壳聚糖具有无毒、生物相容性好、可生物降解以及抗毒抑菌等优点，并且由于壳聚糖存在非常强的分子内和分子间氢键，使其具有良好的成膜性，被认为是制造食品包装材料颇具潜力的原料。但是单一的壳聚糖膜机械强度低，大大影响了其应用范围。因此，研究人员通过多种手段对壳聚糖膜的机械强度进行提升，采用方法大多都是高分子聚合物或无机盐等与壳聚糖复合来增强壳聚糖膜的强度，但这大大降低了壳聚糖膜的生物相容性、可生物降解性等优良特性。
因此，寻找一种能够与壳聚糖复合增加其机械强度，且不影响其生物相容性、生物降解性等优良特性的新物质是十分必要的。
发明内容
本发明第一方面提供了一种双叉犀金龟表皮蛋白，所述蛋白的氨基酸序列包含或由如下序列组成：
a)SEQ ID NO.1所示的氨基酸序列；或，
b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列同一性的功能性同源序列；或
c)在SEQ ID NO.1所示的氨基酸序列中缺失、添加、替换一个或多个氨基酸且具有相同蛋白活性的氨基酸序列。
双叉犀金龟是一种常见的甲壳虫，属于昆虫纲鞘翅目金龟科甲虫。本发明以双叉犀金龟为研究对象，从其CPR家族的RR-2亚族(主要存在于坚硬表皮)中发现了一种新的RR-2亚家族蛋白基因，自定义命名为Td12611，并成功获得该基因的重组蛋白(即上述的双叉犀金龟表皮蛋白，本发明中也命名为Td12611)。Td12611表皮蛋白富含谷氨酰胺的低复杂度序列(Q-rich reign)，具有在常温下发生液液相分离的特性，能够结合壳聚糖与几丁质。
在本发明的一种实施方式中，所述双叉犀金龟表皮蛋白Td12611的氨基酸序列为SEQ ID NO.1所示的氨基酸序列：ESGFSGVQYSNAQSLTPQQTQPSYITAPQPVQPQTPAYQPQSPSFGPPTQVVSQLLLPTNVDRQIQKAAQQIAGPQYVQQIQILQQQELANQAVAAAQRRAMRQRMLAQQAQQQHGGLSGLGGQFGAASSDVYSSQALAQAQTPQQYLQYQPQTARPVARPAPQVYSRPQPQGKDEDLEEYDTPASYKFGFDVTDDQNTNYQNRQEQRDGNKISGSYSVVDSDGFIRTVTYTADPKEGFKAEVVRKPTDIVVKIPTPTPAPKEQYVQNYSAPRGQESKGNVIYQYQ，序列长度为286个氨基酸。
在本发明的一种实施方式中，所述双叉犀金龟表皮蛋白Td12611的氨基酸序列为与SEQ ID NO.1所示的氨基酸序列具有至少80％序列同一性的功能性同源序列。所述功能性同源序列包括但不限于SEQ ID NO.1所示的氨基酸具有约80％或以上、82％或以上、84％或以上、85％或以上、88％或以上、90％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.5％或以上、99.9％或以上同一性的氨基酸序列。
在本发明的一种实施方式中，所述双叉犀金龟表皮蛋白Td12611的氨基酸序列为在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个(例如可以为1-10个，具体地，可以为1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个)氨基酸且具有相同活性的氨基酸序列。
进一步，所述双叉犀金龟表皮蛋白Td12611的氨基酸序列还包含SEQ ID NO.2所示的氨基酸序列：MISSCGVALCYLALVSSVISAPVESGFSGVQYSNAQSLTPQQTQPSYITAPQPVQPQTPAYQPQSPSFGPPTQVVSQLLLPTNVDRQIQKAAQQIAGPQYVQQIQILQQQELANQAVAAAQRRAMRQRMLAQQAQQQHGGLSGLGGQFGAASSDVYSSQALAQAQTPQQYLQYQPQTARPVARPAPQVYSRPQPQGKDEDLEEYDTPASYKFGFDVTDDQNTNYQNRQEQRDGNKISGSYSVVDSDGFIRTVTYTADPKEGFKAEVVRKPTDIVVKIPTPTPAPKEQYVQNYSAPRGQESKGNVIYQYQ(划线部分为信号肽区域)，序列长度为309个氨基酸，其N端包含有长度为23个氨基酸的信号肽区域。
进一步，所述双叉犀金龟表皮蛋白Td12611的氨基酸序列还包含SEQ ID NO.3所示的氨基酸序列：MDHHHHHHESGFSGVQYSNAQSLTPQQTQPSYITAPQPVQPQTPAYQPQSPSFGPPTQVVSQLLLPTNVDRQIQKAAQQIAGPQYVQQIQILQQQELANQAVAAAQRRAMRQRMLAQQAQQQHGGLSGLGGQFGAASSDVYSSQALAQAQTPQQYLQYQPQTARPVARPAPQVYSRPQPQGKDEDLEEYDTPASYKFGFDVTDDQNTNYQNRQEQRDGNKISGSYSVVDSDGFIRTVTYTADPKEGFKAEVVRKPTDIVVKIPTPTPAPKEQYVQNYSAPRGQESKGNVIYQYQ(下划线为组氨酸标签)，序列长度为294个氨基酸，其N端带有组氨酸标签。
本发明新发现的双叉犀金龟表皮蛋白Td12611丰富了昆虫RR-2亚家族表皮蛋白的种类，提供了新的基因资源。而且本发明以新发现的双叉犀金龟表皮蛋白Td12611作为增强剂去制备壳聚糖复合膜，提升了壳聚糖膜的机械强度，具有较大潜在价值，具有十分广阔的应用前景。
本发明第二方面提供了编码所述双叉犀金龟表皮蛋白Td12611的核苷酸序列。
进一步，编码双叉犀金龟表皮蛋白Td12611的核苷酸序列包含或由如下序列组成：
i)SEQ ID NO.4所示的核苷酸序列；或，
ii)SEQ ID NO.4所示的核苷酸序列的互补序列、简并序列或同源序列；或，
iii)在严谨条件下与SEQ ID NO.4所示的核苷酸序列杂交，且能够编码双叉犀金龟表皮蛋白Td12611的核苷酸序列。
在本发明的一种实施方式中，所述编码双叉犀金龟表皮蛋白Td12611的核苷酸序列为SEQ ID NO.4所示的核苷酸序列：GAATCGGGCTTTTCAGGAGTACAGTATTCCAATGCACAATCGTTAACGCCTCAACAGACACAACCCTCGTACATTACAGCCCCTCAGCCGGTGCAACCGCAGACTCCGGCATATCAACCGCAAAGTCCGTCTTTTGGACCCCCCACACAAGTCGTGTCTCAGCTACTTTTGCCTACGAATGTAGATAGACAAATACAGAAAGCTGCCCAACAAATAGCTGGTCCGCAATATGTGCAACAAATTCAAATACTTCAACAACAAGAGCTTGCAAATCAAGCAGTAGCCGCCGCACAAAGACGTGCCATGAGACAAAGAATGCTTGCACAACAGGCTCAACAGCAGCATGGTGGTTTAAGTGGTTTAGGTGGACAGTTCGGAGCAGCGTCGTCGGACGTATACTCCAGCCAGGCATTAGCACAAGCTCAGACACCCCAACAATATCTACAGTACCAACCGCAGACAGCGAGGCCAGTTGCTAGACCAGCACCGCAAGTGTATTCTCGTCCACAGCCTCAGGGAAAAGACGAAGATCTCGAGGAATATGATACGCCCGCATCCTACAAGTTCGGCTTCGACGTAACGGACGATCAAAACACGAATTACCAGAACCGCCAAGAACAACGGGACGGGAACAAAATCTCCGGCAGTTATTCAGTGGTGGATTCGGATGGATTCATTAGAACCGTAACGTATACCGCGGATCCTAAGGAAGGATTCAAAGCCGAGGTCGTCAGAAAACCGACTGATATTGTTGTGAAAATTCCTACACCTACACCAGCACCGAAAGAACAATATGTTCAGAACTACTCTGCGCCTAGAGGGCAAGAATCCAAAGGAAATGTGATTTACCAGTACCAATAA，序列长度为861个碱基，对应于编码具有SEQ ID NO.1所示氨基酸的双叉犀金龟表皮蛋白。
