CN103757050A - 百脉根特异性表达h5n1抗原蛋白的三价ha-ltb融合表达载体 - Google Patents
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Abstract
本发明涉及基因工程领域，具体地，涉及百脉根特异性表达H5N1抗原蛋白的三价HA-LTB融合表达载体。在百脉根植物表达载体的多克隆位点，由2E+35启动子至poly(A)-Nos终止子方向，正向依次插入核苷酸序列如SEQ ID No.2所示的优化HK97HA基因、核苷酸序列如SEQ ID No.4所示的优化HK07HA基因、核苷酸序列如SEQ ID No.6的优化SX06HA基因和核苷酸序列如SEQ ID No.8所示的优化免疫佐剂LTB基因。本发明利用已建立的外源蛋白高水平表达平台进行表达，为广谱H5N1禽流感植物可饲疫苗的研制奠定基础。
Description
技术领域
本发明涉及基因工程领域，具体地，涉及百脉根特异性表达H5N1抗原蛋白的三价HA-LTB融合表达载体。
背景技术
H5N1高致病性禽流感病毒的不断变异和可通过侯鸟迁徙传播等特性，给传统疫苗的研制及免疫途径带来了极大的困难。禽流感病毒具有极高的突变和重组频率，加之其高毒性和潜在感染人的能力，使得禽流感的防控成为一个世界性难题。不断蔓延的疫情给我们再一次敲响了警钟，被动地根据发病病毒亚型进行治疗往往具有滞后性和盲目性，且禽流感病毒具有宿主多样化和随候鸟迁徙等特点，只有建立成熟的疫苗生产平台，研制可广谱免疫中和多种亚型禽流感病毒的植物可饲疫苗，通过在候鸟迁徙地投放，才能从根本上控制甚至消灭禽流感病毒。
禽流感毒株A/Hong Kong/156/97(H5N1)血凝素基因（HA）序列（Genebank No.AF028709.1）、蛋白序列（AAC40508.1），A/common magpie/HongKong/5052/2007(H5N1)血凝素基因（HA）序列（Genebank No.CY036173.1）、蛋白序列（ACJ26242.1），A/chicken/Shanxi/2/2006(H5N1)血凝素基因（HA）（Genebank No.DQ914814.3）、蛋白序列（ABK34764.2）。此3株禽流感血凝素（HA）抗原蛋白包含所有H5HA蛋白的抗原表位，血凝素参与病毒与受体结合、细胞膜融合及进入细胞全过程，是流感病毒表面重要的糖蛋白。大肠杆菌热敏毒素B亚基基因（LT-B）序列（Genebank No.M17873.1）、蛋白序列（AAA98065.1）均来自Genbank（http://www.ncbi.nlm.nih.gov/genbank/）大肠杆菌热敏毒素B亚基（LTB）能刺激肠道上皮细胞，蹭墙黏膜细胞对抗原通透性，从而增强免疫反应，具有高活性、无毒性的特点。
利用全价饲料豆科牧草——百脉根（Lotus corniculatus）作为植物生物反应器生产广谱（可免疫中和所有H5亚型禽流感病毒）禽流感转基因植物可饲用疫苗，是解决这一难题的最有效的途径。通过基因工程技术手段实现抗原蛋白在百脉根中的高水平表达，并在候鸟迁徙地投放转基因植物疫苗，不仅可节省下游纯化的成本，生产低成本的疫苗，更重要的是能够切断禽流感传播途径，实现根本上防控禽流感的目的。
发明内容
本发明的目的是提供百脉根特异性表达H5N1抗原蛋白的三价HA‐LTB融合植物表达载体。
根据本发明的三价HA-LTB融合植物表达载体，其特征在于，在百脉根植物表达载体的多克隆位点，由2E+35启动子至poly(A)-Nos终止子方向，正向依次插入核苷酸序列如SEQ ID No.2所示的优化HK97HA基因、核苷酸序列如SEQ ID No.4所示的优化HK07HA基因、核苷酸序列如SEQ ID No.6的优化SX06HA基因和核苷酸序列如SEQ ID No.8所示的优化免疫佐剂LTB基因。
根据本发明的三价HA‐LTB融合植物表达载体，其中所述百脉根植物表达载体为pCAMBIA2301。
本发明分析H5HA蛋白抗原表位多态性，依据主要抗原位点氨基酸差异、宿主、病毒致病性强弱，选取包含所有突变抗原表位的三株H5N1高致病性禽流感毒株即：HK97HA-A/Hong Kong/156/97（H5N1）、HK07HA-A/common magpie/HongKong/5052/2007（H5N1）和SX06HA-A/chicken/Shanxi/2/2006（H5N1），以其HA为目的抗原蛋白，构建三价HA-LTB融合蛋白，并利用多种生物信息学软件对融合蛋白的理化性质、二级结构及三级结构进行预测，结果显示融合蛋白结构稳定、构象合理，具备了通过构建植物融合表达载体，实现在植物中稳定表达的基本条件。
为进一步提高外源蛋白的表达水平，对目的抗原蛋白基因进行修饰改造。分别对HK97HA、HK07HA、SX06HA和免疫佐剂LTB基因进行密码子优化，同时对各目的基因起始密码子上游序列进行改造，修饰后基因进行全序列合成。
根据本发明的技术方案，构建完整融合三株HA抗原蛋白，并添加免疫佐剂LTB的三价HA-LTB植物安全高效表达载体。同时根据HA中的HA1是病毒与宿主细胞膜表面受体结合部位，起着诱导机体产生抗体的主要作用，在不降低融合蛋白免疫原性的前提下，为了降低植物表达载体的转化难度，构建完整HK97HA融合HK07HA1和SX06HA1，并添加免疫佐剂LTB的三价HA-2HA1-LTB融合植物表达载体。
根据本发明的具体实施方式，为进一步提高外源蛋白的表达水平，对目的抗原蛋白基因进行修饰改造。分别对HK97HA、HK07HA、SX06HA和免疫佐剂LTB基因进行密码子优化，同时对各目的基因起始密码子上游序列进行改造，修饰后基因进行全序列合成。在不改变禽流感血凝素抗原蛋白（HA）及免疫佐剂大肠杆菌热敏毒素B亚基（LTB）氨基酸组成的前提下，对抗原蛋白密码子进行优化，以促进提高外源抗原蛋白在受体植物百脉根中的表达水平。包括：
HK97HA基因的密码子优化
在不改变HK97HA抗原蛋白氨基酸顺序的前提下，对HK97HA基因进行优化，HK97HA基因全长1707bp，优化后共改变碱基1325bp，即替换了77.6%的核苷酸。HK97HA基因优化前后的序列分别如SEQ ID No.1和SEQ ID No.2所示。
