CN113774047B - 鱼源蛋白酶基因及其应用 - Google Patents
鱼源蛋白酶基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种鱼源蛋白酶基因及其应用，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列，或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。本发明的鱼源蛋白酶基因有助于促进鱼类对高蛋白人工饲料的消化吸收能力，提高鱼的生长性能，可以针对性地应用于鱼类养殖中，绿色高效，前景可观。
Description
技术领域
本发明属于动物基因工程技术领域，更具体地，本发明涉及一种鱼源蛋白酶基因，以及包含所述鱼源蛋白酶基因的重组质粒、包含所述重组质粒的酵母重组表达载体，及其在鱼类养殖和提高鱼类消化能力中的应用。
背景技术
鱼类的肠道是食物消化和营养吸收的重要场所。鱼摄食后，食物的营养物质均依赖各种酶类的作用。食物中的蛋白质和多肽需要经过消化液中或肠壁细胞中的蛋白酶的水解，使成为肠细胞所能吸收的氨基酸。因此，肠道蛋白酶的活性高低对鱼的生长代谢起重要作用。
当下鱼类养殖的现状是：在高蛋白人工饲料的持续投喂下，水质环境容易变差，导致鱼消化吸收能力降低，抵抗力下降，当受到外界刺激时便极其容易爆发寄生虫、细菌性及病毒性疾病。目前有较多研究都集中在产酶基因的克隆、酶学性质研究以及工程菌的构建表达，将作为传统免疫增强剂及益生菌的酵母菌作为鱼源蛋白酶的载体，应用于水产养殖少有报道。因此，筛选具有高产蛋白酶活的菌株，分析其蛋白酶相关基因，利用分子生物学手段对相关基因进行克隆表达，在酵母细胞表面展示蛋白质，使蛋白酶基因得以表达，在鱼类养殖过程中对于提高鱼的消化能力、提高生长性能等具有重要的意义。
细菌噬菌体、革兰氏阴性菌和革兰氏阳性菌均可以用于蛋白质的表面展示，但要将它们应用到食品和水产等其他领域，这些微生物存在许多缺点。如果用条件致病菌和致病菌表面展示蛋白质就存在很多潜在的危险，另外噬菌体和病毒大规模培养较为困难。酿酒酵母被认为是表面展示蛋白质的最佳微生物，一是该微生物安全，并且在食品工业，医学中已大量使用；二是酿酒酵母作为表达外源蛋白的宿主已有众多研究和多年历史，它的遗传体系和克隆方法都非常清晰，它能对外源蛋白质进行正确的折叠和糖基化；三是酿酒酵母有一整套分泌胞外蛋白质的机制，其展示的酶通过天然方式固定在细胞表面，对酶没有损伤。同时酿酒酵母培养方法简单可行，经济廉价，便于大规模生产。
发明内容
基于此，本发明的目的之一是提供一种鱼源蛋白酶基因及鱼源蛋白酶基因的表达蛋白，在提高鱼类消化能力中的应用，所述基因及其表达蛋白可以提高鱼对高蛋白人工饲料的消化能力，提高鱼的生长性能。
实现上述发明目的的具体技术方案如下：
鱼源蛋白酶基因在提高鱼类对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列，或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。
鱼源蛋白酶基因的表达蛋白在提高鱼类对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的表达蛋白的氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6所示；或如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸，但蛋白活性相同。
本发明还提供了一种插入有鱼源蛋白酶基因的开放阅读框的重组质粒和插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体。
实现上述发明目的的具体技术方案如下：
插入有鱼源蛋白酶基因的开放阅读框的重组质粒，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列，或为与SEQ IDNo.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ IDNo.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。
在其中一些实施例中，所述重组质粒具有如SEQ ID No.13、SEQ ID No.14或SEQID No.15所示的核苷酸序列。
插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列，或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。
所述酵母重组表达载体优选为插入有开放阅读框具有如SEQ ID No.1所示的核苷酸序列，或与SEQ ID No.1互补配对的核苷酸序列，或编码氨基酸序列如SEQ ID No.2的核苷酸序列的鱼源蛋白酶基因。
所述酵母重组表达载体优选为插入有序列如SEQ ID No.13所示的重组质粒。
本发明还提供了上述插入有鱼源蛋白酶基因的开放阅读框的重组质粒、插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体在鱼类养殖、提高鱼类对高蛋白人工饲料消化能力中的应用。
插入有鱼源蛋白酶基因的开放阅读框的重组质粒、插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体在鱼类养殖中的应用。
插入有鱼源蛋白酶基因的开放阅读框的重组质粒、插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体在提高鱼类对高蛋白人工饲料消化能力中的应用。
本发明还提供了一种提高鱼类对高蛋白人工饲料消化能力的生物制剂。
实现上述发明目的的具体技术方案如下：
一种提高鱼类对高蛋白人工饲料消化能力的生物制剂，其活性成份来源于插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体，或其活性成分含有鱼源蛋白酶基因的生物制品，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ IDNo.5所示的核苷酸序列，或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。
所述生物制剂的活性成份优选为：来源于插入有开放阅读框具有如SEQ ID No.1所示的核苷酸序列，或为与SEQ ID No.1互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2的核苷酸序列的鱼源蛋白酶基因的酵母重组表达载体；或来源于插入有序列如SEQ ID No.13所示的重组质粒的酵母重组表达载体；或其活性成分含有开放阅读框具有如SEQ ID No.1所示的核苷酸序列，或为与SEQ ID No.1互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2的核苷酸序列的鱼源蛋白酶基因。
本发明还提供了一种提高鱼类对高蛋白人工饲料消化能力的方法。
实现上述发明目的的具体技术方案如下：
一种提高鱼类对高蛋白人工饲料消化能力的方法，所述方法包括：对鱼类投喂提高鱼类消化能力的生物制剂，所述生物制剂其活性成份来源于插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体，或其活性成分含有鱼源蛋白酶基因的生物制品，所述鱼源蛋白酶基因的开放阅读框具有如SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示的核苷酸序列，或为与SEQ ID No.1、SEQ ID No.3或SEQ ID No.5互补配对的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2、SEQ ID No.4或SEQ ID No.6的核苷酸序列。
与现有技术相比，本发明具有以下有益效果：
本发明的发明人从加州鲈的肠道中筛选纯化三株高产蛋白酶菌株，通过基因注释汇总分析，选取开放阅读框编号分别是ORF32、ORF436、ORF160的三个蛋白酶基因，分别插入pyd1-GFP载体中，构建得到重组质粒，再将含蛋白酶基因的重组质粒转化至酿酒酵母EBY100中，得到酵母重组表达载体；将酵母重组表达载体与基础鱼饲料混合制成生物制剂，对鱼类进行28天投喂，发现含3种酵母重组表达载体的生物制剂可以明显降低饵料系数、提升鱼类的增重率和特定生长率，并且提高鱼肠道酶活，因此，有助于促进鱼类对高蛋白人工饲料的消化吸收能力，提高鱼的生长性能，尤其是含重组质粒pyd1-GFP-ORF32的酵母重组表达载体的生物制剂，效果最为明显。本发明的鱼源蛋白酶基因可以针对性地应用于鱼类养殖中，绿色高效，前景可观。
附图说明
图1为本发明鱼源产蛋白酶基因应用的技术路线图。
图2为本发明实施例1中产蛋白酶菌株筛选纯化图，其中a为菌株P1、P2纯化图，b为菌株D1纯化图。
图3为本发明实施例1中PCR扩增电泳图；其中，泳道1、2、3分别为基因ORF160、基因ORF32和基因ORF436的目的条带。
图4为本发明实施例1中未含重组质粒的酿酒酵母EBY100经诱导后的荧光检测图，其中a为明场图片，b为暗场图片，c为明暗场叠加图片。
图5为本发明实施例1中含重组质粒pyd1-GFP-ORF32的酵母重组表达载体经诱导后的荧光检测图，其中a为明场图片，b为暗场图片，c为明暗场叠加图片。
图6为本发明实施例1中含重组质粒pyd1-GFP-ORF436的酵母重组表达载体经诱导后的荧光检测图，其中a为明场图片，b为暗场图片，c为明暗场叠加图片。
图7为本发明实施例1中含重组质粒pyd1-GFP-ORF160的酵母重组表达载体经诱导后的荧光检测图，其中a为明场图片，b为暗场图片，c为明暗场叠加图片。
图8为本发明实施例2中在显微镜400倍视野下计数细菌计数板中总酵母数的方法示意图。
图9为本发明实施例2中对照组和实验组的加州鲈的增重率的显著性分析图。
图10为本发明实施例2中对照组和实验组的加州鲈的特定生长率的显著性分析图。
图11为本发明实施例2中对照组和实验组的加州鲈的饵料系数的显著性分析图。
图12为本发明实施例3中对照组和实验组的加州鲈肠道胃蛋白酶活性显著性分析图。
图13为本发明实施例3中对照组和实验组的加州鲈肠道胰蛋白酶活性显著性分析图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
请参考图1，为本发明鱼源产蛋白酶基因应用的技术路线图，包括筛选鱼源产蛋白酶菌株、筛选产蛋白酶基因、构建包含产蛋白酶基因的重组质粒、将重组质粒转入酿酒酵母中，经半乳糖诱导表达，进行加州鲈养殖试验等，以检验筛选得到的产蛋白酶基因的效果。
以下结合具体实施例和附图对本发明作进一步详细的说明。
实施例1 鱼源产蛋白酶菌株筛选、产蛋白酶基因筛选及重组质粒构建
本实施例包括：从加州鲈肠道筛选产蛋白酶菌株，再从菌株中分析筛选产蛋白酶基因，构建包含筛选得到的产蛋白酶基因的重组质粒，具体包括以下步骤：
(1)、产蛋白酶菌株的筛选