在本发明的一种实施方式中，所述编码双叉犀金龟表皮蛋白Td12611的核苷酸序列为SEQ ID NO.4所示的核苷酸序列按照碱基互补配对原则形成的互补序列，互补序列可以为具有编码表皮蛋白Td12611功能的不完全互补序列或完全互补序列。
在本发明的一种实施方式中，所述编码表皮蛋白Td12611的核苷酸序列为SEQ IDNO.4所示的核苷酸序列的简并序列。简并序列是指改变SEQ ID NO.4核苷酸某个或多个核苷酸序列后，改变的核苷酸序列位置对应编码的氨基酸种类不变，不会影响核苷酸序列的编码功能和表达水平。
在本发明的一种实施方式中，所述编码表皮蛋白Td12611的核苷酸序列为SEQ IDNO.4所示的核苷酸序列的同源序列。所述同源核苷酸序列包括在SEQ ID NO.4所示的核苷酸序列中添加和/或取代和/或缺失一个或几个核苷酸而生成的可编码具有表皮蛋白Td12611相同活性的突变基因、等位基因或衍生物。
进一步优选同源序列为与SEQ ID NO.4所示的核苷酸序列约80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1％或以上、99.2％或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性且具备编码表皮蛋白Td12611功能的多核苷酸。
在本发明的一种实施方式中，所述编码表皮蛋白Td12611的核苷酸序列为在严谨条件下与SEQ ID NO.4的核苷酸序列杂交，且能够编码表皮蛋白Td12611的核苷酸序列。示例性地，所述“严谨条件”是指探针将与其靶序列杂交至可探测程度超过与其它序列杂交(如至少2倍于背景)的条件。严谨条件具有序列依赖性，且因环境的不同而不同。通过控制杂交和/或洗涤条件的严谨性，可以鉴定与探针100％互补的靶序列。可选择地，可以调节严谨条件以允许一些序列错配，使得探测到较低程度的同一性。
进一步，所述编码双叉犀金龟表皮蛋白Td12611的核苷酸序列为SEQ ID NO.5所示的核苷酸序列：ATGATTAGTAGTTGTGGTGTAGCGCTGTGTTATCTTGCGCTGGTGTCTAGTGTCATTTCTGCACCAGTAGAATCGGGCTTTTCAGGAGTACAGTATTCCAATGCACAATCGTTAACGCCTCAACAGACACAACCCTCGTACATTACAGCCCCTCAGCCGGTGCAACCGCAGACTCCGGCATATCAACCGCAAAGTCCGTCTTTTGGACCCCCCACACAAGTCGTGTCTCAGCTACTTTTGCCTACGAATGTAGATAGACAAATACAGAAAGCTGCCCAACAAATAGCTGGTCCGCAATATGTGCAACAAATTCAAATACTTCAACAACAAGAGCTTGCAAATCAAGCAGTAGCCGCCGCACAAAGACGTGCCATGAGACAAAGAATGCTTGCACAACAGGCTCAACAGCAGCATGGTGGTTTAAGTGGTTTAGGTGGACAGTTCGGAGCAGCGTCGTCGGACGTATACTCCAGCCAGGCATTAGCACAAGCTCAGACACCCCAACAATATCTACAGTACCAACCGCAGACAGCGAGGCCAGTTGCTAGACCAGCACCGCAAGTGTATTCTCGTCCACAGCCTCAGGGAAAAGACGAAGATCTCGAGGAATATGATACGCCCGCATCCTACAAGTTCGGCTTCGACGTAACGGACGATCAAAACACGAATTACCAGAACCGCCAAGAACAACGGGACGGGAACAAAATCTCCGGCAGTTATTCAGTGGTGGATTCGGATGGATTCATTAGAACCGTAACGTATACCGCGGATCCTAAGGAAGGATTCAAAGCCGAGGTCGTCAGAAAACCGACTGATATTGTTGTGAAAATTCCTACACCTACACCAGCACCGAAAGAACAATATGTTCAGAACTACTCTGCGCCTAGAGGGCAAGAATCCAAAGGAAATGTGATTTACCAGTACCAATAA。序列长度为930个碱基，对应于编码具有SEQ ID NO.2所示氨基酸的双叉犀金龟表皮蛋白。
进一步，所述编码双叉犀金龟表皮蛋白Td12611的核苷酸序列为SEQID NO.6所示的核苷酸序列：CCATGGATCACCACCACCACCACCACGAATCGGGCTTTTCAGGAGTACAGGAATCGGGCTTTTCAGGAGTACAGTATTCCAATGCACAATCGTTAACGCCTCAACAGACACAACCCTCGTACATTACAGCCCCTCAGCCGGTGCAACCGCAGACTCCGGCATATCAACCGCAAAGTCCGTCTTTTGGACCCCCCACACAAGTCGTGTCTCAGCTACTTTTGCCTACGAATGTAGATAGACAAATACAGAAAGCTGCCCAACAAATAGCTGGTCCGCAATATGTGCAACAAATTCAAATACTTCAACAACAAGAGCTTGCAAATCAAGCAGTAGCCGCCGCACAAAGACGTGCCATGAGACAAAGAATGCTTGCACAACAGGCTCAACAGCAGCATGGTGGTTTAAGTGGTTTAGGTGGACAGTTCGGAGCAGCGTCGTCGGACGTATACTCCAGCCAGGCATTAGCACAAGCTCAGACACCCCAACAATATCTACAGTACCAACCGCAGACAGCGAGGCCAGTTGCTAGACCAGCACCGCAAGTGTATTCTCGTCCACAGCCTCAGGGAAAAGACGAAGATCTCGAGGAATATGATACGCCCGCATCCTACAAGTTCGGCTTCGACGTAACGGACGATCAAAACACGAATTACCAGAACCGCCAAGAACAACGGGACGGGAACAAAATCTCCGGCAGTTATTCAGTGGTGGATTCGGATGGATTCATTAGAACCGTAACGTATACCGCGGATCCTAAGGAAGGATTCAAAGCCGAGGTCGTCAGAAAACCGACTGATATTGTTGTGAAAATTCCTACACCTACACCAGCACCGAAAGAACAATATGTTCAGAACTACTCTGCGCCTAGAGGGCAAGAATCCAAAGGAAATGTGATTTACCAGTACCAATAAGAATTC(下划线为酶切位点)，序列长度为917个碱基，对应于编码具有SEQ IDNO.3所示氨基酸的双叉犀金龟表皮蛋白。