HK97优化前序列SEQ ID No.1
ATGGAAAGAACAGTGCTTCTTCTTGCAACAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAATGTTACTGTTACACATGCCCAAGACATACTGGAAAGGACACACAACGGGAAGCTCTGCGATCTAAATGGAGTGAAGCCTCTCATTTTGAGGGATTGTAGTGTAGCTGGATGGCTCCTCGGAAACCCTATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTTACATAGTGGAGAAGGCCAGTCCAGCCAATGACCTCTGTTATCCAGGGAATTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAGATCATCCCCAAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAATAAAGAGGAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGGGTTCACCATCCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAATCCAACCACCTACATTTCCGTTGGAACATCAACACTGAACCAGAGATTGGTTCCAGAAATAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCCATCAATTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCATTCCACAACATACACCCCCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGACTCAGAAATACCCCTCAAAGAGAGAGAAGAAGAAAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTTGCTACTCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTAACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTGGAAAGGAGGATAGAGAATTTAAACAAGAAGATGGAAGACGGATTCCTAGATGTCTGGACTTACAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTCGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAATGGTTGTTTCGAATTCTATCACAAATGTGATAATGAATGTATGGAAAGTGTAAAAAACGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCAAGACTAAACAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATGGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCTTTATGGATGTGCTCCAATGGATCGTTACAATGCAGAATTTGCATTTAAATTTGTGAGTTCAGATTGTAGTTAAAAACACCCT
HK97优化后序列SEQ ID No.2
GAAAGAACAGTTCTATTGTTAGCTACTGTTTCTCTAGTTAAGTCTGATCAGATTTGTATTGGTTATCATGCAAATAATTCTACTGAACAGGTAGACACCATTATGGAAAAGAATGTCACAGTTACTCATGCTCAAGACATTCTTGAAAGAACTCACAATGGTAAGCTTTGTGATCTGAATGGTGTCAAGCCTCTAATTCTCAGAGATTGCTCTGTGGCAGGTTGGCTATTAGGTAATCCAATGTGTGACGAATTTATCAATGTGCCTGAATGGAGTTACATAGTTGAAAAAGCTAGTCCAGCAAACGATTTGTGCTATCCTGGTAATTTCAATGATTATGAAGAACTTAAACACTTATTGTCCAGGATCAACCACTTTGAAAAAATTCAGATAATCCCCAAATCTTCATGGTCAAACCATGATGCCAGCTCTGGTGTGAGTAGTGCTTGCCCGTACTTAGGTAGAAGTTCTTTCTTCCGCAATGTAGTCTGGTTGATCAAGAAGAATAGCGCGTATCCTACCATCAAAAGATCCTATAATAATACTAATCAAGAAGATCTGCTTGTTCTCTGGGGTGTTCATCATCCTAATGATGCAGCTGAACAGACAAAGTTGTACCAAAACCCCACCACATATATTAGTGTCGGTACTTCCACACTCAACCAAAGACTTGTGCCCGAAATAGCCACTAGACCGAAAGTTAATGGTCAATCAGGTCGCATGGAATTTTTCTGGACTATATTGAAACCAAACGATGCTATCAATTTCGAATCTAATGGTAACTTTATTGCCCCAGAATATGCATATAAGATTGTTAAGAAGGGTGACAGTACAATTATGAAATCAGAACTTGAATATGGTAACTGTAACACAAAATGCCAGACTCCAATGGGTGCCATCAACTCTAGCATGCCATTCCATAACATCCACCCTCTTACCATAGGTGAATGCCCAAAGTACGTGAAGTCTAATAGGCTTGTGTTGGCTACTGGTTTGAGGAACACACCTCAACGTGAAAGGAGGAGGAAGAAGAGGGGTCTTTTTGGTGCGATTGCTGGTTTCATTGAAGGTGGTTGGCAGGGTATGGTCGATGGTTGGTATGGTTACCACCACTCCAATGAACAAGGTAGCTGCTACTCAGCAGATAAAGAAAGCACCCAAAAGGCAATCGATGGTGTAACCAACAAAGTGAATTCAATCATTAACAAGATGAATACCCAGTTCGAAGCTGTAGGTAGGGAATTTAACAATCTGGAAAGACGTATAGAAAATCTCAACAAGAAGATGGAAGATGGTTTTTTGGATGTCTGGACATACAATGCCGAACTTCTTGTGCTCATGGAAAATGAAAGAACACTCGACTTTCATGATTCCAACGTGAAAAACCTTTACGACAAAGTGAGGTTGCAGTTGCGTGACAACGCTAAGGAATTGGGTAACGGTTGTTTCGAATTTTACCATAAATGTGATAACGAATGCATGGAATCTGTTAAGAACGGTACTTACGACTATCCTCAGTATAGCGAAGAAGCAAGACTGAACCGCGAAGAAATTTCAGGTGTTAAACTAGAATCAATGGGTACTTATCAAATACTGTCAATTTACTCTACCGTTGCATCCTCTCTCGCTCTCGCTATAATGGTTGCCGGTCTGAGCCTGTGGATGTGTTCCAATGGTTCATTACAATGTAGAATTTGTATT
HK07HA基因的密码子优化
在不改变HK07HA抗原蛋白氨基酸顺序的前提下，对HK07HA基因进行优化，HK97HA基因全长1704bp，优化后共改变碱基1311bp，即替换了76.9%的核苷酸。HK07HA基因优化前后的序列分别如SEQ ID No.3和SEQ ID No.4所示
HK07优化前序列SEQ ID No.3
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCATATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGACACACAACGGGAAGCTCTGCGATCTAAATGGAGTGAAGCCTCTGATTTTAAAAGATTGTAGTGTAGCAGGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCAGAATGGTCTTACATAGTAGAGAAGGCCAATCCAGCCAATGACCTCTGTTACCCAGGGAATTTCAACGATTATGAAGAATTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATACAGATCATCCCCAAAGATTCTTGGTCAGATCATGAAGCCTCATTGGGGGTGAGCTCAGCATGTCCATACCAGGGAAATTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGGGCAATGCATACCCAACAATAAAGAAAAGCTACAATAATACCAACCAAGAAGATCTCTTGGTACTGTGGGGGATTCACCATCCTAATGATGAGGCAGAGCAGACAAGGCTCTATCAAAACCCAACCACCTATATTTCCATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATAGATTTCTTCTGGACAATTTTAAAACCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGAGACTCAACAATTATGAAAAGTGAAGTGGAATATGGTAACTGCAACACCAGGTGTCAGACTCCGATGGGGGCGATAAACTCTAGTATGCCATTCCACAACATACACCCTCTCACCATAGGAGAATGTCCCAAATATGTGAAATCAAACAAATTAGTCCTTGCGACTGGGCTCAGAAATAGTCCTCAAAGAGAGAGAAGAAGAAAAAGAGGACTGTTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCACAGCAATGAGCAGGGGAGTGGATATGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGACGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTAGGAAGGGAATTTAATAACTTAGAGAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTCCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCAGACTACAGCTTAGGGATAATGCAAAAGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATGTGATAATGAATGTATGGAAAGTGTAAGAAACGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCAAGATTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGTACTGGCAATCATGGTGGCTGGTCTATCCTCATGGATGTGCTCCAACGGGTCGTTACAATGCAGAATTTGCATTTAA
HK07优化后序列SEQ ID No.4