通过产蛋白酶筛选固体培养基(配方为：蛋白胨1g/100mL，酵母提取物0.5g/mL，氯化钠1g/mL，技术琼脂粉1.5g/100mL，脱脂奶粉1g/100mL，pH7.2)对加州鲈肠道菌株进行筛选，观察菌落周围是否有透明产酶圈。如果有透明的产酶圈出现，说明是产蛋白酶菌株，测定产酶菌株的产酶圈和菌落的直径比值，在三轮筛选后，初步选定3株产酶活性较高(产酶圈直径/菌落圈直径较大)的产蛋白酶菌株作为候选菌株。
(2)、产蛋白酶菌株的纯化
用上述产蛋白酶筛选培养基进行菌株纯化(图2为产酶菌株经纯化后的平板图，a为菌株P1和P2纯化后的图，b为菌株D1纯化后的图)，经过六轮纯化后，将3株候选菌株进行细菌基因组denovo测序，通过16SrRNA测序，3株候选菌株分别为普通变形杆菌(编号D1，GenBank:CP023965.1，Proteus vulgaris)、维氏气单胞菌(P1，GenBank:CP058912.1，Aeromonas veronii)和豚鼠气单胞菌(P2，GenBank:AP022013.1，Aeromonas caviae)。在6个数据库(NR，Swiss-prot，Pfam，eggNOG，GO和KEGG)中得到相关基因的注释结果。通过基因注释汇总分析：与蛋白酶相关的基因共有68个。
(3)、产蛋白酶基因的筛选
将上述68个与蛋白酶相关的基因序列使用DNAMAN进行多序列的比对分析其同源性，选取研究报道较多的金属蛋白酶基因和丝氨酸蛋白酶基因进行后续重组质粒的构建工作，其开放阅读框编号分别是ORF32、ORF436、ORF160。
ORF32(SprT family zinc-dependent metalloprotease)
该基因是豚鼠气单胞菌(P2)基因组染色体上筛选到的基因，开放阅读框ORF编号32，全长540bp，核苷酸序列如SEQ ID No.1所示，编码氨基酸179个，氨基酸序列如SEQ IDNo.2所示，是金属蛋白酶家族中的一种锌依赖性金属蛋白酶基因。金属蛋白酶广泛存在生物有机体中，参与很多重要的生理过程，如消化、血管生成和细胞浸润、转移等。越来越多的研究显示金属蛋白酶参与了机体炎症反应的多个方面，作为降解细胞外基质的重要蛋白，金属蛋白酶对调节创伤愈合同样有重要的作用。因此调控金属蛋白酶的活性和功能具有很大的临床应用价值。
核酸序列长度Nucleotide Length＝540bp，去掉终止密码子扩增基因片段长度共537bp，序列如下(SEQ ID No.1)：
ATGTCTGCCACCAGAGCCCAACTCGACGCTTCCCAGCTCCTGCTGCTGCACCAGCGGGTCGACGCCTGTTTCGCGCAGGCGGAGGCACGCCTCGGCCGCCCCTTCCCGCGCCCGCAGATCCACTGCAACATGCGGGGCCGGGCGGCAGGGTCTGCTCGGCTGCAAACCTGGGAGCTGCGTTTCAATCCGGCGCTCTATCAGGCCAATCAGCAGGCGTTTCTCAGGGAAGTGGTGCCCCACGAGGTGGCGCACCTGCTGGTCTATGCGCTCTGGGGAGAGGGGCGCGGCAAGAGCCGGGTACTGCCCCACGGTCGCCAGTGGCAGTCGGTGATGCGGGATCTGTTCGGTCTCGAACCCAGCACCACCCACAGCTTTGATCTGGGGGTGCTGGCCCAGCGCACCTTCGTGTATGCCTGCGCCTGCCAGCAGCATCCCCTCTCGGTGCGCCGCCACAACAAGGTGATGCGCGGCGAGGCCCGCTATCACTGCCGCCGCTGTCGCCAGCCCCTGGTGTGGCAGCGCGACACGACGGCGGATTGA
其编码的氨基酸序列如下(SEQ ID No.2)：
MSATRAQLDASQLLLLHQRVDACFAQAEARLGRPFPRPQIHCNMRGRAAGSARLQTWELRFNPALYQANQQAFLREVVPHEVAHLLVYALWGEGRGKSRVLPHGRQWQSVMRDLFGLEPSTTHSFDLGVLAQRTFVYACACQQHPLSVRRHNKVMRGEARYHCRRCRQPLVWQRDTTAD
ORF436(metalloprotease TldD)
该基因是普通变形杆菌(D1)基因组染色体上筛选到的目的基因，开放阅读框ORF编号436，全长1446bp，核苷酸序列如SEQ ID No.3所示，编码氨基酸481个，氨基酸序列如SEQ ID No.4所示。同样是一个金属蛋白酶的基因。
核酸序列长度Nucleotide Length＝1446bp，去掉终止密码子扩增基因片段长度共1443bp，序列如下(SEQ ID No.3)：
ATGAGTTTAGCTGTTGTCAGCGAAAGTCTGTTGGAAGCAAACAAACTTAGTTTAGATGATTTAGCATCAACACTAGAGCAGCTTGCACAGCGTCAAATTGATTATGGTGATCTTTATTTTCAGTCAAGTTATCACGAGGCTTGGAGCCTTGATGATCAGATTATTAAAGATGGCTCTTACAATATTGATCAAGGTGTTGGTGTTAGAGCAATTTACGGTGAAAAAACCGGTTTTGCTTATGCTGACCAACTAACGCTTAACGCACTTAACCAAAGTGCTCATGCTGCACGAAGTATTGTTCAGGCTAAAGGTAATGGCCGTATCCATACTTTAGGAGCTATTCAACATTCTCCGCTATACAGCTTAAATGATCCTCTGCAAAGCCTTTCTCGTGAAGAGAAAATTGCATTATTGCATGAGGTAGATAAAGTCGCTCGTGCTGAAGATAAACGCGTTAAACAAGTTAATGCGTCATTAACTGGTGTTTATGAGCATGTGCTGGTTGCAGCAACCGATGGTACGTTCGCCGCTGATGTGCGTCCTTTAGTTCGCCTTTCTGTCAGCGTGCTGGTGGAAGAAGATGGCAAACGTGAGCGTGGCGCAAGTGGTGGCGGTGGTCGTTTTGGTTATGACTATTTTTTAACTAAAGTGGATGGTGAAAGCCATGCAGTCACTTATGCTCGTGAAGCAGTACGTATGGCATTAGTGAATTTATCAGCGATTGCAGCACCAGCAGGAACAATGCCTGTGGTATTAGGTGCAGGATGGCCAGGTGTATTATTGCATGAAGCTGTGGGTCATGGTTTAGAAGGTGATTTCAACCGCCGTGAAACCTCTGTATTTTCTGGTCGCCTTGGTGAGAAAGTTACTTCTGAGCTTTGTACGATTGTTGATGATGGTACTCTTGAAGGCCGTCGAGGCTCTGTTGCTATCGACGATGAAGGTGTTCCGGGTCAATACAATGTCTTAATCGAAAACGGCATCTTAAAAGGCTATATGCAAGATAAGATGAATGCACGTTTAATGGGTGTTTCACCAACAGGAAATGGTCGTCGTGAGTCTTATGCACATCTTCCTATGCCTCGTATGACAAACACTTATATGTTAGCAGGCAAATCTTCGCCTGAAGAAATTATTACTAGCGTTGATCGCGGTATTTACGCACCAAACTTTGGTGGCGGTCAGGTTGATATCACATCAGGTAAATTTGTTTTCTCAACCTCAGAAGCTTATTTAATCGAGAATGGAAAAATAACAAAACCAATTAAAGGGGCAACTCTGATTGGTTCAGGTATTGAAGCCATGCAACAGGTCTCTATGGTGGGAAATGATCTCGCTTTAGATAAAGGAGTGGGCGTTTGTGGTAAAGAAGGACAAAGCCTCCCTGTTGGTGTCGGTCAACCTACGTTGAAGCTTGATAAGATCACCGTAGGCGGTACTGCTTAA
其编码的蛋白长度Protein Length＝481aa，氨基酸序列如下(SEQ ID No.4)：
MSLAVVSESLLEANKLSLDDLASTLEQLAQRQIDYGDLYFQSSYHEAWSLDDQIIKDGSYNIDQGVGVRAIYGEKTGFAYADQLTLNALNQSAHAARSIVQAKGNGRIHTLGAIQHSPLYSLNDPLQSLSREEKIALLHEVDKVARAEDKRVKQVNASLTGVYEHVLVAATDGTFAADVRPLVRLSVSVLVEEDGKRERGASGGGGRFGYDYFLTKVDGESHAVTYAREAVRMALVNLSAIAAPAGTMPVVLGAGWPGVLLHEAVGHGLEGDFNRRETSVFSGRLGEKVTSELCTIVDDGTLEGRRGSVAIDDEGVPGQYNVLIENGILKGYMQDKMNARLMGVSPTGNGRRESYAHLPMPRMTNTYMLAGKSSPEEIITSVDRGIYAPNFGGGQVDITSGKFVFSTSEAYLIENGKITKPIKGATLIGSGIEAMQQVSMVGNDLALDKGVGVCGKEGQSLPVGVGQPTLKLDKITVGGTA
ORF160(rhomboid family intramembrane serine protease)
该基因是普通变形杆菌(D1)基因组染色体上筛选到的基因，开放阅读框ORF编号160，全长585bp，核苷酸序列如SEQ ID No.5所示，编码氨基酸194个，氨基酸序列如SEQ IDNo.6所示，该基因是扁菱形蛋白家族内膜丝氨酸蛋白酶基因，它是一类以丝氨酸为活性中心的重要蛋白水解酶，在生物有机体中发挥着广泛而又重要的作用。研究显示，丝氨酸蛋白酶在胚胎发育、细胞分化、组织重建和血管形成中有着重要作用，以其降解、消化和凝血效果最为显著。
核酸序列长度Nucleotide Length＝585bp，去掉终止密码子扩增基因片段长度共582bp，序列如下(SEQ ID No.5)：
ATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTAACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAGAACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTACTCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATTATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTTGTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCTATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATTGCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAATAG
其编码的蛋白长度Protein Length＝194aa，氨基酸序列如下(SEQ ID No.6)：
MDKIWFKKRLTFLGGLTIILVLLQLINSLLPISLLQWGIIPRTGEGLIGIFIAPFIHGSWSHLFSNLLPLLILSFLSMTQSLREYVLSSIFIIIVSGLLVWIFGRNAVHVGASGWIFGLWSLLIAHAFTRRKIIDIVIALFVLFYYGSMAYGLIPGQLGVSTESHISGVIAGLLYAWCARKLIRRKSRVVEVAK
(4)、基因序列信号肽分析及含酶切位点引物设计
经SignalP 5.0对上述三条目的氨基酸序列进行信号肽分析，三条目的氨基酸序列均无明显信号肽。以pYD1-GFP载体序列为前提，设计含有酶切位点的引物(如表1，分别为针对ORF32基因的SEQ ID No.7和SEQ ID No.8，针对ORF436基因的SEQ ID No.9和SEQ IDNo.10，针对ORF160基因的SEQ ID No.11和SEQ ID No.12)。
使用天根细菌基因组DNA提取试剂盒(货号DP302)提取三株细菌(细菌编号D1、P1、P2)的DNA，使用表1引物对提取的DNA进行扩增，电泳结果如图3所示，结果表明，扩增得到的条带大小符合预期，为目的基因ORF32、ORF436和ORF160。
表1 含酶切位点引物序列
(5)、重组质粒构建
使用AXYGEN PCR纯化试剂盒对PCR产物进行纯化回收，使用TAKARA快切酶BamH I和EcoR I分别对ORF32、ORF436、ORF160以及载体pYD1-GFP(GFP为绿色应荧光蛋白基因，载体序列如SEQ ID No.16所示，载体已于实验前构建好，保存于申请人实验室)分别进行双酶切：于30℃用BamH I酶切1.5h后于37℃用EcoR I酶切1.5h。酶切体系50uL：快切酶各1.25uL，buffer5uL，纯化后片段全加进去，不足用ddH2O补够50uL。
使用AXYGEN PCR纯化试剂盒回收产物进行目的片段和载体酶切后的纯化回收。分别测浓度，计算目的片段与载体连接使用量，按载体摩尔浓度：插入目的片段摩尔浓度比＝1:3，并加入TAKARA的T4 DNA连接酶与对应buffer组成20uL体系，于16℃金属浴连接过夜。
取10uL连接产物加入TOP10感受态细胞(TOP10感受态细胞为申请人实验室长期备有)，冰浴放置30min，42℃水浴80s，冰浴2min，加入800uL的LB液体(配方为：蛋白胨1g/100mL，酵母提取物0.5g/mL，氯化钠1g/mL，pH7.2)37℃复苏1h，取200uL涂布氨苄抗性LB固体平板。37℃培养12h后，挑取5个单克隆进行摇菌送北京擎科生物科技有限公司进行测序验证。确定插入序列正确后，提取质粒，剩余菌液以15％终浓度的甘油进行菌的保种，-20℃备用。重组质粒pyd1-GFP-ORF32、pyd1-GFP-ORF436和pyd1-GFP-ORF160的序列分别如SEQID No.13、SEQ ID No.14、SEQ ID No.15所示。
pyd1-GFP-ORF32重组质粒(SEQ ID No.13)，其中下划线部分为ORF32基因片段
GGTGATCGTCCGACTAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGTCTGCCACCAGAGCCCAACTCGACGCTTCCCAGCTCCT GCTGCTGCACCAGCGGGTCGACGCCTGTTTCGCGCAGGCGGAGGCACGCCTCGGCCGCCCCTTCCCGCGCCCGCAGA TCCACTGCAACATGCGGGGCCGGGCGGCAGGGTCTGCTCGGCTGCAAACCTGGGAGCTGCGTTTCAATCCGGCGCTC TATCAGGCCAATCAGCAGGCGTTTCTCAGGGAAGTGGTGCCCCACGAGGTGGCGCACCTGCTGGTCTATGCGCTCTG GGGAGAGGGGCGCGGCAAGAGCCGGGTACTGCCCCACGGTCGCCAGTGGCAGTCGGTGATGCGGGATCTGTTCGGTC TCGAACCCAGCACCACCCACAGCTTTGATCTGGGGGTGCTGGCCCAGCGCACCTTCGTGTATGCCTGCGCCTGCCAG CAGCATCCCCTCTCGGTGCGCCGCCACAACAAGGTGATGCGCGGCGAGGCCCGCTATCACTGCCGCCGCTGTCGCCA GCCCCTGGTGTGGCAGCGCGACACGACGGCGGATGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGGCAGAATTTTTT
pyd1-GFP-ORF436重组质粒(SEQ ID No.14)，其中下划线部分为ORF436基因片段
TGTCTGACAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGAGTTTAGCTGTTGTCAGCGAAAGTCTGTTGGAAGCAAACAAACTT AGTTTAGATGATTTAGCATCAACACTAGAGCAGCTTGCACAGCGTCAAATTGATTATGGTGATCTTTATTTTCAGTC AAGTTATCACGAGGCTTGGAGCCTTGATGATCAGATTATTAAAGATGGCTCTTACAATATTGATCAAGGTGTTGGTG TTAGAGCAATTTACGGTGAAAAAACCGGTTTTGCTTATGCTGACCAACTAACGCTTAACGCACTTAACCAAAGTGCT CATGCTGCACGAAGTATTGTTCAGGCTAAAGGTAATGGCCGTATCCATACTTTAGGAGCTATTCAACATTCTCCGCT ATACAGCTTAAATGATCCTCTGCAAAGCCTTTCTCGTGAAGAGAAAATTGCATTATTGCATGAGGTAGATAAAGTCG CTCGTGCTGAAGATAAACGCGTTAAACAAGTTAATGCGTCATTAACTGGTGTTTATGAGCATGTGCTGGTTGCAGCA ACCGATGGTACGTTCGCCGCTGATGTGCGTCCTTTAGTTCGCCTTTCTGTCAGCGTGCTGGTGGAAGAAGATGGCAA ACGTGAGCGTGGCGCAAGTGGTGGCGGTGGTCGTTTTGGTTATGACTATTTTTTAACTAAAGTGGATGGTGAAAGCC ATGCAGTCACTTATGCTCGTGAAGCAGTACGTATGGCATTAGTGAATTTATCAGCGATTGCAGCACCAGCAGGAACA ATGCCTGTGGTATTAGGTGCAGGATGGCCAGGTGTATTATTGCATGAAGCTGTGGGTCATGGTTTAGAAGGTGATTT CAACCGCCGTGAAACCTCTGTATTTTCTGGTCGCCTTGGTGAGAAAGTTACTTCTGAGCTTTGTACGATTGTTGATG ATGGTACTCTTGAAGGCCGTCGAGGCTCTGTTGCTATCGACGATGAAGGTGTTCCGGGTCAATACAATGTCTTAATC GAAAACGGCATCTTAAAAGGCTATATGCAAGATAAGATGAATGCACGTTTAATGGGTGTTTCACCAACAGGAAATGG TCGTCGTGAGTCTTATGCACATCTTCCTATGCCTCGTATGACAAACACTTATATGTTAGCAGGCAAATCTTCGCCTG AAGAAATTATTACTAGCGTTGATCGCGGTATTTACGCACCAAACTTTGGTGGCGGTCAGGTTGATATCACATCAGGT AAATTTGTTTTCTCAACCTCAGAAGCTTATTTAATCGAGAATGGAAAAATAACAAAACCAATTAAAGGGGCAACTCT GATTGGTTCAGGTATTGAAGCCATGCAACAGGTCTCTATGGTGGGAAATGATCTCGCTTTAGATAAAGGAGTGGGCG TTTGTGGTAAAGAAGGACAAAGCCTCCCTGTTGGTGTCGGTCAACCTACGTTGAAGCTTGATAAGATCACCGTAGGC GGTACTGCTGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG
pyd1-GFP-ORF160重组质粒(SEQ ID No.15)，其中下划线部分为ORF160基因片段
CGTCCGACAGCAAGGCAGCCCCATAAACACACAGTATGTTTTTAAGCTTCTGCAGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGTACCAGGATCCATGGATAAAATTTGGTTTAAAAAAAGACTCACTTTTCTTGGTGGGTTA ACTATCATATTAGTATTACTTCAACTAATTAACTCACTACTCCCCATCTCTCTTCTTCAATGGGGCATTATTCCAAG AACAGGTGAAGGTCTAATTGGTATTTTTATTGCGCCTTTCATTCATGGATCTTGGTCTCATCTATTTAGTAATCTAC TCCCGCTTCTTATTCTTAGCTTTTTATCCATGACCCAATCTCTACGAGAATATGTGTTATCCAGTATATTTATCATT ATCGTAAGCGGTTTATTAGTTTGGATTTTTGGACGAAATGCTGTTCACGTTGGTGCAAGTGGATGGATTTTTGGGTT GTGGTCTTTGCTTATTGCTCACGCTTTTACTCGACGTAAAATCATCGATATTGTGATCGCACTCTTTGTTCTATTCT ATTATGGATCAATGGCCTACGGATTAATCCCAGGACAATTAGGTGTATCAACAGAATCACATATTTCAGGTGTTATT GCAGGGCTACTTTATGCATGGTGTGCAAGAAAGCTAATTCGCCGTAAAAGCCGAGTAGTAGAAGTGGCTAAAGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGGGTGGTGGTGGTTCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCCCTACGGCAGCTGACCCTGAAGTCATCTGCCCACCGGCAGCTGCCGTGCCTGGCCCACCTCGGACACCCTGACT
(6)、酵母感受态制备、转化与阳性单克隆筛选
使用酵母化学感受态细胞制备试剂盒(货号CAT#:81109-20，北京天恩泽基因科技有限公司)，进行酿酒酵母EBY100的感受态细胞制备及转化。制备及转化方法参考试剂盒说明书。