进一步，在本发明提供的技术方案的基础上，提供了用于检测所述编码双叉犀金龟表皮蛋白的核苷酸序列的引物。
在本发明的一种优选地实施方式中，所述引物包括上游克隆引物P1和/或下游克隆引物P2；其中，所述上游克隆引物P1的核苷酸序列如SEQ ID NO.7所示：5’-ATGATTAGTAGTTGTGGTGTAGCGCT-3’；所述下游克隆引物P2的核苷酸序列如SEQ ID NO.8所示：5’-TTATTGGTACTGGTAAATCACATTTCCTTTGG-3’。
在本发明的一种优选地实施方式中，所述引物包括上游连接引物P3和/或下游连接引物P4；其中，所述上游连接引物P3的核苷酸序列如SEQ ID NO.9所示：5’-CCATGGATCACCACCACCACCACCACGAATCGGGCTTTTCAGGAGT ACAG-3’；所述下游连接引物P4的核苷酸序列如SEQ ID NO.10所示：5’-GAATTCTCATTGGTACTGGTAAATCACATTTCC-3’。
本发明第三方面提供了所述的双叉犀金龟表皮蛋白，或所述的编码双叉犀金龟的核苷酸序列在提升壳聚糖或其类似物的性能方面的应用。
具体的，双叉犀金龟Td12611重组蛋白作为增强剂在制备壳聚糖复合膜上的应用，即Td12611重组蛋白可以单独作为增强剂开发出相应的产品应用于提升壳聚糖膜的机械强度。
所述双叉犀金龟表皮蛋白Td12611的应用还包括以下几个方面：(1)表皮蛋白Td12611氨基酸序列或至少部分氨基酸序列的多肽可能在去除或替代某些氨基酸之后仍有生物活性甚至有新的生物学活性，或者提高了产量或优化了蛋白动力学特征或其他致力于得到的性质；(2)涉及表皮蛋白Td12611及相关截短体、突变体、多肽的生物合成；(3)涉及表皮蛋白Td12611与壳聚糖或其类似物复合方面的应用。
所述的编码核苷酸序列的应用包括以下几个方面：(1)本发明所提供的核苷酸序列或至少部分核苷酸序列被修饰或突变，修饰或突变的途径包括插入、缺失，聚合酶链式反应(PCR)，易错PCR，不同序列的重新连接，序列的不同部分或与其他来源的同源序列进行定向进化，或通过化学试剂诱变等。(2)本发明所提供的核苷酸序列或至少部分核苷酸序列的克隆基因通过合适的表达体系在外源宿主中表达以得到相应的表皮蛋白或其他更高的生物活性或产量。(3)本发明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以通过遗传重组来构建重组质粒以获得新型生物合成途径，也可以通过插入、置换、缺失或失活进而获得新型生物合成途径。
本发明第四方面提供了载体，所述载体导入了所述的核苷酸序列。
对于载体的种类不作具体限定，可以根据需要选择适合的载体。例如载体包括但不限于pET28a、pcdna3.1、pUC18、pBR322、pUC19、pGEX2T或pESC-Ura，优选pET28a。
本发明第五方面提供了遗传工程化的宿主细胞，其包含所述的氨基酸序列，或包含所述的核苷酸序列，或导入了所述的载体。
进一步，所述的宿主细胞包括植物细胞、动物细胞或微生物细胞；
进一步，所述微生物细胞包括但不限于大肠杆菌、假单孢菌、芽孢杆菌、酵母细胞中的至少一种。优选大肠杆菌BL21(DE3)。
进一步，在体内和体外将编码所述表皮蛋白的核苷酸序列，或所述的重组质粒，或所述的载体引入宿主细胞的方法包括但不限于聚乙二醇(PEG)转化、电穿孔、脂质转染、热休克、磷酸钙沉淀、病毒介导和显微注射。
本发明第六方面提供了制备所述表皮蛋白的方法，包括以下步骤：将所述的编码双叉犀金龟表皮蛋白的核苷酸序列，或所述的载体导入宿主细胞中表达获得所述表皮蛋白；和/或，采用所述的遗传工程化的宿主细胞表达获得所述表皮蛋白。
在本发明一种优选地实施方式中，制备所述表皮蛋白的方法，包括以下步骤：
步骤一：合成td12611核苷酸序列
(1)提取双叉犀金龟预蛹期头壳组织总RNA，利用反转录合成cDNA；
(2)以cDNA为模板，利用克隆引物P1和克隆引物P2，进行PCR扩增；
(3)将PCR产物进行琼脂糖凝胶电泳，进行目的基因回收；获得合成SEQ ID NO.5所示的核苷酸序列；
(4)将PCR产物连接到克隆载体T-vector上，通过测序验证扩增片段的正确性；
(5)以(4)中测序成功的质粒为模板，设计连接引物P1和连接引物P2进行PCR扩增，扩增产物去除N端23个残基信号肽区域，并引入N端组氨酸标签；
(6)将(4)中的PCR产物进行琼脂糖凝胶电泳，进行目的基因回收，获得合成SEQ IDNO.6所示的核苷酸序列。
步骤二：构建pET28a-td12611的重组载体及重组表达基因工程菌
(1)将SEQ ID NO.6所示的核苷酸序列与pET28a原核表达载体分别用NcoI/EcorI两种限制性内切酶进行双酶切，双酶切产物经纯化回收后用T4DNA连接酶进行连接，得到重组表达载体连接产物；
(2)重组表达载体的转化，将重组表达载体连接产物用化学转化法转入大肠杆菌感受态细胞(E.coli DH5α)，获得克隆单菌落；
(3)挑取多个单克隆菌落接种于含卡那霉素的LB培养液中，培养振荡过夜，取菌液进行菌落PCR鉴定，取阳性克隆菌株进行质粒提取并送样测序，获得测序正确的重组表达载体pET28a-td12611。
步骤三：蛋白表达与纯化
(1)将测序正确的重组质粒转入大肠杆菌感受态细胞，获得重组表达工程菌株。
(2)将获得重组表达工程菌株单菌落接种于含有卡那霉素的LB液体培养基中，过夜培养；将过夜培养的菌液接种于含有卡那霉素的LB液体培养基中进行扩大培养。破碎菌体，离心后收集上清液。
(3)将收集的上清液用滤膜除去杂质，上清过镍离子亲和层析柱；纯化蛋白，对纯化后的蛋白进行聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹试验(western blot，WB)检测，获得纯化的重组Td12611蛋白，其氨基酸序列如SEQ ID NO.3所示。
本发明第七方面提供了一种复合材料，包括所述的表皮蛋白，以及任选地其它组分和/或辅助组分。
优选所述复合材料包括但不限于表皮蛋白Td12611与壳聚糖和/或其类似物组成的复合材料(例如复合膜)，表皮蛋白Td12611团聚形成的凝胶等。
进一步，所述复合材料为所述的表皮蛋白与壳聚糖和/或其类似物复合形成的薄膜材料；更优选为所述的表皮蛋白Td12611与壳聚糖复合形成的薄膜材料。
进一步，所述的表皮蛋白Td12611与壳聚糖复合形成的薄膜材料中表皮蛋白的浓度小于1％，例如蛋白浓度可以为但不限于0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％。
本发明采用上述技术方案具有以下有益效果：
(1)本发明提供了一种新发现的双叉犀金龟CPR家族RR-2亚族的表皮蛋白，丰富了昆虫RR-2亚家族表皮蛋白的种类，为研究RR-2亚家族表皮蛋白提供了新的基因资源。
(2)本发明提供的双叉犀金龟金龟表皮蛋白Td12611，绿色环保，生物相容性高，可以基于蛋白本身性质为生物材料提供原料，与壳聚糖和/或其类似物组成的复合材料，提升了单独壳聚糖膜的机械强度，具有较大潜在价值。
附图说明
图1所示为实施例1获得的表皮蛋白Td12611的SDS-PAGE电泳和Western-bloting结果。
图2所示为实施例2中Td12611蛋白与几丁质结合SDS-PAGE电泳检测结果图和相对灰度分析统计图。其中，A为SDS-PAGE电泳图，T：用于几丁质结合实验的总蛋白，F：未与几丁质结合的蛋白，E：与几丁质结合的蛋白；Maker与图1相同。B为A中电泳条带灰度值分析统计图。