GAAAAGATCGTTTTGCTATTTGCTATCGTGAGTCTTGTGAAATCAGATCATATCTGTATCGGTTACCATGCCAACAATTCTACAGAACAGGTTGACACCATAATGGAAAAGAATGTTACTGTGACACATGCACAGGACATCTTGGAAAAGACACATAATGGTAAGCTTTGTGACCTTAATGGTGTGAAACCCCTTATTCTTAAGGATTGTTCAGTTGCTGGTTGGCTTCTCGGTAATCCGATGTGCGATGAATTCATTAACGTTCCAGAATGGTCTTATATAGTTGAAAAAGCCAACCCTGCCAACGACCTGTGTTACCCAGGTAATTTTAATGATTATGAAGAACTTAAACATCTCCTTTCTCGCATCAATCACTTCGAAAAAATTCAAATAATTCCCAAGGATTCATGGTCTGATCACGAAGCATCCTTAGGTGTTTCCTCTGCATGCCCTTACCAGGGTAACTCCTCATTCTTCAGAAATGTGGTGTGGTTGATAAAGAAAGGTAACGCTTACCCTACAATAAAGAAGAGCTACAACAATACCAATCAGGAAGATTTACTGGTCTTGTGGGGTATTCATCACCCAAACGACGAAGCTGAACAAACTAGGCTTTACCAGAATCCAACCACTTATATTTCAATTGGTACATCCACCCTAAACCAAAGGCTAGTCCCTAAGATAGCGACTCGCTCAAAAGTTAATGGTCAATCCGGTAGAATTGATTTTTTCTGGACTATACTTAAGCCAAACGACGCTATTAATTTCGAATCAAACGGTAACTTTATCGCTCCCGAATACGCATACAAGATCGTCAAAAAGGGTGATTCAACTATAATGAAAAGCGAAGTCGAATATGGTAATTGCAACACTAGGTGTCAAACTCCTATGGGTGCTATAAACAGTAGCATGCCATTCCACAATATTCATCCGTTGACAATCGGTGAATGTCCTAAGTACGTGAAATCTAACAAGTTGGTCCTCGCTACTGGTTTGAGAAATTCTCCACAAAGAGAAAGGCGTAGGAAAAGGGGTCTTTTTGGTGCCATCGCAGGTTTTATTGAAGGTGGTTGGCAAGGTATGGTGGATGGTTGGTACGGTTATCATCACTCCAACGAACAGGGTAGTGGTTATGCGGCAGATAAGGAATCAACTCAAAAAGCTATAGATGGTGTAACAAATAAGGTCAATAGTATTATTGACAAAATGAATACCCAATTCGAAGCCGTGGGTCGTGAATTCAACAACTTGGAAAGAAGAATCGAAAATCTGAACAAGAAAATGGAAGATGGTTTTTTAGATGTTTGGACCTATAATGCAGAACTCCTCGTTTTGATGGAAAACGAACGCACATTGGATTTTCATGACAGCAATGTAAAGAACCTATATGATAAAGTTAGATTACAACTGAGGGATAATGCAAAAGAACTGGGTAATGGTTGCTTTGAATTTTATCACAAGTGTGATAATGAATGCATGGAATCTGTAAGAAACGGTACTTACGACTATCCTCAGTATTCTGAAGAAGCTAGACTGAAGCGTGAAGAAATTTCTGGTGTTAAGCTCGAAAGTATTGGTACCTATCAGATTTTATCTATTTATAGCACTGTAGCTAGCTCTCTCGTTCTCGCAATTATGGTGGCGGGTCTTAGTTCCTGGATGTGCAGTAACGGTTCATTGCAGTGTAGAATCTGCATT
SX06HA基因的密码子优化
同样在不改变SX06HA抗原蛋白氨基酸顺序的前提下，对SX06HA基因进行优化，SX06HA基因全长1704bp，优化后共改变碱基1313bp，即替换了77.0%的核苷酸。SX06HA基因优化前后的序列分别如SEQ ID No.5和SEQ ID No.6所示
SX06优化前序列SEQ ID No.5
AGCAAAAGCAGGGGTTCAATCTGTCAAAATGGAGAAAATAGTGCTTCTTCTTGCAATAATCGGTCTTGTTAAAAGTGATCAGATTTGCGTTGGTTACCATGCAAACAACTCAACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCTCAAGACATACTGGAGAAGACACACAACGGGAAGCTCTGCAACCTAGATGGAGTGAAGCCTCTAATTTTGAAAGATTGTAGTGTAGCTGGATGGCTCCTCGGAAACCCAATGTGTGACGAATTTCTCAATGTGTCGGAATGGTCTTACATAGTGGAGAAGGCCAGTCCAGCCAATGGCCTCTGTTACCCAGGGGATTTCAATGACTATGAAGAACTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTAAGATCATCCCCAAAAGTTCTTGGTCCAATCATGAAGCCTCAGGGGTGAGCTCAGCATGTTCCTATCTGGGGAAGCCCTCCTTTTTCAGAAATTTGGTATGGCTTATCAAAAAGAATAATACATACCCACCAATAAAGGTGAACTACACCAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGGATTCACCATCCCAACGATGAGACAGAGCAGGTAAAGATCTATCAAAACCCAACCACCTATATTTCCGTTGGAACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCCAAAGTGAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCTATCAATTTCGATAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAGCGATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAATTCTAGTATGCCATTCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTCGCGACTGGACTCAGAAATGCCCCTCAAAGAGAGGGAGGAAGAAGAAAAAGAGGACTATTTGGAGCCATAGCAGGGTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGATACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAATCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAATAACTTAGAAAGGAGAATAGAAAATTTAAACAAAAAGATGGAGGACGGATTCCTAGATGTCTGGACTTATAACGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGAAAAGGTCCGACTGCAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTACCACAAATGTGATAATGAATGTATGGAAAGTGTAAAAAACGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCAAGACTAAACAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATGGTAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCATTGGCAATCATGGTGGCTGGTCTATCTTTATGGATGTGCTCCAATGGATCGTTACAATGCAGAATTTGCATTTGAATTTGTGAGTTCAGATTGTAGTTAAAAACACCCTTGTTTCTACT
SX06优化后序列SEQ ID No.6
GAAAAGATTGTTCTCCTGCTCGCAATCATAGGTTTAGTGAAAAGTGACCAGATATGTGTTGGTTACCATGCTAATAACAGCACTGAACAAGTGGATACAATCATGGAAAAGAATGTGACCGTCACTCACGCACAAGACATTCTGGAAAAGACCCATAATGGTAAACTTTGCAACTTGGATGGTGTTAAGCCATTAATACTCAAAGATTGCTCAGTCGCCGGTTGGCTTCTCGGTAATCCAATGTGTGATGAATTTCTTAACGTATCAGAATGGAGTTACATTGTTGAAAAGGCAAGTCCTGCCAATGGTCTTTGTTACCCCGGTGATTTCAACGATTATGAAGAATTGAAACATCTGTTGAGCAGAATTAACCACTTTGAAAAGATCAAGATTATTCCAAAGAGTTCTTGGTCAAATCATGAAGCTTCCGGTGTTAGTAGCGCATGTTCTTACCTCGGTAAGCCGTCCTTTTTCAGAAACCTCGTATGGCTCATTAAAAAGAATAACACTTATCCTCCTATTAAAGTGAATTATACCAATACAAACCAAGAAGATCTATTAGTTTTGTGGGGTATTCACCATCCCAACGATGAAACAGAACAAGTGAAGATATATCAGAATCCAACTACCTACATTTCAGTCGGTACCTCAACTTTAAATCAAAGACTAGTCCCTAAGATTGCTACTAGAAGTAAAGTTAATGGTCAATCTGGTAGGATGGAATTCTTTTGGACTATTCTGAAGCCAAATGATGCCATAAACTTTGACTCCAACGGTAACTTCATCGCACCTGAATATGCTTATAAGATCGTGAAGAAAGGTGATAGCGCTATTATGAAATCTGAACTTGAATACGGTAATTGCAACACAAAGTGTCAAACTCCCATGGGTGCTATAAATTCTTCTATGCCTTTTCACAACATTCATCCACTTACAATCGGTGAATGTCCTAAATACGTGAAGTCAAATAGACTTGTTTTAGCTACAGGTCTGAGAAATGCACCACAAAGGGAAGGTGGTAGAAGGAAGAGGGGTCTTTTCGGTGCTATTGCCGGTTTTATCGAAGGTGGTTGGCAGGGTATGGTTGACGGTTGGTATGGTTACCATCATTCCAATGAACAAGGTTCCGGTTATGCAGCTGACAAAGAATCCACCCAGAAGGCCATAGATGGTATAACTAACAAAGTTAACTCCATAATCGATAAGATGAATACACAATTCGAAGCTGTTGGTAGAGAATTCAATAACTTGGAACGCAGGATCGAAAACTTGAACAAGAAAATGGAAGATGGTTTTTTAGACGTCTGGACTTACAACGCGGAACTTTTGGTGCTAATGGAAAACGAACGCACATTGGACTTCCACGATTCTAATGTCAAAAATTTGTACGAAAAAGTTCGTCTTCAGTTGAGAGACAACGCGAAAGAACTAGGTAATGGTTGTTTTGAATTCTATCACAAGTGCGATAACGAATGTATGGAATCAGTAAAAAATGGTACTTACGATTATCCGCAGTATAGTGAAGAAGCACGTCTCAATAGGGAAGAAATTTCTGGTGTAAAGCTGGAATCTATGGTTACATATCAGATTCTTTCAATTTATTCTACCGTGGCCAGCTCTTTGGCACTAGCTATCATGGTGGCTGGTCTGTCTTTGTGGATGTGCAGCAATGGTTCACTTCAGTGCAGGATCTGCATC
LTB基因的密码子优化