使用YNB-CAA-葡萄糖培养基(配方如表3)对pyd1-GFP-ORF32重组质粒、pyd1-GFP-ORF436重组质粒、pyd1-GFP-ORF160重组质粒进行扩培，扩培后提取pyd1-GFP-ORF32重组质粒、pyd1-GFP-ORF436重组质粒、pyd1-GFP-ORF160重组质粒，通过YNB转化固体培养基(配方如表2)分别转入酿酒酵母EBY100感受态细胞，待48h后长出单克隆，挑取单克隆于YNB-CAA-葡萄糖培养基(配方如表3)进行扩培。送测序，鉴定成功后保种。
取保种的菌株，在YNB转化固体培养基上划线，30℃，培养48h，待长出明显单菌落，挑菌至液体YNB-CAA葡萄糖培养基(50mL离心管，含20mL培养基)中，30℃，培养48h，然后常温6000g离心10min，把菌体转100mL至液体YNB-CAA葡萄糖培养基(锥形瓶)中，30℃，培养24h，此时菌液OD值1.5-2.0之间，常温6000g离心10min后重悬于等体积的YNB-CAA半乳糖诱导液体培养基(配方如表4)，以1:2稀释转入1L锥形瓶中(内含300mL YNB-CAA半乳糖诱导液体培养基)，在20℃恒温摇床，200rpm培养72h，进行诱导。
表2 YNB转化固体培养基(转化用)
| 组份 | 用量1L | 100mL |
| 葡萄糖 | 20g | 2g |
| YNB | 6.7g | 0.67g |
| Leucine(亮氨酸) | 0.1g | 0.01g |
| 琼脂 | 15g | 1.5g |
| ddH2O | To1L | To100mL |
注：115℃高压灭菌30min。其中Leucine不能高压灭菌，配备1％溶液，80℃助溶后，0.22um过滤除菌，然后每90mL培养基加入10mL。
表3 液体YNB-CAA葡萄糖培养基(挑单克隆及扩培用)
| 组份 | 用量1L | 100mL |
| 葡萄糖 | 20g | 2g |
| YNB | 6.7g | 0.67g |
| 酸水解酪蛋白 | 5g | 0.5g |
| ddH2O | To1L | To100mL |
注：0.22um过滤除菌，或115℃高压灭菌30min。
表4 液体YNB-CAA半乳糖诱导培养基
| 组份 | 用量1L | 100mL |
| 2％半乳糖 | 20g | 2g |
| YNB | 6.7g | 0.67g |
| 酸水解酪蛋白 | 5g | 0.5g |
| ddH2O | To900mL | To90mL |
注：配制20％半乳糖溶液，0.22um过滤除菌，每90mL培养基加入10mL。
诱导结束后，通过伯乐荧光细胞成像仪ZOE进行菌液荧光检测，通过明场、暗场及明暗场叠加检测，结果表明，与酿酒酵母EBY100相比(见图4中的a、b和c)，含重组质粒pyd1-GFP-ORF32的酵母重组表达载体(见图5中的a、b和c)、含重组质粒pyd1-GFP-ORF436的酵母重组表达载体(见图6中的a、b和c)、含重组质粒pyd1-GFP-ORF160的酵母重组表达载体(见图7中的a、b和c)，经诱导后，均有绿色荧光。
实施例2 三个含重组质粒的酵母重组表达载体对养殖鱼类生长性能的影响
本实施例是使用实施例1的三个含重组质粒的酵母重组表达载体进行试验的，具体包括以下步骤：
1、试验分组
使用含400L水体(平均水温26℃-28℃)的大蓝桶进行加州鲈动物试验，设置三个实验组和一个对照组，每组90尾鱼，共设3个平行，即每个平行30尾鱼，以下实施例中分别用对照组1、对照组2、对照组3、实验组1(ORF32-1、ORF32-2、ORF32-3)、实验组2(ORF436-1、ORF436-2、ORF436-3)、实验组3(ORF160-1、ORF160-2、ORF160-3)表示。
2、酵母计数及菌量设定
将实施例1中含重组质粒pyd1-GFP-ORF32的酵母重组表达载体，含重组质粒pyd1-GFP-ORF436的酵母重组表达载体，含重组质粒pyd1-GFP-ORF160的酵母重组表达载体稀释100倍后，置于Auvon Helber Thoma细菌计数板(计数板标注一个小方格体积为V＝1/400mm2*0.02mm＝5*10-8cm3)，在显微镜400倍视野下计数细菌计数板16个中方格总酵母数M(如图8所示)，计数时，只计算落在方格内的酵母数，若酵母出现出芽生殖，芽体小于母细胞一半时为1个酵母细胞，否则计数为2个酵母细胞。则每mL酵母个数N＝(M*100)/(16*16*5*10-8)CFU/ml。其中：100为菌液稀释倍数；16分别为中方格个数和小方格个数；CFU为Colony-Forming Units的缩写，即菌落形成单位。
按每尾鱼每餐摄入2*108个重组酵母菌(即酵母重组表达载体)，将重组酵母菌均匀喷洒添加至基础料(珠海海龙生物科技有限公司生产的加州鲈鱼膨化配合饲料2#料)中，自然风干30min后进行投喂。投喂含喷洒重组酵母菌的饲料量必须小于每次投喂量以保证鱼完全摄食含喷洒重组酵母菌的饲料，再以基础料补足，以保证投喂量。
3、投喂方式
每组投喂类型、投喂频率如表5所示。
表5 各组的投喂方式
4、各组投喂量统计
28天后，统计各组投喂量，如表6所示。
表6 投喂量统计
5、四周饵料系数、增重率和特定生长率的统计
28天后，对四周饵料系数等进行了统计，结果如表7所示。
表7 四周饵料系数统计
28天后，对各组鱼的增重率和特定生长率进行了统计，结果如表8所示。
表8 各平行鱼的增重率、特定生长率及饵料系数统计
注：饵料系数＝总投饵量/(末重-始重)×100％，WGR＝(末重-初重)/初重*100％，SGR＝(ln末重-ln初重)/养殖时间*100％。
各组鱼的增重率、特定生长率和饵料系数显著性分析分别如图9、图10、图11所示。
从表7、8和图9～11可以看出：加州鲈鱼28天养殖实验，饵料系数：对照组＞实验组3＞实验组2＞实验组1，增重率：实验组1＞实验组2＞实验组3＞对照组，特定生长率：实验组1＞实验组2＞实验组3＞对照组，其中，实验组1的饵料系数、增重率与特定生长率与对照组相比，都达到了显著性差异。本实施例的结果说明：对加州鲈投喂含重组质粒pyd1-GFP-ORF32的酵母重组表达载体的饲料、含重组质粒pyd1-GFP-ORF436的酵母重组表达载体的饲料、含重组质粒pyd1-GFP-ORF160的酵母重组表达载体的饲料，能明显提升加州鲈的生长性能，体现在降低饵料系数，提高增重率和特定生长率，尤其是对加州鲈投喂含重组质粒pyd1-GFP-ORF32的酵母重组表达载体的饲料，饵料系数最低，增重率和特定生长率最高。
实施例3 三个含重组质粒的酵母重组表达载体对鱼类蛋白酶活性的影响
采用实施例2的分组方式、投喂方式，进行加州鲈养殖，28天后，每个组每个平行取3尾鱼的肠道组织进行混样匀浆稀释5倍后进行酶活性的测定。采用Solarbio胃蛋白酶活性检测试剂盒(货号：BC2325规格：100T/48S)测定各组鱼的胃蛋白酶活，采用Solarbio胰蛋白酶活性检测试剂盒(货号：BC2315规格：100T/96S)测定各组鱼的胰蛋白酶活。酶活测定操作步骤和酶活计算公式均参考试剂盒说明书。
1、养殖28天后，加州鲈鱼肠道胃蛋白酶活性检测(胃蛋白酶试剂盒)
按样本质量计算酶活性：37℃下每克组织每分钟催化血红蛋白水解成1umol酪氨酸为一个酶活单位。
计算公式：胃蛋白酶酶活(U/g)＝(ΔA÷ε÷d÷V反总)÷(WxV样÷V提)÷T＝0.786÷ΔA÷Wx*稀释倍数
ΔA＝A测定管-A对照管，其中，A对照管：按照试剂盒的说明，血红蛋白和相关试剂反应后的吸光值；A测定管：血红蛋白和相关试剂以及样品反应后的吸光值；
Wx：样本质量，0.1g；V反总：反应总体积，0.22mL；V提：粗酶液总体积，1mL；T：催化反应时间，10min；V样：加入样本体积，0.02mL；V液：液体体积，0.1mL；ε：酪氨酸吸光系数，1.4μmol-1·mL·cm-1；d：光程，1cm。稀释倍数：5。测定结果如表9所示。
表9 各组鱼肠道胃蛋白酶活性检测结果
2、养殖28天后，加州鲈鱼肠道胰蛋白酶活性检测(胰蛋白酶试剂盒)
按样本质量计算酶活性：1mL体系下，37℃下每克组织每分钟催化253nm处吸光值增加0.001为一个酶活单位。
计算公式：胰蛋白酶酶活(U/g)＝(ΔA测定-ΔA空白)÷0.001÷(W×V1÷V2)÷T×(V3÷V4)＝105×(ΔA测定-ΔA空白)÷Wx*稀释倍数
Wx：样本质量，0.1g；V1：加入反应体系中粗酶液体积，2μL＝0.002mL；V2：粗酶液总体积，1mL；T：反应时间，1min；V3：反应总体积，198μL+2μL＝200μL＝0.2mL；V4：1mL体系。稀释倍数：5。测定结果如表10所示。
表10 各组鱼肠道胰蛋白酶活性检测结果
3、肠道酶活显著性分析
使用SPSS分析软件，在工具栏中选择分析-比较平均值-单因素ANOVA检验。对表9和表10中肠道胃蛋白酶活和胰蛋白酶活的结果进行显著性分析，结果如表11、图12和图13所示。
表11 肠道酶活显著性分析结果
从表11、图12和图13可以看出，实验组1～3的胃蛋白酶活和胰蛋白酶活均高于对照组，其中，实验组1与对照组相比，可以显著提高胃蛋白酶活性(P＜0.5)，实验组1和实验组3与对照组相比，可以显著提高胰蛋白酶活性(P＜0.5)。本实施例的结果说明：对加州鲈投喂含重组质粒pyd1-GFP-ORF32的酵母重组表达载体的饲料、含重组质粒pyd1-GFP-ORF436的酵母重组表达载体的饲料、含重组质粒pyd1-GFP-ORF160的酵母重组表达载体的饲料，能提升加州鲈的肠道胃蛋白酶活和胰蛋白酶活，其中，加州鲈投喂含重组质粒pyd1-GFP-ORF32的酵母重组表达载体的饲料，提高胃蛋白酶和胰蛋白酶活性的效果最为显著。
综上可知，转入重组质粒ORF32、ORF436、ORF160的酿酒酵母EBY100重组表达菌体，按一定量添加到饲料中，可以降低饵料系数，提高生长性能，提高鱼肠道酶活。其中转入重组质粒ORF32的酿酒酵母EBY100重组表达载体综合效果最佳。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广东省农业科学院动物卫生研究所
<120> 鱼源蛋白酶基因及其应用
<130> 1
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 540
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtctgcca ccagagccca actcgacgct tcccagctcc tgctgctgca ccagcgggtc 60
gacgcctgtt tcgcgcaggc ggaggcacgc ctcggccgcc ccttcccgcg cccgcagatc 120
cactgcaaca tgcggggccg ggcggcaggg tctgctcggc tgcaaacctg ggagctgcgt 180
ttcaatccgg cgctctatca ggccaatcag caggcgtttc tcagggaagt ggtgccccac 240
gaggtggcgc acctgctggt ctatgcgctc tggggagagg ggcgcggcaa gagccgggta 300