图3所示为实施例3中Td12611蛋白液液相分离特性表征结果图。其中，A为Td12611蛋白发生相分离的宏观现象，B为显微镜下观察到的相分离现象，C为Td12611蛋白溶液随温度变化的浊度变化曲线。
图4所示为实施例4中不同浓度Td12611蛋白-壳聚糖复合膜极限拉伸强度和杨氏模量统计图。
具体实施方式
本发明中，自定义“Td12611”可以表示双叉犀金龟的CPR家族RR-2亚族表皮蛋白，或表皮蛋白基因，或编码表皮蛋白的核苷酸序列，具体所指的含义可以结合上下文判断。
本发明中，术语“核苷酸”以本领域技术人员理解的一般含义。
本发明中，术语“氨基酸”是指任何氨基酸(标准和非标准氨基酸二者)，包括但不限于α-氨基酸、β-氨基酸、γ-氨基酸和δ-氨基酸。适合的氨基酸的实例包括但不限于丙氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、丝氨酸、酪氨酸、精氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、蛋氨酸、苯基丙氨酸、苏氨酸、色氨酸和缬氨酸。
本发明中，所述的“严谨条件”可为低严谨条件、中严谨条件、高严谨条件中的任一种。“低严谨条件”，例如，为5×SSC、5×Denhardt溶液、0.5％SDS、50％甲酰胺、32℃的条件。“中严谨条件”，例如，为5×SSC、5×Denhardt溶液、0.5％SDS、50％甲酰胺、42℃的条件。“高严谨条件”，例如，为5×SSC、5×Denhardt溶液、0.5％SDS、50％甲酰胺、50℃的条件。在上述条件中，越提高温度，越能期待高效地获得具有高同源性的DNA。影响杂交严谨性的因素可为温度、探针浓度、探针长度、离子强度、时间、盐浓度等多种因素，本领域技术人员通过适宜选择这些因素，可实现同样的严谨条件。
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下面结合具体实施例详细描述本发明，这些实施例用于理解而不是限制本发明。
实施例1Td12611基因的克隆及表达纯化
一.合成编码Td12611的核苷酸序列
1.双叉犀金龟头壳总RNA提取
(1)取双叉犀金龟预蛹期头壳组织样品，液氮速冻2-3min，加入研钵中，加液氮研磨，直至呈现出均一的粉末。
(2)向1.5mL离心管中加入Trizol试剂1mL，取约100mg研磨好的样品加入离心管，室温静置20min。
(3)吸取200μL氯仿加入到(2)中的离心管中，在振荡器上充分震荡混匀，室温静置5min。然后4℃、12000rpm，离心20min，用枪头吸取200μL上清，加入到新的1.5mL离心管中。
(4)用枪头吸取500μL异丙醇加入到(4)中的离心管，反复颠倒10次。将离心管放入-20℃冰箱冷冻，促进RNA沉淀。
(5)将(4)中离心管于4℃、12000rpm离心20min。弃上清。用RNase-free H2O和无水乙醇配置75％乙醇清洗沉淀，清洗后于4℃、12000rpm条件下离心5min，弃上清，此步骤重复两次。
(6)室温静置20min，加入50μLRNase-free H2O，使RNA沉淀充分溶解。
2.反转录合成双叉犀金龟头壳组织cDNA
以步骤1提取的RNA作为反转录模板，反转录得到cDNA。10μL反应体系中，分别加入RNA 500ng，Oligo dT Primer 0.5μL，5×PrimeScript Buffer2μL，PromeScript EnzymeMix I 0.5μL，Random 6mers 0.5μL，补充RNase-free Water至10μL。反应条件：37℃，1h；85℃，5s；4℃保存。
3.合成双叉犀金龟表皮蛋白基因td12611
(1)以步骤2获得的cDNA为模板，利用克隆引物P1和克隆引物P2，进行PCR扩增；
克隆引物P1：5’-ATGATTAGTAGTTGTGGTGTAGCGCT-3’(SEQ ID NO.7)；
克隆引物P2：5’-TTATTGGTACTGGTAAATCACATTTCCTTTGG-3’(SEQ ID NO.8)；
PCR反应体系：1μL cDNA模板，20μL 2×PrimerSTAR Max DNA Polymerase，2μL克隆引物P1，2μL克隆引物P2，补充ddH2O(双蒸水)至总反应体系为40μL；
PCR反应条件：①95℃，5min；②95℃，10s；③60℃，10s；④72℃，10s；⑤重复步骤②-④32个循环；⑥72℃，10min；
(2)将PCR产物进行1％琼脂糖凝胶电泳，用TaKaRa MiniBEST Agarose Gel DNAExtraction Kit进行目的基因回收，获得合成SEQ ID NO.5所示的核苷酸序列；
(3)将PCR产物连接到克隆载体T-vector上，通过测序验证扩增片段的正确性；
(4)以步骤(3)中测序成功的质粒为模板，设计连接引物P3和连接引物P4进行PCR扩增；P3引物中分别引入NcoI位点和6×His标签，P4引物中引入EcorI酶切位点，利用P3/P4引物扩增出来的基因片段去除了N端23个残基的信号肽区域并在N端引入组氨酸标签；
连接引物P3：5’-CCATGGATCACCACCACCACCACCACGAATCGGGCTTTTCAGGAGTACAG-3’(SEQ ID NO.9)；
连接引物P4：5’-GAATTCTCATTGGTACTGGTAAATCACATTTCC-3’(SEQ ID NO.10)；
PCR反应体系：1μL模板，20μL 2×PrimerSTAR Max DNA Polymerase，2μL连接引物P3，2μL连接引物P4，补充ddH2O(双蒸水)至总反应体系为40μL；
PCR反应条件：①95℃，5min；②95℃，10s；③60℃，10s；④72℃，10s；⑤重复步骤②-④32个循环；⑥72℃，10min；
(5)将步骤(4)中的PCR产物进行1％琼脂糖凝胶电泳，用TaKaRaMiniBEST AgaroseGel DNA Extraction Kit进行目的基因回收，获得合成序列表SEQ ID NO.6所示的核苷酸序列。
二、构建重组载体和重组表达基因工程菌
表达重组载体的载体骨架为pET28a原核表达载体；重组表达工程菌株为大肠杆菌BL21(DE3)。
(1)将SEQ ID NO.6所示的核苷酸序列与pET28a原核表达载体分别用NcoI/EcorI两种限制性内切酶进行双酶切，双酶切产物经纯化回收后用T4DNA连接酶进行连接；连接体系为0.5μL酶切后的pET28a，7μL酶切后的td12611基因片段，7.5μL TaKaRa Ligation Mix；反应条件为16℃，1h，获得重组表达载体连接产物；
(2)重组表达载体的转化，将重组表达载体连接产物与大肠杆菌感受态细胞(E.coli DH5α)混匀，冰浴30min，然后42℃水浴50s，取出后再次冰浴2min；加入1mL SOC培养基与37℃摇床中培养1h，取200μL菌液均匀涂布在含50μg/mL卡那霉素的LB固体培养基上，37℃过夜培养，获得克隆单菌落；
(3)挑取多个单克隆菌落接种于含50μg/mL卡那霉素的LB培养液中，在37℃、200rpm条件振荡过夜，取菌液进行菌落PCR鉴定，取阳性克隆菌株进行质粒提取并送样测序，获得测序正确的重组表达载体pET28a-td12611。