同样在不改变LTB抗原蛋白氨基酸顺序的前提下，对LTB基因进行优化。LTB基因优化前后的序列分别如SEQ ID No.7和SEQ ID No.8所示。
LTB优化前序列SEQ ID No.7
ATGAATAAAGTAAAATGTTATGTTTTATTTACGGCGTTACTATCCTCTCTATATGCACACGGAGCTCCCCAGACTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGAAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAGAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGAAAAACTAG
LTB优化后序列SEQ ID No.8
AATAAGGTTAAATGCTATGTGCTGTTCACCGCCCTTCTAAGCTCTTTGTATGCTCATGGTGCACCTCAAACAATCACTGAACTCTGTTCCGAATACAGAAACACCCAGATTTACACTATCAATGACAAGATCCTTTCATATACTGAAAGTATGGCAGGTAAAAGGGAAATGGTGATTATTACCTTTAAGTCAGGTGAAACATTTCAAGTTGAAGTACCTGGTTCTCAACACATAGATAGTCAGAAGAAAGCTATTGAAAGAATGAAAGATACACTCAGGATCACTTACTTAACAGAAACTAAGATTGATAAGTTGTGTGTCTGGAACAATAAAACCCCAAATAGCATTGCTGCGATATCCATGAAGAAC
根据本发明的技术方案，优选对起始密码子侧翼序列的修饰，对所选用的禽流感血凝素基因及大肠杆菌热敏毒素B亚基基因起始密码子上下游序列进行优化。在经密码子优化的HK97HA、HK07HA、SX06HA和LTB基因起始密码子前分别添加AAAAAAA(A/C)A基因序列，起始密码子后添加甘氨酸密码子（GCU）。
根据本发明的具体实施方式，获得三价HA-LTB融合植物表达载体为pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-LTB-poly(A)-Nos-MARs，其结构如图1所示。
根据本发明的技术方案，优选通过添加多种载体元件优化植物表达载体，例如通过添加多种顺势作用元件，如消除位置效应的Mars序列、增强子Ω序列、内质网滞留信号的KDEL序列及可有效终止转录的加长poly(A)序列等，对植物表达载体进行改造。
本发明首先通过添加顺式作用元件，优化表达载体，建立了高水平表达外源抗原蛋白生产平台。通过抗原位点多态性分析筛选获得了3株禽流感病毒血凝素基因，构建包含所有突变抗原位点三价融合蛋白，构建三价融合植物表达载体，利用已建立的外源蛋白高水平表达平台进行表达，为广谱H5N1禽流感植物可饲疫苗的研制奠定基础。
附图说明
图1实施例1构建的三价融合载体示意图。
图2显示HK97HA-Linker PCR鉴定图和酶切鉴定，M.DNA marker+/-：质粒/空白1-2.阳性菌株M.DNA marker1.KpnI/BamHI酶切结果。
图3显示HK07HA-Linker PCR鉴定和酶切鉴定，M.DNA marker+/-：质粒/空白1-2.阳性菌株M.DNA marker1.BamHI/ApaI酶切结果。
图4显示SX06HA-Linker PCR鉴定和酶切鉴定，M.DNA marker+/-：质粒/空白1-2.阳性菌株M.DNA marker1.ApaI/PstI酶切结果。
图5显示LTB PCR鉴定图和LTB-poly(A)-Nos-Mar酶切鉴定，M.DNA marker+/-：质粒/空白1-3.阳性菌株M.DNA marker1.HindIII/PstI酶切结果。
图6显示图2-17百脉根的遗传转化，1.种子2.七日龄子叶3.侵染4.选择培养，5.分化培养6.抗性植株。
具体实施方式
1、植物材料
受体植物：豆科牧草百脉根栽培品种——里奥（Lotus corniculatus L.CV.Leo）。
2菌种及载体
2.1菌种
大肠杆菌DH5α，根癌农杆菌LBA4404
2.2质粒及载体
pCAMBIA2301、pMD19-T商业载体购于Takara生物公司
pMD-2E+35S：将CaMV35S启动子的-46至-105序列中含有增强子元件重复串联，构建双35S启动子
pMD-Ω：富含TTAAC序列的68bp的Ω元件
pMD-Nos-poly(A)：长度达到100bp的poly(A)与NOS串联序列pCAMBIA2301-PMI-MHA-MLTB-MARs植物融合表达载体：在载体pCAMBIA1302基础上，将Hygromycin基因替换为PMI基因，将经过人工改造的禽流感血凝素MHA基因和大肠杆菌热敏毒素B亚基基因MLTB，插入载体，构建了以PMI为筛选标记的植物高效融合表达载体pCAMBIA1302-PMI-MHA-MLTB-MARs。
2.3试剂
EcoR I、Hind III、Mlu I等限制性内切酶、T4DNA连接酶、Ex Taq Polymerase、LA Taq Polymerase购自Takara生物公司；DNA Marker购自北京全式金生物技术有限公司（TransGen Biotech）；高纯质粒小量制备试剂盒、通用植物总RNA提取试剂盒购自百泰克生物公司（Bioteke）；反转录试剂盒购于Invitrogen公司；Gel/PCRExtraction kit购自Biomiga生物公司；大肠杆菌热敏毒素B亚基酶联免疫分析试剂盒购自R&D system。标准重组LTB蛋白和兔抗CT血清购自sigma，碱性磷酸酶标记的羊抗兔IgG和其他试剂购自promega等公司
B5培养基、MS培养基购自美国Phyto Technology生物技术公司，YEB培养基、LB培养基依照Sambrook（Sambrook，2001）等所述配制；植物凝胶（phytagel）、甘露糖购自北京拜尔迪生物公司（Biodee），1mg/ml的BSA标准品购自GeneStar公司。
2.4基因及蛋白序列