ctgccccacg gtcgccagtg gcagtcggtg atgcgggatc tgttcggtct cgaacccagc 360
accacccaca gctttgatct gggggtgctg gcccagcgca ccttcgtgta tgcctgcgcc 420
tgccagcagc atcccctctc ggtgcgccgc cacaacaagg tgatgcgcgg cgaggcccgc 480
tatcactgcc gccgctgtcg ccagcccctg gtgtggcagc gcgacacgac ggcggattga 540
<210> 2
<211> 179
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Ala Thr Arg Ala Gln Leu Asp Ala Ser Gln Leu Leu Leu Leu
1 5 10 15
His Gln Arg Val Asp Ala Cys Phe Ala Gln Ala Glu Ala Arg Leu Gly
20 25 30
Arg Pro Phe Pro Arg Pro Gln Ile His Cys Asn Met Arg Gly Arg Ala
35 40 45
Ala Gly Ser Ala Arg Leu Gln Thr Trp Glu Leu Arg Phe Asn Pro Ala
50 55 60
Leu Tyr Gln Ala Asn Gln Gln Ala Phe Leu Arg Glu Val Val Pro His
65 70 75 80
Glu Val Ala His Leu Leu Val Tyr Ala Leu Trp Gly Glu Gly Arg Gly
85 90 95
Lys Ser Arg Val Leu Pro His Gly Arg Gln Trp Gln Ser Val Met Arg
100 105 110
Asp Leu Phe Gly Leu Glu Pro Ser Thr Thr His Ser Phe Asp Leu Gly
115 120 125
Val Leu Ala Gln Arg Thr Phe Val Tyr Ala Cys Ala Cys Gln Gln His
130 135 140
Pro Leu Ser Val Arg Arg His Asn Lys Val Met Arg Gly Glu Ala Arg
145 150 155 160
Tyr His Cys Arg Arg Cys Arg Gln Pro Leu Val Trp Gln Arg Asp Thr
165 170 175
Thr Ala Asp
<210> 3
<211> 1446
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgagtttag ctgttgtcag cgaaagtctg ttggaagcaa acaaacttag tttagatgat 60
ttagcatcaa cactagagca gcttgcacag cgtcaaattg attatggtga tctttatttt 120
cagtcaagtt atcacgaggc ttggagcctt gatgatcaga ttattaaaga tggctcttac 180
aatattgatc aaggtgttgg tgttagagca atttacggtg aaaaaaccgg ttttgcttat 240
gctgaccaac taacgcttaa cgcacttaac caaagtgctc atgctgcacg aagtattgtt 300
caggctaaag gtaatggccg tatccatact ttaggagcta ttcaacattc tccgctatac 360
agcttaaatg atcctctgca aagcctttct cgtgaagaga aaattgcatt attgcatgag 420
gtagataaag tcgctcgtgc tgaagataaa cgcgttaaac aagttaatgc gtcattaact 480
ggtgtttatg agcatgtgct ggttgcagca accgatggta cgttcgccgc tgatgtgcgt 540
cctttagttc gcctttctgt cagcgtgctg gtggaagaag atggcaaacg tgagcgtggc 600
gcaagtggtg gcggtggtcg ttttggttat gactattttt taactaaagt ggatggtgaa 660
agccatgcag tcacttatgc tcgtgaagca gtacgtatgg cattagtgaa tttatcagcg 720
attgcagcac cagcaggaac aatgcctgtg gtattaggtg caggatggcc aggtgtatta 780
ttgcatgaag ctgtgggtca tggtttagaa ggtgatttca accgccgtga aacctctgta 840
ttttctggtc gccttggtga gaaagttact tctgagcttt gtacgattgt tgatgatggt 900
actcttgaag gccgtcgagg ctctgttgct atcgacgatg aaggtgttcc gggtcaatac 960
aatgtcttaa tcgaaaacgg catcttaaaa ggctatatgc aagataagat gaatgcacgt 1020
ttaatgggtg tttcaccaac aggaaatggt cgtcgtgagt cttatgcaca tcttcctatg 1080
cctcgtatga caaacactta tatgttagca ggcaaatctt cgcctgaaga aattattact 1140
agcgttgatc gcggtattta cgcaccaaac tttggtggcg gtcaggttga tatcacatca 1200
ggtaaatttg ttttctcaac ctcagaagct tatttaatcg agaatggaaa aataacaaaa 1260
ccaattaaag gggcaactct gattggttca ggtattgaag ccatgcaaca ggtctctatg 1320
gtgggaaatg atctcgcttt agataaagga gtgggcgttt gtggtaaaga aggacaaagc 1380
ctccctgttg gtgtcggtca acctacgttg aagcttgata agatcaccgt aggcggtact 1440
gcttaa 1446
<210> 4
<211> 481
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ser Leu Ala Val Val Ser Glu Ser Leu Leu Glu Ala Asn Lys Leu
1 5 10 15
Ser Leu Asp Asp Leu Ala Ser Thr Leu Glu Gln Leu Ala Gln Arg Gln
20 25 30
Ile Asp Tyr Gly Asp Leu Tyr Phe Gln Ser Ser Tyr His Glu Ala Trp
35 40 45
Ser Leu Asp Asp Gln Ile Ile Lys Asp Gly Ser Tyr Asn Ile Asp Gln
50 55 60
Gly Val Gly Val Arg Ala Ile Tyr Gly Glu Lys Thr Gly Phe Ala Tyr
65 70 75 80
Ala Asp Gln Leu Thr Leu Asn Ala Leu Asn Gln Ser Ala His Ala Ala
85 90 95
Arg Ser Ile Val Gln Ala Lys Gly Asn Gly Arg Ile His Thr Leu Gly
100 105 110
Ala Ile Gln His Ser Pro Leu Tyr Ser Leu Asn Asp Pro Leu Gln Ser
115 120 125
Leu Ser Arg Glu Glu Lys Ile Ala Leu Leu His Glu Val Asp Lys Val
130 135 140
Ala Arg Ala Glu Asp Lys Arg Val Lys Gln Val Asn Ala Ser Leu Thr
145 150 155 160
Gly Val Tyr Glu His Val Leu Val Ala Ala Thr Asp Gly Thr Phe Ala
165 170 175
Ala Asp Val Arg Pro Leu Val Arg Leu Ser Val Ser Val Leu Val Glu
180 185 190
Glu Asp Gly Lys Arg Glu Arg Gly Ala Ser Gly Gly Gly Gly Arg Phe
195 200 205
Gly Tyr Asp Tyr Phe Leu Thr Lys Val Asp Gly Glu Ser His Ala Val
210 215 220
Thr Tyr Ala Arg Glu Ala Val Arg Met Ala Leu Val Asn Leu Ser Ala
225 230 235 240
Ile Ala Ala Pro Ala Gly Thr Met Pro Val Val Leu Gly Ala Gly Trp
245 250 255
Pro Gly Val Leu Leu His Glu Ala Val Gly His Gly Leu Glu Gly Asp
260 265 270
Phe Asn Arg Arg Glu Thr Ser Val Phe Ser Gly Arg Leu Gly Glu Lys
275 280 285
Val Thr Ser Glu Leu Cys Thr Ile Val Asp Asp Gly Thr Leu Glu Gly
290 295 300
Arg Arg Gly Ser Val Ala Ile Asp Asp Glu Gly Val Pro Gly Gln Tyr
305 310 315 320
Asn Val Leu Ile Glu Asn Gly Ile Leu Lys Gly Tyr Met Gln Asp Lys
325 330 335
Met Asn Ala Arg Leu Met Gly Val Ser Pro Thr Gly Asn Gly Arg Arg
340 345 350
Glu Ser Tyr Ala His Leu Pro Met Pro Arg Met Thr Asn Thr Tyr Met
355 360 365
Leu Ala Gly Lys Ser Ser Pro Glu Glu Ile Ile Thr Ser Val Asp Arg
370 375 380