(4)将测序正确的重组质粒与大肠杆菌感受态细胞(E.coli BL21DE3)混合，冰浴30min，随后42℃水浴热激50s，取出后再冰浴2min。随后加入1mL SOC培养基于37℃，200rpm条件下培养1h，取200μl菌液均匀涂布于含50μg/mL卡那霉素的LB固体培养基上，37℃静置过夜，获得重组表达工程菌株。
三、Td12611蛋白表达与纯化
(1)将获得重组表达工程菌株单菌落接种于20mL含有浓度为50mg/L卡娜霉素的LB液体培养基中，37℃过夜培养；将过夜培养的菌液接种于1000mL含有50mg/L卡那霉素的LB液体培养基中进行扩大培养。OD 600达到0.5-0.6后加入终浓度为0.5mM的异丙基-β-D硫代半乳糖苷(IPTG)诱导培养5h；诱导后的菌液经5000rpm离心10min收集菌体，加入150mL的缓冲液(Tris-HCl,500mM NaCl,pH 8.0)重悬菌体；用高压匀浆机在4℃，600bar条件下破碎菌液，反复四次。然后12000×g，离心10min，收集上清液。
(2)将收集的上清液用0.22μm滤膜除去杂质，上清过镍离子亲和层析柱；用AKTA蛋白纯化仪对Td12611蛋白进行洗涤(20mM Tris，500mM NaCl，50mM咪唑，pH 8.0)和洗脱(20mM Tris，500mM NaCl，200mM咪唑，pH 8.0)，对洗脱下来的组分进行聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹试验(western blot)检测，检测结果见图1。最终获得纯度较高的重组Td12611蛋白，其具体氨基酸序列如SEQ ID NO.3所示。
实施例2检测Td12611蛋白与几丁质结合能力
实验目的：检测Td12611重组蛋白与α-几丁质(α-chitin)、β-几丁质(β-chitin)、壳聚糖(chitosan)和胶体几丁质(colloidal chitin)4种类型几丁质的结合能力。
实验过程：几丁质结合实验在体外进行，采用将蛋白与不同类型的几丁质混合结合并洗脱的方法。具体实验操作如下：将纯化后的蛋白透析到结合缓冲液(20mM Tris，500mM NaCl，pH 8.0)中，构建200μL反应体系，使蛋白终浓度为0.5mg/mL，几丁质终浓度为2mg/mL。反应在4℃条件下进行，Td12611蛋白与不同类型几丁质在2mL离心管中持续颠倒混匀2h，反应结束后，12000rpm/min离心10min，将上清取出记为未结合几丁质的蛋白。在沉淀中加入相当于反应体系5倍体积的漂洗缓冲液(20mM Tris，500mM NaCl，pH 8.0)，将沉淀重悬并颠倒混匀5min进行漂洗后12000rpm/min离心5min弃上清，重复漂洗3次。最后将沉淀加入200μL的结合缓冲液后对沉淀进行重悬，取同样体积的上清和沉淀重悬液进行SDS-PAGE检测蛋白与不同类型几丁质的结合情况。
结果分析：利用Image J灰度分析软件将SDS-PAGE结果进行灰度扫描量化，将用于结合实验的蛋白条带灰度值作为背景，进行反应体系上清液及沉淀重悬液蛋白条带相对灰度计算，以此表征蛋白与几丁质的结合能力。Td12611重组蛋白与几丁质结合能力检测结果见图2。
实验结论：Td12611重组蛋白与α-几丁质、β-几丁质、壳聚糖和胶体几丁质4种类型的几丁质均能结合，而且从相对灰度分析可以看出Td12611重组蛋白与壳聚糖的结合能力最强。
实施例3检测Td12611蛋白的液液相分离特性
实验过程：首先，用肉眼观察在不同温度下Td12611蛋白溶液产生液液相分离(Liquid-liquid phase separation，LLPS)的宏观情况。其次，用光学显微镜观察蛋白溶液形成相分离的微观情况。最后，采用Bio-Logic MOS-500圆二色谱仪的自动变温系统对Td12611重组蛋白在5-45℃温度范围内的浊度情况进行测量。使用蛋白溶液的浓度为5mg/mL，测量浊度的波长为300nm，每5℃设置一个梯度，共9个平行，每个平行测量三次取平均值。用蛋白溶液的相对浊度(Relative Turbidity)代表该蛋白产生液液相分离的情况，液液相分离表征结果详见图3。
实验结果，图3的结果表明Td12611重组蛋白可以发生液液相分离。其中图3A为肉眼观察的宏观现象，4℃时蛋白溶液澄清，温度升高到25℃时肉眼可明显观察到团聚体，溶液明显变浑浊，再降温到4℃时蛋白溶液再次变澄清。图3B中在显微镜下观察到液液相分离的Td12611重组蛋白形成大小不一的液滴。图3C变化曲线图显示出Td12611蛋白在10-15℃条件下开始发生相分离，形成团聚体。随温度升高团聚体含量不断增加，当温度达到30℃时蛋白团聚达到饱和状态。
实施例4Td12611蛋白-壳聚糖复合膜及其性能检测
一、制备Td12611蛋白-壳聚糖复合膜
将终浓度为20mg/mL的壳聚糖(脱乙酰度99％)溶于1％的乙酸溶液中，用0.22μm的滤膜过滤去除杂质，然后取3mL壳聚糖放入10mL离心管中，分别加入占壳聚糖质量的0.5％、1％、2％和5％的Td12611蛋白，用不加蛋白的壳聚糖作为对照，涡旋震荡充分混匀后4000g/min离心去除气泡，然后倒入平皿中均匀平铺，放入烘箱中充分干燥后取3mL浓度为1M的NaOH溶液中和复合膜1min，然后用大量去离子水洗涤复合膜至中性，再放入烘箱中使复合膜充分干燥后，用镊子将复合膜撕下，获得蛋白含量不同的Td12611蛋白-壳聚糖复合膜。
二、检测Td12611蛋白-壳聚糖复合膜的机械性能
将Td12611蛋白-壳聚糖复合膜与纯壳聚糖膜裁成约35.0mm×7.0mm的长条，同时在长条两段粘附约7.0mm×7.0mm的砂纸以便后续拉伸实验。最后对长条的长宽厚度进行测量，利用E43.104型微机控制电子万能试验机进行膜的拉力机械性能测试，获得的数据拟合成拉力-应变曲线，分析曲线得到复合膜的拉力机械性能参数。用X轴的最大值表征膜材料的延伸率，用Y轴的最大值表征膜材料的极限拉伸强度，用曲线弹性形变部分的斜率计算膜材料的杨氏模量，以此来表征膜的刚性。
对Td12611蛋白-壳聚糖复合膜力学性质表征结果详见图4，数据表明，蛋白浓度为0.5％和1％条件下，膜的拉伸强度和刚度得到显著提升，蛋白溶液浓度大于1％时，膜的拉伸强度和刚度没有明显变化。
实验结果表明，少量的Td12611重组蛋白的加入对壳聚糖膜材料的机械性能提升有所贡献。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中国农业科学院深圳农业基因组研究所
<120> 双叉犀金龟表皮蛋白，编码核苷酸序列及其应用
<130> 2020
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 286
<212> PRT
<213> Unknown
<220>
<223> 双叉犀金龟表皮蛋白Td12611的氨基酸序列1
<400> 1
Glu Ser Gly Phe Ser Gly Val Gln Tyr Ser Asn Ala Gln Ser Leu Thr
1 5 10 15
Pro Gln Gln Thr Gln Pro Ser Tyr Ile Thr Ala Pro Gln Pro Val Gln
20 25 30
Pro Gln Thr Pro Ala Tyr Gln Pro Gln Ser Pro Ser Phe Gly Pro Pro
35 40 45
Thr Gln Val Val Ser Gln Leu Leu Leu Pro Thr Asn Val Asp Arg Gln