禽流感毒株A/Hong Kong/156/97(H5N1)血凝素基因（HA）序列（Genebank No.AF028709.1）、蛋白序列（AAC40508.1），A/common magpie/HongKong/5052/2007(H5N1)血凝素基因（HA）序列（Genebank No.CY036173.1）、蛋白序列（ACJ26242.1），A/chicken/Shanxi/2/2006(H5N1)血凝素基因（HA）（Genebank No.DQ914814.3）、蛋白序列（ABK34764.2）以及大肠杆菌热敏毒素B亚基基因（LT-B）序列（Genebank No.M17873.1）、蛋白序列（AAA98065.1）均来自Genbank（http://www.ncbi.nlm.nih.gov/genbank/）。
实施例1三价完整HA-LTB融合植物表达载体的构建
1、植物表达载体的构建过程
分别对HK97HA、HK07HA、SX06HA和免疫佐剂LTB基因进行密码子优化，同时对各目的基因起始密码子上游序列进行改造，修饰后基因进行全序列合成。在不改变禽流感血凝素抗原蛋白（HA）及免疫佐剂大肠杆菌热敏毒素B亚基（LTB）氨基酸组成的前提下，对抗原蛋白密码子进行优化，以促进提高外源抗原蛋白在受体植物百脉根中的表达水平。优化后的HK97HA、HK07HA、SX06HA和免疫佐剂LTB基因序列如SEQ ID No.2、4、6、8所示。
PCR反应
Mar-2E+35S-Ω和poly(A)-Nos-Mar长片段PCR反应条件：94℃预变性5min，然后94℃变性30sec，62℃退火30sec，72℃延伸1min，35个循环，最后72℃延伸10min。
HK97HA、HK07HA和SX06HA基因PCR检测反应条件：94℃预变性5min，然后94℃变性30sec，62℃退火30sec，72℃延伸45sec，30个循环，最后72℃延伸10min。
回收PCR产物胶，提取质粒，然后酶切。EcoRI和Kpn I双酶切37℃反应1小时，Kpn I和BamHI采用分步酶切，先加入BamHI，30℃反应1小时后，再加入Kpn I，37℃反应1小时，Apa I和BamH I采用分步酶切，先加入BamHI，30℃反应1小时后，再加入Apa I，37℃反应1小时，Apa I和Pst I双酶切，37℃反应1小时，Pst I和Asc I双酶切，37℃反应1小时，Asc I和HindIII双酶切，37℃反应1小时。
连接
经过修饰改造的抗原蛋白基因：HK97HA、HK07HA、SX06HA和LTB由上海生物工程有限公司进行全序列合成，并以pMD-HK97HA-Linker、pMD-HK07HA-Linker、pMD-SX06HA-Linker、pMD-LTB、的形式提供。然后转化大肠杆菌及农杆菌感受态细胞，并PCR鉴定大肠杆菌阳性菌落。
2、植物表达载体的构建结果
（1）pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-poly(A)-Nos-MARs载体的构建
原有载体pCAMBIA2301-PMI-2E+35S-Ω-MHA-MLTB-KDEL-poly(A)-Nos-MARs和pMD-HK97HA-Linker经Kpn I和BamHI双酶切，分别回收目的片段，T4连接酶连接后转化大肠杆菌，经PCR及酶切验证（如图2所示），将验证结果正确的质粒进行测序，获得载体pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-poly(A)-Nos-MARs。
（2）
pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-poly(A)-Nos-MARs载体的构建
将载体pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-poly(A)-Nos-MARs和pMD-HK07HA-Linker经BamHI和Apa I双酶切，分别回收目的片段，T4连接酶连接后转化大肠杆菌，经PCR及酶切验证（如图3所示），将验证结果正确的质粒进行测序，获得载体pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-poly(A)-Nos-MARs。
（3）
pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-poly(A)-Nos-MARs载体的构建
载体pCAMBIA2301-Mar-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker和pMD-SX06HA-Linker经Apa I和Pst I双酶切，分别回收目的片段，T4连接酶连接后转化大肠杆菌，经PCR及酶切验证（如图4），将验证结果正确的质粒进行测序，获得载体pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-poly(A)-Nos-MARs。
（4）
pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-LTB-poly(A)-Nos-MARs载体的构建
将载体pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-poly(A)-Nos-MARs和pMD-LTB经Pst I和Asc I双酶切，分别回收目的片段，T4连接酶连接后转化大肠杆菌，PCR扩增LTB基因，PstI和HindIII双酶切得到LTB-poly(A)-Nos-Mar片段，结果如图5。将验证结果正确的质粒进行测序，获得载体
pCAMBIA2301-PMI-2E+35S-Ω-HK97HA-Linker-HK07HA-Linker-SX06HA-Linker-LTB-poly(A)-Nos-MARs（如图1）。
3、百脉根的遗传转化
参照现有以磷酸甘露糖为筛选标记的百脉根安全高效遗传转化体系，以百脉根七日龄苗子叶（含下胚轴）为外植体，农杆菌介导法转化百脉根，经暗培养，分化培养和选择培养，最终获得抗性植株，遗传转化过程如图6.