Gly Ile Tyr Ala Pro Asn Phe Gly Gly Gly Gln Val Asp Ile Thr Ser
385 390 395 400
Gly Lys Phe Val Phe Ser Thr Ser Glu Ala Tyr Leu Ile Glu Asn Gly
405 410 415
Lys Ile Thr Lys Pro Ile Lys Gly Ala Thr Leu Ile Gly Ser Gly Ile
420 425 430
Glu Ala Met Gln Gln Val Ser Met Val Gly Asn Asp Leu Ala Leu Asp
435 440 445
Lys Gly Val Gly Val Cys Gly Lys Glu Gly Gln Ser Leu Pro Val Gly
450 455 460
Val Gly Gln Pro Thr Leu Lys Leu Asp Lys Ile Thr Val Gly Gly Thr
465 470 475 480
Ala
<210> 5
<211> 585
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggataaaa tttggtttaa aaaaagactc acttttcttg gtgggttaac tatcatatta 60
gtattacttc aactaattaa ctcactactc cccatctctc ttcttcaatg gggcattatt 120
ccaagaacag gtgaaggtct aattggtatt tttattgcgc ctttcattca tggatcttgg 180
tctcatctat ttagtaatct actcccgctt cttattctta gctttttatc catgacccaa 240
tctctacgag aatatgtgtt atccagtata tttatcatta tcgtaagcgg tttattagtt 300
tggatttttg gacgaaatgc tgttcacgtt ggtgcaagtg gatggatttt tgggttgtgg 360
tctttgctta ttgctcacgc ttttactcga cgtaaaatca tcgatattgt gatcgcactc 420
tttgttctat tctattatgg atcaatggcc tacggattaa tcccaggaca attaggtgta 480
tcaacagaat cacatatttc aggtgttatt gcagggctac tttatgcatg gtgtgcaaga 540
aagctaattc gccgtaaaag ccgagtagta gaagtggcta aatag 585
<210> 6
<211> 194
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Asp Lys Ile Trp Phe Lys Lys Arg Leu Thr Phe Leu Gly Gly Leu
1 5 10 15
Thr Ile Ile Leu Val Leu Leu Gln Leu Ile Asn Ser Leu Leu Pro Ile
20 25 30
Ser Leu Leu Gln Trp Gly Ile Ile Pro Arg Thr Gly Glu Gly Leu Ile
35 40 45
Gly Ile Phe Ile Ala Pro Phe Ile His Gly Ser Trp Ser His Leu Phe
50 55 60
Ser Asn Leu Leu Pro Leu Leu Ile Leu Ser Phe Leu Ser Met Thr Gln
65 70 75 80
Ser Leu Arg Glu Tyr Val Leu Ser Ser Ile Phe Ile Ile Ile Val Ser
85 90 95
Gly Leu Leu Val Trp Ile Phe Gly Arg Asn Ala Val His Val Gly Ala
100 105 110
Ser Gly Trp Ile Phe Gly Leu Trp Ser Leu Leu Ile Ala His Ala Phe
115 120 125
Thr Arg Arg Lys Ile Ile Asp Ile Val Ile Ala Leu Phe Val Leu Phe
130 135 140
Tyr Tyr Gly Ser Met Ala Tyr Gly Leu Ile Pro Gly Gln Leu Gly Val
145 150 155 160
Ser Thr Glu Ser His Ile Ser Gly Val Ile Ala Gly Leu Leu Tyr Ala
165 170 175
Trp Cys Ala Arg Lys Leu Ile Arg Arg Lys Ser Arg Val Val Glu Val
180 185 190
Ala Lys
<210> 7
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgcggatcca tgtctgccac cagagccca 29
<210> 8
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccggaattca tccgccgtcg tgtcgcgct 29
<210> 9
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgcggatcca tgagtttagc tgttgtcag 29
<210> 10
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccggaattca gcagtaccgc ctacggtga 29
<210> 11
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cgcggatcca tggataaaat ttggtttaa 29
<210> 12
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccggaattct ttagccactt ctactactc 29
<210> 13
<211> 1028
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtgatcgtc cgactagcaa ggcagcccca taaacacaca gtatgttttt aagcttctgc 60
aggctagtgg tggtggtggt tctggtggtg gtggttctgg tggtggtggt tctgctagca 120
tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag gtaccaggat 180
ccatgtctgc caccagagcc caactcgacg cttcccagct cctgctgctg caccagcggg 240
tcgacgcctg tttcgcgcag gcggaggcac gcctcggccg ccccttcccg cgcccgcaga 300
tccactgcaa catgcggggc cgggcggcag ggtctgctcg gctgcaaacc tgggagctgc 360
gtttcaatcc ggcgctctat caggccaatc agcaggcgtt tctcagggaa gtggtgcccc 420
acgaggtggc gcacctgctg gtctatgcgc tctggggaga ggggcgcggc aagagccggg 480
tactgcccca cggtcgccag tggcagtcgg tgatgcggga tctgttcggt ctcgaaccca 540
gcaccaccca cagctttgat ctgggggtgc tggcccagcg caccttcgtg tatgcctgcg 600
cctgccagca gcatcccctc tcggtgcgcc gccacaacaa ggtgatgcgc ggcgaggccc 660
gctatcactg ccgccgctgt cgccagcccc tggtgtggca gcgcgacacg acggcggatg 720
aattctgcag atatccagca cagtggcggc cgctcgaggg tggtggtggt tcaatggtga 780
gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 840
taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 900
tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 960
ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcaggcag 1020
aatttttt 1028
<210> 14
<211> 1781
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tgtctgacag caaggcagcc ccataaacac acagtatgtt tttaagcttc tgcaggctag 60
tggtggtggt ggttctggtg gtggtggttc tggtggtggt ggttctgcta gcatgactgg 120
tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggtaccag gatccatgag 180
tttagctgtt gtcagcgaaa gtctgttgga agcaaacaaa cttagtttag atgatttagc 240
atcaacacta gagcagcttg cacagcgtca aattgattat ggtgatcttt attttcagtc 300
aagttatcac gaggcttgga gccttgatga tcagattatt aaagatggct cttacaatat 360
tgatcaaggt gttggtgtta gagcaattta cggtgaaaaa accggttttg cttatgctga 420
ccaactaacg cttaacgcac ttaaccaaag tgctcatgct gcacgaagta ttgttcaggc 480
taaaggtaat ggccgtatcc atactttagg agctattcaa cattctccgc tatacagctt 540
aaatgatcct ctgcaaagcc tttctcgtga agagaaaatt gcattattgc atgaggtaga 600