50 55 60
Ile Gln Lys Ala Ala Gln Gln Ile Ala Gly Pro Gln Tyr Val Gln Gln
65 70 75 80
Ile Gln Ile Leu Gln Gln Gln Glu Leu Ala Asn Gln Ala Val Ala Ala
85 90 95
Ala Gln Arg Arg Ala Met Arg Gln Arg Met Leu Ala Gln Gln Ala Gln
100 105 110
Gln Gln His Gly Gly Leu Ser Gly Leu Gly Gly Gln Phe Gly Ala Ala
115 120 125
Ser Ser Asp Val Tyr Ser Ser Gln Ala Leu Ala Gln Ala Gln Thr Pro
130 135 140
Gln Gln Tyr Leu Gln Tyr Gln Pro Gln Thr Ala Arg Pro Val Ala Arg
145 150 155 160
Pro Ala Pro Gln Val Tyr Ser Arg Pro Gln Pro Gln Gly Lys Asp Glu
165 170 175
Asp Leu Glu Glu Tyr Asp Thr Pro Ala Ser Tyr Lys Phe Gly Phe Asp
180 185 190
Val Thr Asp Asp Gln Asn Thr Asn Tyr Gln Asn Arg Gln Glu Gln Arg
195 200 205
Asp Gly Asn Lys Ile Ser Gly Ser Tyr Ser Val Val Asp Ser Asp Gly
210 215 220
Phe Ile Arg Thr Val Thr Tyr Thr Ala Asp Pro Lys Glu Gly Phe Lys
225 230 235 240
Ala Glu Val Val Arg Lys Pro Thr Asp Ile Val Val Lys Ile Pro Thr
245 250 255
Pro Thr Pro Ala Pro Lys Glu Gln Tyr Val Gln Asn Tyr Ser Ala Pro
260 265 270
Arg Gly Gln Glu Ser Lys Gly Asn Val Ile Tyr Gln Tyr Gln
275 280 285
<210> 2
<211> 309
<212> PRT
<213> Unknown
<220>
<223> 双叉犀金龟表皮蛋白Td12611的氨基酸序列2
<400> 2
Met Ile Ser Ser Cys Gly Val Ala Leu Cys Tyr Leu Ala Leu Val Ser
1 5 10 15
Ser Val Ile Ser Ala Pro Val Glu Ser Gly Phe Ser Gly Val Gln Tyr
20 25 30
Ser Asn Ala Gln Ser Leu Thr Pro Gln Gln Thr Gln Pro Ser Tyr Ile
35 40 45
Thr Ala Pro Gln Pro Val Gln Pro Gln Thr Pro Ala Tyr Gln Pro Gln
50 55 60
Ser Pro Ser Phe Gly Pro Pro Thr Gln Val Val Ser Gln Leu Leu Leu
65 70 75 80
Pro Thr Asn Val Asp Arg Gln Ile Gln Lys Ala Ala Gln Gln Ile Ala
85 90 95
Gly Pro Gln Tyr Val Gln Gln Ile Gln Ile Leu Gln Gln Gln Glu Leu
100 105 110
Ala Asn Gln Ala Val Ala Ala Ala Gln Arg Arg Ala Met Arg Gln Arg
115 120 125
Met Leu Ala Gln Gln Ala Gln Gln Gln His Gly Gly Leu Ser Gly Leu
130 135 140
Gly Gly Gln Phe Gly Ala Ala Ser Ser Asp Val Tyr Ser Ser Gln Ala
145 150 155 160
Leu Ala Gln Ala Gln Thr Pro Gln Gln Tyr Leu Gln Tyr Gln Pro Gln
165 170 175
Thr Ala Arg Pro Val Ala Arg Pro Ala Pro Gln Val Tyr Ser Arg Pro
180 185 190
Gln Pro Gln Gly Lys Asp Glu Asp Leu Glu Glu Tyr Asp Thr Pro Ala
195 200 205
Ser Tyr Lys Phe Gly Phe Asp Val Thr Asp Asp Gln Asn Thr Asn Tyr
210 215 220
Gln Asn Arg Gln Glu Gln Arg Asp Gly Asn Lys Ile Ser Gly Ser Tyr
225 230 235 240
Ser Val Val Asp Ser Asp Gly Phe Ile Arg Thr Val Thr Tyr Thr Ala
245 250 255
Asp Pro Lys Glu Gly Phe Lys Ala Glu Val Val Arg Lys Pro Thr Asp
260 265 270
Ile Val Val Lys Ile Pro Thr Pro Thr Pro Ala Pro Lys Glu Gln Tyr
275 280 285
Val Gln Asn Tyr Ser Ala Pro Arg Gly Gln Glu Ser Lys Gly Asn Val
290 295 300
Ile Tyr Gln Tyr Gln
305
<210> 3
<211> 294
<212> PRT
<213> Unknown
<220>
<223> 双叉犀金龟表皮蛋白Td12611的氨基酸序列3
<400> 3
Met Asp His His His His His His Glu Ser Gly Phe Ser Gly Val Gln
1 5 10 15
Tyr Ser Asn Ala Gln Ser Leu Thr Pro Gln Gln Thr Gln Pro Ser Tyr
20 25 30
Ile Thr Ala Pro Gln Pro Val Gln Pro Gln Thr Pro Ala Tyr Gln Pro
35 40 45
Gln Ser Pro Ser Phe Gly Pro Pro Thr Gln Val Val Ser Gln Leu Leu
50 55 60
Leu Pro Thr Asn Val Asp Arg Gln Ile Gln Lys Ala Ala Gln Gln Ile
65 70 75 80
Ala Gly Pro Gln Tyr Val Gln Gln Ile Gln Ile Leu Gln Gln Gln Glu
85 90 95
Leu Ala Asn Gln Ala Val Ala Ala Ala Gln Arg Arg Ala Met Arg Gln