4、转基因百脉根的分子检测
PCR检测所需引物如以下表1所示
表1本章使用PCR引物
获得百脉根抗性植株后，提取其基因组DNA，分别进行HK97HA、HK07HA、SX06HA、LTB、PMI基因PCR检测，结果显示获得转基因阳性百脉根植株。
为验证PCR阳性百脉根植株中HK97HA、HK07HA、SX06HA、LTB在转录水平的表达，提取转基因植株总RNA，按照Invitrogen公司反转录试剂盒说明书进行反转录，并对cDNA进行PCR检测，结果说明HK97HA、HK07HA、SX06HA、LTB基因在mRNA水平成功表达。
5、ELISA检测
5.3.1溶液的配制
蛋白提取液（pH8.0）：PBS+EDTA+0.001%PMSF
包含137mmol/L NaCl，2.7mmol/L KCl，10mmol/L Na2HPO4，2mmol/L KH2PO4，5mmol/LEDTA，0.001%PMSF。即用800ml水溶解8g NaCl，0.2g KCl，2.98gNa2HPO4·12H2O，0.24g KH2PO4，1.861g EDTA，0.01g PMSF。用HCl调pH至8.0，加水至1L，分装后121℃，20min保存于室温。
考马斯亮蓝G-250染料试剂：10mg G250先溶于5ml95%乙醇中，再添加10ml85%磷酸，最后加水定容至100ml（可保存一个月）。
5.3.2百脉根总可溶性蛋白的提取
超净台内取100mg转基因百脉根叶片放入2.0ml Ep管中，迅速置于液氮内，打样机上破碎，加入500μl蛋白提取液，室温12000rpm离心10min，取上清，即为百脉根总可溶性蛋白粗提液，可用于考染定量。
5.3.3牛血清白蛋白标准曲线绘制
按照下表对0.1mg/ml BSA蛋白标准品进行梯度稀释，稀释后各取5μl与250μl G250染料混匀，用酶标仪测定其在595nm下的吸光值，并依据吸光值绘制标准曲线。
5.3.4百脉根总可溶性蛋白的定量
取各样本蛋白粗提液各5μl与250μl G250染料混匀，用酶标仪测定其在595nm下的吸光值，并依据牛血清白蛋白标准曲线计算各样品总可溶性蛋白含量。
5.3.5转基因百脉根中大肠杆菌热敏毒素B亚基（LTB）蛋白的定量
转基因百脉根中大肠杆菌热敏毒素B亚基（LTB）蛋白的定量依照大肠杆菌热敏毒素B亚基酶联免疫试剂盒说明书操作。
ELISA结果表明HK97HA、HK07HA、SX06HA、LTB蛋白融合不影响各自蛋白的免疫活性，分别在百脉根中达到一定的表达量。
6、免疫实验
6.1实验动物
24只7周龄BALB/c雌性小鼠，体重16～22g,分为4组，每组6只（购自北京维通利华实验动物有限公司）。
6.2试验材料
1）PBS溶液（pH7.2）：NaCl8g,KCl0.2g,Na2HPO41.44g,KH2PO40.24g,蒸馏水定容1L，调pH值7.2。
2）50ug/30ul牛血清白蛋白（BSA≥99.9％）（购自sigma公司）：BSA溶于pH7.2的磷酸缓冲盐液（PBS）中，4℃，8000g，离心20min，取上清，滤膜过滤，配成50ug/30ul的浓度，－20℃保存备用。
3)含BSA50ug的各种百脉根可溶性总蛋白提取液：30ul蛋白提取液中加入BSA50ug，溶解混匀。
6.3试验方法
小鼠24只，随机分为4组，每组6只,以非免疫鼠血清作为0组，30ulPBS经鼻免疫的小鼠组为第1组、30ul含BSA50ug的PBS经鼻免疫的小鼠组为第2组、30ul含BSA50ug加非转基因百脉根可溶性蛋白提取液经鼻免疫的小鼠组为第3组、30ul含BSA50ug加转野生型表达载体pC234ACKLTAB百脉根可溶性蛋白提取液经鼻免疫的小鼠组为第4组，30ul含BSA50ug加转本植物表达载体pC234ANLTAB百脉根可溶性蛋白提取液经鼻免疫的小鼠组为第5组，在0、7、14天各经鼻腔免疫小鼠，21天后颈动脉采血，ELISA检测抗体水平。
ELISA检测抗体方法：
用含50ul BSA（30ug/ml）、碳酸盐50mM，pH9.6的包被缓冲液包被96孔玻板过夜，再用PBS-Tween20清洗缓冲液洗3次，用1％明胶封闭板，清洗后每孔中加入稀释样品50ul，室温温育过夜，再用清洗缓冲液清洗，加入50ul800倍稀释的辣根过氧化酶标羊抗鼠IgG抗体（购自sigma公司），室温放置2小时，清洗后，50ulTMB-H2O2(购自Bio-rid公司)显色30min，1MH2SO410ul终止，405nm检测。试验结果表明，采用本转基因百脉根的可溶性蛋白提取液作为疫苗免疫小鼠，其血清中抗BSA血清IGg显著升高，证明本发明转基因植物有增强免疫应答的作用，具有良好的免疫效果。
Claims (2)
1.一种异性表达H5N1抗原蛋白的三价HA-LTB融合表达载体，其特征在于，在百脉根植物表达载体的多克隆位点，由2E+35启动子至poly(A)-Nos终止子方向，正向依次插入核苷酸序列如SEQ ID No.2所示的优化HK97HA基因、核苷酸序列如SEQ ID No.4所示的优化HK07HA基因、核苷酸序列如SEQ ID No.6的优化SX06HA基因和核苷酸序列如SEQ ID No.8所示的优化免疫佐剂LTB基因。
2.根据权利要求1所述的异性表达H5N1抗原蛋白的三价HA‐LTB融合表达载体，其特征在于，所述百脉根植物表达载体为pCAMBIA2301。
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| 张占路 等: "百脉根表达H5N1亚型禽流感血凝素的研究", 《中国农业科学》, vol. 41, no. 1, 10 January 2008 (2008-01-10) * |
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