taaagtcgct cgtgctgaag ataaacgcgt taaacaagtt aatgcgtcat taactggtgt 660
ttatgagcat gtgctggttg cagcaaccga tggtacgttc gccgctgatg tgcgtccttt 720
agttcgcctt tctgtcagcg tgctggtgga agaagatggc aaacgtgagc gtggcgcaag 780
tggtggcggt ggtcgttttg gttatgacta ttttttaact aaagtggatg gtgaaagcca 840
tgcagtcact tatgctcgtg aagcagtacg tatggcatta gtgaatttat cagcgattgc 900
agcaccagca ggaacaatgc ctgtggtatt aggtgcagga tggccaggtg tattattgca 960
tgaagctgtg ggtcatggtt tagaaggtga tttcaaccgc cgtgaaacct ctgtattttc 1020
tggtcgcctt ggtgagaaag ttacttctga gctttgtacg attgttgatg atggtactct 1080
tgaaggccgt cgaggctctg ttgctatcga cgatgaaggt gttccgggtc aatacaatgt 1140
cttaatcgaa aacggcatct taaaaggcta tatgcaagat aagatgaatg cacgtttaat 1200
gggtgtttca ccaacaggaa atggtcgtcg tgagtcttat gcacatcttc ctatgcctcg 1260
tatgacaaac acttatatgt tagcaggcaa atcttcgcct gaagaaatta ttactagcgt 1320
tgatcgcggt atttacgcac caaactttgg tggcggtcag gttgatatca catcaggtaa 1380
atttgttttc tcaacctcag aagcttattt aatcgagaat ggaaaaataa caaaaccaat 1440
taaaggggca actctgattg gttcaggtat tgaagccatg caacaggtct ctatggtggg 1500
aaatgatctc gctttagata aaggagtggg cgtttgtggt aaagaaggac aaagcctccc 1560
tgttggtgtc ggtcaaccta cgttgaagct tgataagatc accgtaggcg gtactgctga 1620
attctgcaga tatccagcac agtggcggcc gctcgagggt ggtggtggtt caatggtgag 1680
caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 1740
aaacggccac aagttcagcg tgtccggcga gggcgagggc g 1781
<210> 15
<211> 999
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgtccgacag caaggcagcc ccataaacac acagtatgtt tttaagcttc tgcaggctag 60
tggtggtggt ggttctggtg gtggtggttc tggtggtggt ggttctgcta gcatgactgg 120
tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggtaccag gatccatgga 180
taaaatttgg tttaaaaaaa gactcacttt tcttggtggg ttaactatca tattagtatt 240
acttcaacta attaactcac tactccccat ctctcttctt caatggggca ttattccaag 300
aacaggtgaa ggtctaattg gtatttttat tgcgcctttc attcatggat cttggtctca 360
tctatttagt aatctactcc cgcttcttat tcttagcttt ttatccatga cccaatctct 420
acgagaatat gtgttatcca gtatatttat cattatcgta agcggtttat tagtttggat 480
ttttggacga aatgctgttc acgttggtgc aagtggatgg atttttgggt tgtggtcttt 540
gcttattgct cacgctttta ctcgacgtaa aatcatcgat attgtgatcg cactctttgt 600
tctattctat tatggatcaa tggcctacgg attaatccca ggacaattag gtgtatcaac 660
agaatcacat atttcaggtg ttattgcagg gctactttat gcatggtgtg caagaaagct 720
aattcgccgt aaaagccgag tagtagaagt ggctaaagaa ttctgcagat atccagcaca 780
gtggcggccg ctcgagggtg gtggtggttc aatggtgagc aagggcgagg agctgttcac 840
cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 900
gtccggcgag ggcgagggcg atgcccctac ggcagctgac cctgaagtca tctgcccacc 960
ggcagctgcc gtgcctggcc cacctcggac accctgact 999
<210> 16
<211> 5729
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60
cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120
acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180
ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240
ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300
taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc 360
ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420
ctctatactt taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac 480
gactcactat agggaatatt aagctaattc tacttcatac attttcaatt aagatgcagt 540
tacttcgctg tttttcaata ttttctgtta ttgcttcagt tttagcacag gaactgacaa 600
ctatatgcga gcaaatcccc tcaccaactt tagaatcgac gccgtactct ttgtcaacga 660
ctactatttt ggccaacggg aaggcaatgc aaggagtttt tgaatattac aaatcagtaa 720
cgtttgtcag taattgcggt tctcacccct caacaactag caaaggcagc cccataaaca 780
cacagtatgt ttttaagctt ctgcaggcta gtggtggtgg tggttctggt ggtggtggtt 840
ctggtggtgg tggttctgct agcatgactg gtggacagca aatgggtcgg gatctgtacg 900
acgatgacga taaggtacca ggatccagtg tggtggaatt ctgcagatat ccagcacagt 960
ggcggccgct cgagggtggt ggtggttcaa tggtgagcaa gggcgaggag ctgttcaccg 1020
gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 1080
ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 1140
ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 1200
gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 1260
aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 1320
ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 1380
tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 1440
tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 1500
acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 1560
acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 1620
accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 1680
ctctcggcat ggacgagctg tacaagttcg aaggtaagcc tatccctaac cctctcctcg 1740
gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat 1800
ctgataacaa cagtgtagat gtaacaaaat cgactttgtt cccactgtac ttttagctcg 1860
tacaaaatac aatatacttt tcatttctcc gtaaacaaca tgttttccca tgtaatatcc 1920
ttttctattt ttcgttccgt taccaacttt acacatactt tatatagcta ttcacttcta 1980
tacactaaaa aactaagaca attttaattt tgctgcctgc catatttcaa tttgttataa 2040