100 105 110
Arg Met Leu Ala Gln Gln Ala Gln Gln Gln His Gly Gly Leu Ser Gly
115 120 125
Leu Gly Gly Gln Phe Gly Ala Ala Ser Ser Asp Val Tyr Ser Ser Gln
130 135 140
Ala Leu Ala Gln Ala Gln Thr Pro Gln Gln Tyr Leu Gln Tyr Gln Pro
145 150 155 160
Gln Thr Ala Arg Pro Val Ala Arg Pro Ala Pro Gln Val Tyr Ser Arg
165 170 175
Pro Gln Pro Gln Gly Lys Asp Glu Asp Leu Glu Glu Tyr Asp Thr Pro
180 185 190
Ala Ser Tyr Lys Phe Gly Phe Asp Val Thr Asp Asp Gln Asn Thr Asn
195 200 205
Tyr Gln Asn Arg Gln Glu Gln Arg Asp Gly Asn Lys Ile Ser Gly Ser
210 215 220
Tyr Ser Val Val Asp Ser Asp Gly Phe Ile Arg Thr Val Thr Tyr Thr
225 230 235 240
Ala Asp Pro Lys Glu Gly Phe Lys Ala Glu Val Val Arg Lys Pro Thr
245 250 255
Asp Ile Val Val Lys Ile Pro Thr Pro Thr Pro Ala Pro Lys Glu Gln
260 265 270
Tyr Val Gln Asn Tyr Ser Ala Pro Arg Gly Gln Glu Ser Lys Gly Asn
275 280 285
Val Ile Tyr Gln Tyr Gln
290
<210> 4
<211> 861
<212> DNA
<213> Unknown
<220>
<223> 编码双叉犀金龟表皮蛋白Td12611的核苷酸序列1
<400> 4
gaatcgggct tttcaggagt acagtattcc aatgcacaat cgttaacgcc tcaacagaca 60
caaccctcgt acattacagc ccctcagccg gtgcaaccgc agactccggc atatcaaccg 120
caaagtccgt cttttggacc ccccacacaa gtcgtgtctc agctactttt gcctacgaat 180
gtagatagac aaatacagaa agctgcccaa caaatagctg gtccgcaata tgtgcaacaa 240
attcaaatac ttcaacaaca agagcttgca aatcaagcag tagccgccgc acaaagacgt 300
gccatgagac aaagaatgct tgcacaacag gctcaacagc agcatggtgg tttaagtggt 360
ttaggtggac agttcggagc agcgtcgtcg gacgtatact ccagccaggc attagcacaa 420
gctcagacac cccaacaata tctacagtac caaccgcaga cagcgaggcc agttgctaga 480
ccagcaccgc aagtgtattc tcgtccacag cctcagggaa aagacgaaga tctcgaggaa 540
tatgatacgc ccgcatccta caagttcggc ttcgacgtaa cggacgatca aaacacgaat 600
taccagaacc gccaagaaca acgggacggg aacaaaatct ccggcagtta ttcagtggtg 660
gattcggatg gattcattag aaccgtaacg tataccgcgg atcctaagga aggattcaaa 720
gccgaggtcg tcagaaaacc gactgatatt gttgtgaaaa ttcctacacc tacaccagca 780
ccgaaagaac aatatgttca gaactactct gcgcctagag ggcaagaatc caaaggaaat 840
gtgatttacc agtaccaata a 861
<210> 5
<211> 930
<212> DNA
<213> Unknown
<220>
<223> 编码双叉犀金龟表皮蛋白Td12611的核苷酸序列2
<400> 5
atgattagta gttgtggtgt agcgctgtgt tatcttgcgc tggtgtctag tgtcatttct 60
gcaccagtag aatcgggctt ttcaggagta cagtattcca atgcacaatc gttaacgcct 120
caacagacac aaccctcgta cattacagcc cctcagccgg tgcaaccgca gactccggca 180
tatcaaccgc aaagtccgtc ttttggaccc cccacacaag tcgtgtctca gctacttttg 240
cctacgaatg tagatagaca aatacagaaa gctgcccaac aaatagctgg tccgcaatat 300
gtgcaacaaa ttcaaatact tcaacaacaa gagcttgcaa atcaagcagt agccgccgca 360
caaagacgtg ccatgagaca aagaatgctt gcacaacagg ctcaacagca gcatggtggt 420
ttaagtggtt taggtggaca gttcggagca gcgtcgtcgg acgtatactc cagccaggca 480
ttagcacaag ctcagacacc ccaacaatat ctacagtacc aaccgcagac agcgaggcca 540
gttgctagac cagcaccgca agtgtattct cgtccacagc ctcagggaaa agacgaagat 600
ctcgaggaat atgatacgcc cgcatcctac aagttcggct tcgacgtaac ggacgatcaa 660
aacacgaatt accagaaccg ccaagaacaa cgggacggga acaaaatctc cggcagttat 720
tcagtggtgg attcggatgg attcattaga accgtaacgt ataccgcgga tcctaaggaa 780
ggattcaaag ccgaggtcgt cagaaaaccg actgatattg ttgtgaaaat tcctacacct 840
acaccagcac cgaaagaaca atatgttcag aactactctg cgcctagagg gcaagaatcc 900
aaaggaaatg tgatttacca gtaccaataa 930
<210> 6
<211> 917
<212> DNA
<213> Unknown
<220>
<223> 编码双叉犀金龟表皮蛋白Td12611的核苷酸序列3
<400> 6
ccatggatca ccaccaccac caccacgaat cgggcttttc aggagtacag gaatcgggct 60
tttcaggagt acagtattcc aatgcacaat cgttaacgcc tcaacagaca caaccctcgt 120
acattacagc ccctcagccg gtgcaaccgc agactccggc atatcaaccg caaagtccgt 180
cttttggacc ccccacacaa gtcgtgtctc agctactttt gcctacgaat gtagatagac 240
aaatacagaa agctgcccaa caaatagctg gtccgcaata tgtgcaacaa attcaaatac 300
ttcaacaaca agagcttgca aatcaagcag tagccgccgc acaaagacgt gccatgagac 360
aaagaatgct tgcacaacag gctcaacagc agcatggtgg tttaagtggt ttaggtggac 420
agttcggagc agcgtcgtcg gacgtatact ccagccaggc attagcacaa gctcagacac 480
cccaacaata tctacagtac caaccgcaga cagcgaggcc agttgctaga ccagcaccgc 540
aagtgtattc tcgtccacag cctcagggaa aagacgaaga tctcgaggaa tatgatacgc 600
ccgcatccta caagttcggc ttcgacgtaa cggacgatca aaacacgaat taccagaacc 660
gccaagaaca acgggacggg aacaaaatct ccggcagtta ttcagtggtg gattcggatg 720
gattcattag aaccgtaacg tataccgcgg atcctaagga aggattcaaa gccgaggtcg 780
tcagaaaacc gactgatatt gttgtgaaaa ttcctacacc tacaccagca ccgaaagaac 840
aatatgttca gaactactct gcgcctagag ggcaagaatc caaaggaaat gtgatttacc 900
agtaccaata agaattc 917
<210> 7
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> 上游克隆引物P1
<400> 7
atgattagta gttgtggtgt agcgct 26
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 下游克隆引物P2
<400> 8
ttattggtac tggtaaatca catttccttt gg 32
<210> 9
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> 上游连接引物P3
<400> 9
ccatggatca ccaccaccac caccacgaat cgggcttttc aggagtacag 50
<210> 10
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 下游连接引物P4
<400> 10
gaattctcat tggtactggt aaatcacatt tcc 33
Claims (10)
1.一种双叉犀金龟表皮蛋白，其特征在于，所述蛋白的氨基酸序列包含或由如下序列组成：
a)SEQ ID NO.1所示的氨基酸序列；或，
b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列同一性的功能性同源序列；或
c)在SEQ ID NO.1所示的氨基酸序列中缺失、添加、替换一个或多个氨基酸且具有相同蛋白活性的氨基酸序列。
2.根据权利要求1所述的表皮蛋白，其特征在于，所述b)和/或c)的氨基酸序列包含SEQID NO.2所示的氨基酸序列；
优选地，所述b)和/或c)的氨基酸序列包含SEQ ID NO.3所示的氨基酸序列。
3.编码权利要求1或2所述的表皮蛋白的核苷酸序列。
4.根据权利要求3所述的编码表皮蛋白的核苷酸序列，其特征在于，所述核苷酸序列包含或由如下序列组成：
i)SEQ ID NO.4所示的核苷酸序列；或，
ii)SEQ ID NO.4所示的核苷酸序列的互补序列、简并序列或同源序列(优选同源性80％以上)；或，
iii)在严谨条件下与SEQ ID NO.4所示的核苷酸序列杂交，且能够编码所述表皮蛋白的核苷酸序列。
5.根据权利要求4所述的编码表皮蛋白的核苷酸序列，其特征在于，所述ii)和/或iii)的核苷酸序列包含SEQ ID NO.5所示的核苷酸序列；
优选地，所述ii)和/或iii)的核苷酸序列包含SEQ ID NO.6所示的核苷酸序列。
6.权利要求1或2所述的表皮蛋白，或权利要求3-5任一项所述的编码核苷酸序列在提升壳聚糖或其类似物的性能(优选机械性能)方面的应用。
7.载体，其特征在于，包含权利要求3-5任一项所述的核苷酸序列。
8.遗传工程化的宿主细胞，其特征在于，其包含权利要求1或2所述的氨基酸序列，或包含权利要求3-5任一项所述的核苷酸序列，或导入了权利要求7所述的载体；
优选地，所述宿主细胞包括植物细胞、动物细胞或微生物细胞中的至少一种；
优选地，所述微生物细胞包括大肠杆菌、假单孢菌、芽孢杆菌、酵母细胞中的至少一种。
9.制备权利要求1或2所述表皮蛋白的方法，其特征在于，包括以下步骤：
将权利要求3-5任一项所述的核苷酸序列，或权利要求7所述的载体导入宿主细胞中表达获得所述表皮蛋白；和/或，
采用权利要求8所述的遗传工程化的宿主细胞表达获得所述表皮蛋白。
10.一种复合材料，其特征在于，包括权利要求1或2所述的表皮蛋白；
优选地，所述复合材料还包括壳聚糖和/或其类似物；
优选地，所述复合材料为所述的表皮蛋白与壳聚糖和/或其类似物复合形成的薄膜材料；优选薄膜材料中表皮蛋白的浓度小于1％。
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| Title |
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| 卜云飞;刘田;陈磊;杨青;: "亚洲玉米螟表皮蛋白CPR9的表达纯化及与几丁质的相互作用", 中国生物防治学报, no. 01 * |
| 李智;甘进锋;蒙炳超;黄科;李春峰;何宁佳;周泽扬;: "家蚕表皮基因BmGCP2的克隆及表达谱分析", 蚕业科学, no. 03 * |
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| CN116063746B (zh) * | 2022-08-18 | 2024-04-02 | 中国农业科学院深圳农业基因组研究所 | 提升壳聚糖材料机械性能的方法及制备的壳聚糖复合材料 |
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