attcctataa tttatcctat tagtagctaa aaaaagatga atgtgaatcg aatcctaaga 2100
gaattgggca agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct 2160
tagcattttt gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac 2220
ctccgcttac atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg 2280
gcgtcagtcc accagctaac ataaaatgta agctctcggg gctctcttgc cttccaaccc 2340
agtcagaaat cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca 2400
agggaataaa cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg 2460
gaaatacgag tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact 2520
catctccgtg cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct 2580
ccttaggttg attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat 2640
atgcttttac aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat 2700
tgggcacaca tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct 2760
ctgtgctctg caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa 2820
cagacatact ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc 2880
accaatgccc tccctcttgg ccctctcctt ttcttttttc gaccgaattt cttgaagacg 2940
aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 3000
ggacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat ggaataattt 3060
gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta gaaagtaaat 3120
aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt 3180
caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat taaatagata 3240
tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta 3300
cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc aagataaaag 3360
gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca aaaactattt 3420
tttctttaat ttcttttttt actttctatt tttaatttat atatttatat taaaaaattt 3480
aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact tttcggggaa 3540
atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3600
tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3660
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3720
acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3780
acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3840
ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg 3900
ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3960
caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 4020
ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 4080
aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 4140
aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 4200
tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 4260
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4320
cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4380
ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggca 4440
gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4500
agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4560
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4620
cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4680
cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4740
cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4800
tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4860
tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4920
ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4980
aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 5040
cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag 5100
ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 5160
agcttccagg ggggaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 5220
ttgagcgtcg atttttgtga tgctcgtcag gggggccgag cctatggaaa aacgccagca 5280
acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 5340
cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5400
gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5460
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 5520
ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt 5580
aggcacccca ggctttacac tttatgcttc cggctcctat gttgtgtgga attgtgagcg 5640
gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc ggaattaacc 5700
ctcactaaag ggaacaaaag ctggctagt 5729
Claims (5)
1.鱼源蛋白酶基因在提高加州鲈对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的开放阅读框为SEQ ID No.1所示的核苷酸序列，或为编码氨基酸序列如SEQ IDNo.2的核苷酸序列。
2.鱼源蛋白酶基因的表达蛋白在提高加州鲈对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的表达蛋白的氨基酸序列如SEQ ID No.2所示。
3.插入有鱼源蛋白酶基因的开放阅读框的重组质粒在提高加州鲈对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的开放阅读框为SEQ ID No.1所示的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2的核苷酸序列。
4.插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体在提高加州鲈对高蛋白人工饲料消化能力中的应用，所述鱼源蛋白酶基因的开放阅读框为SEQ ID No.1所示的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2的核苷酸序列。
5.一种提高加州鲈对高蛋白人工饲料消化能力的方法，其特征在于，所述方法包括：对加州鲈投喂生物制剂，所述生物制剂的活性成份来源于插入有鱼源蛋白酶基因的开放阅读框的酵母重组表达载体，或活性成分含有鱼源蛋白酶基因的生物制品，所述鱼源蛋白酶基因的开放阅读框为SEQ ID No.1所示的核苷酸序列，或为编码氨基酸序列如SEQ ID No.2的核苷酸序列。
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