CN119506295A 一种前列腺特异性膜抗原的制备方法 （上海皓鸿生物医药科技有限公司）

本发明提供了一种前列腺特异性膜抗原的2述多核苷酸的核苷酸序列如SEQIDNO:2.如权利要求1所述的多核苷酸，其特征在于，所述多核苷酸编码氨基酸序列如SEQ3[0004]PSMA在前列腺组织良、恶性鉴别中优于PSA（前列腺特异性抗原且临床分期越[0009]在另一优选例中，所述多核苷酸编码氨基酸序列如SEQIDNO:5所示的PSMA蛋4(b2)使用Superdex200pg层析柱对所述PSMA蛋白进行纯化处理，从而得到高纯度(iv)将所述培养物离心后收集上清液，过滤纯化后得到前列腺特异性膜抗原重其中，所述前列腺特异性膜抗原蛋白对应的核酸构建体序列如SEQIDNO:2所5[0041]图7显示了Superdex200pg层析电泳图谱。主峰部分目的蛋白蛋白纯度达到95%发明最优的人PSMA编码序列(SEQIDNo:2)，可实现PSMA产量的显著提升(与野生型编码6是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分44_750位氨基酸残基是该蛋白的胞外部分，是该蛋白的主要功能结构部分。PSMA的第44_750位的氨基酸序列如SEQIDNO:白，而人源的宿主细胞的翻译系统已经是与人野生型编码序列非常匹配的(或经自然进化7[0055]一旦获得了本发明的优化的编码序列，就可以用重组法来大批量地获得有关序得到有关序列。[0057]应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物[0062]本领域的技术人员熟知的方法能用于构建含人PSMA编码DNA序列和合适的转录/[0064]包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适8动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的并且经分离纯化后可获得浓度为约0.87mg[0076](e)本发明相对于现有的同为真核表达体系表达的进行件，例如Sambrook等人，分子克隆：实验室手册(NewYork:ColdSpringHarbor9AAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTAGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTAGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCAAGAGCAGCAACGAGGCCACCAACATCACCCCCAAGCACAACATGAAGGCCTTCCTGGACGAGCTGAAGGCCGAGAACATCAAGAAGTTCCTGTACAACTTCACACAGATCCCCCACCTGGCCGGCACCGAGCAGAACTTTCAGCTGGCCAAGCAGATTCAGAGCCAATGGAAGGAGTTCGGCCTCGACTCCGTGGAGCTGGCCCACTACGACGTGCTGCTGAGCTACCCCAACAAGACCCACCCCAACTACATCAGCATCATCAACGAGGACGGCAACGAGATCTTCAACACAAGCCTGTTCGAGCCCCCTCCCCCCGGCTACGAGAACGTGAGCGACATCGTGCCCCCCTTCAGCGCCTTCAGCCCCCAAGGCATGCCCGAGGGCGACCTGGTGTACGTGAACTACGCTAGAACCGAGGACTTCTTCAAGCTGGAGAGAGACATGAAGATCAACTGCAGCGGCAAGATCGTGATCGCTAGATACGGCAAGGTGTTCAGAGGCAACAAGGTGAAGAACGCTCAGCTGGCCGGCGCCAAGGGCGTGATCCTGTACAGCGACCCCGCCGACTACTTCGCCCCCGGCGTGAAGAGCTACCCCGACGGCTGGAACCTGCCCGGCGGGGGCGTGCAGAGAGGCAACATCCTGAACCTGAACGGCGCCGGCGACCCCCTGACCCCCGGCTACCCCGCCAACGAGTACGCCTACAGAAGAGGCATCGCCGAGGCCGTGGGCCTGCCTAGCATCCCCGTGCACCCCATCGGCTACTACGACGCTCAGAAACTGCTGGAGAAGATGGGCGGCAGCGCCCCCCCCGACAGCAGCTGGAGAGGCAGCCTGAAGGTGCCCTACAACGTGGGCCCCGGCTTCACCGGCAACTTCAGCACACAGAAGGTGAAGATGCACATCCACAGCACCAACGAGGTGACAAGAATCTACAACGTGATCGGCACCCTGAGAGGCGCCGTGGAGCCCGACAGATACGTGATCCTGGGCGGCCACAGAGACAGCTGGGTGTTCGGCGGCATCGACCCTCAGAGCGGCGCCGCCGTGGTGCACGAGATCGTGCGGAGCTTCGGCACCCTCAAAAAGGAGGGCTGGAGACCTAGAAGAACCATCCTGTTCGCTAGCTGGGACGCCGAGGAGTTTGGCCTCCTCGGCAGCACCGAGTGGGCCGAGGAGAACAGCAGACTGCTGCAAGAGAGAGGCGTGGCCTACATCAACGCCGACAGCAGCATCGAGGGCAACTACACCCTGAGAGTGGACTGCACCCCCCTGATGTACAGCCTGGTGCACAACCTGACCAAGGAGCTGAAGAGCCCCGACGAGGGCTTCGAGGGCAAGAGCCTGTACGAGAGCTGGACCAAGAAGAGCCCTAGCCCCGAGTTCAGCGGCATGCCTAGAATCAGCAAGCTGGGCAGCGGCAACGACTTCGAGGTGTTCTTTCAGAGACTGGGCATCGCTAGCGGCAGAGCTAGATACACCAAGAACTGGGAGACCAACAAGTTCAGCGGCTACCCCCTGTACCACAGCGTGTACGAGACCTACGAGCTGGTGGAGAAGTTCTACGACCCCATGTTCAAGTACCACCTGACCGTGGCCCAAGTGAGAGGCGGCATGGTGTTCGAGCTGGCCAACAGCATCGTGCTGCCCTTCGACTGCAGAGACTACGCCGTGGTGCTGAGAAAGTACGCCGACAAGATCTACAGCATCAGCATGAAGCACCCCCAAGAGATGAAGACCTACAGCGTGAGCTTCGACAGCCTGTTCAGCGCCGTGAAGAACTTCACCGAGATCGCTAGCAAGTTCAGCGAGAGACTGCAAGACTTCGACAAGAGCAACCCCATCGTGCTGAGAATGATGAACGATCAGCTGATGTTCCTGGAGAGAGCCTTCATCGACCCCCTGGGCCTGCCCGACAGACCCTTCTACAGACACGTGATCTACGCCCCTAGCAGCCACAACAAGTACGCCGGCGAGAGCTTCCCCGGCATCTACGACGCCCTGTTCGACATCGAGAGCAAGGTGGACCCTAGCAAGGCCTGGGGCGAGGTGAAGAGACAGATCTACGTGGCCGCCTTCACCGTGCAAGCCGCTGCCGAGACCCTGAGCGAGGTGGCCAAAAGCAGCAATGAAGCCACCAATATTACCCCGAAACATAACATGAAAGCCTTTCTGGATGAACTGAAAGCCGAGAACATTAAAAAGTTTCTGTACAACTTTACGCAGATCCCGCATCTGGCAGGCACCGAACAGAATTTTCAGCTGGCAAAACAAATTCAGAGCCAGTGGAAAGAATTTGGTCTGGATAGCGTTGAACTGGCCCATTATGATGTTCTGCTGAGCTATCCGAATAAAACCCATCCGAACTATATCAGCATCATCAATGAAGATGGCAACGAGATTTTTAACACCAGCCTGTTTGAACCGCCTCCGCCTGGTTATGAAAATGTTAGCGATATTGTTCCGCCTTTTAGCGCATTTAGTCCGCAGGGTATGCCGGAAGGTGATCTGGTTTATGTTAATTATGCACGCACCGAGGATTTCTTTAAACTGGAACGTGATATGAAGATTAACTGCAGCGGCAAAATTGTGATTGCCCGTTATGGTAAAGTTTTCCGTGGCAACAAAGTTAAAAATGCACAGCTGGCAGGCGCAAAAGGTGTTATTCTGTATAGCGATCCGGCAGATTATTTTGCACCGGGTGTGAAAAGCTATCCGGATGGTTGGAATCTGCCTGGTGGTGGTGTTCAGCGTGGTAATATTCTGAATCTGAATGGTGCCGGTGATCCGCTGACACCGGGTTATCCGGCAAATGAATATGCCTATCGTCGTGGTATTGCAGAAGCAGTTGGTCTGCCGAGCATTCCGGTTCATCCGATTGGTTATTATGATGCACAGAAACTGCTGGAAAAGATGGGTGGTAGCGCACCGCCTGATAGCAGCTGGCGTGGTAGCCTGAAAGTTCCGTATAATGTTGGTCCGGGTTTTACCGGCAATTTTAGCACCCAGAAAGTGAAAATGCATATCCATAGCACCAATGAAGTGACCCGTATCTATAATGTTATTGGCACCCTGCGTGGTGCAGTTGAACCGGATCGTTATGTTATTTTAGGTGGTCATCGTGATAGCTGGGTTTTTGGTGGTATTGATCCGCAGAGCGGTGCAGCAGTTGTTCATGAAATTGTTCGTAGCTTTGGTACGCTGAAGAAAGAAGGTTGGCGTCCGCGTCGTACCATTCTGTTTGCAAGCTGGGATGCAGAAGAATTCGGCCTGCTGGGTAGCACCGAATGGGCAGAAGAAAATAGCCGTCTGCTGCAAGAACGTGGTGTTGCCTATATTAACGCAGATAGCAGCATTGAAGGTAATTATACACTGCGTGTTGATTGTACACCGCTGATGTATAGCCTGGTTCATAATCTGACCAAAGAACTGAAAAGTCCGGATGAAGGTTTTGAAGGCAAAAGCCTGTATGAAAGCTGGACCAAAAAGAGCCCGAGTCCGGAATTTAGCGGTATGCCTCGTATTAGCAAATTAGGTAGCGGCAATGATTTTGAGGTTTTCTTTCAGCGTCTGGGTATTGCAAGCGGTCGTGCACGTTATACCAAAAATTGGGAAACCAATAAATTCAGCGGCTATCCGCTGTATCATAGCGTTTATGAAACATACGAACTGGTGGAAAAATTCTACGACCCGATGTTCAAATATCATCTGACCGTTGCACAGGTTCGTGGTGGTATGGTTTTCGAACTGGCAAATAGCATTGTTCTGCCGTTTGATTGTCGTGATTATGCCGTTGTGCTGCGTAAATATGCCGATAAAATCTATAGCATCAGCATGAAACATCCGCAAGAGATGAAAACCTATAGCGTTAGCTTTGATAGCCTGTTTAGTGCCGTGAAAAACTTCACCGAAATTGCCAGCAAATTTAGCGAACGTCTGCAGGATTTTGATAAAAGCAATCCGATCGTTCTGCGCATGATGAATGATCAACTGATGTTTCTGGAACGCGCATTTATCGATCCGCTGGGTCTGCCTGATCGTCCGTTTTATCGTCATGTGATTTATGCACCGAGCAGCCATAACAAATATGCGGGTGAATCATTTCCGGGTATTTATGATGCCCTGTTTGATATCGAAAGCAAAGTTGATCCGAGCAAAGCATGGGGTGAAGTTAAACGTCAGATTTATGTTGCAGCATTTACCGTTCAGGCAGCAGCAGAAACCCTGAGCGAAGTTGCAAAAAGTTCAAATGAAGCTACAAACATAACTCCGAAACACAACATGAAAGCCTTTCTGGATGAGTTGAAGGCGGAGAACATTAAAAAGTTTCTGTACAACTTCACCCAGATTCCGCACCTGGCCGGTACTGAGCAGAATTTTCAGCTGGCTAAGCAGATCCAGAGCCAGTGGAAAGAGTTCGGCCTGGATAGCGTGGAACTGGCGCATTATGATGTTCTGCTGAGCTATCCGAACAAGACCCATCCGAATTACATCAGCATTATTAACGAAGATGGCAACGAGATCTTTAACACCAGCCTGTTCGAGCCACCACCGCCGGGTTACGAAAACGTGAGCGATATTGTCCCGCCGTTTAGCGCGTTTTCGCCGCAAGGTATGCCGGAAGGCGACCTGGTGTACGTGAATTATGCCCGCACCGAGGACTTCTTCAAGCTTGAGAGGGATATGAAGATCAACTGCAGCGGAAAAATTGTGATCGCGCGTTATGGTAAAGTGTTCCGCGGCAATAAGGTGAAGAACGCCCAACTGGCGGGCGCGAAAGGTGTTATCCTGTACTCGGACCCAGCTGACTACTTCGCTCCGGGTGTTAAGTCTTATCCGGACGGCTGGAATCTTCCGGGTGGTGGCGTTCAGCGTGGCAACATCTTGAATCTGAATGGTGCAGGGGATCCGCTGACGCCTGGTTACCCGGCTAATGAGTATGCGTATCGTCGTGGCATCGCGGAAGCAGTCGGTTTGCCGTCTATCCCGGTGCACCCCATCGGTTACTATGACGCTCAAAAACTGCTGGAAAAAATGGGTGGAAGTGCACCACCGGACTCTAGCTGGCGCGGTAGCCTCAAGGTCCCGTATAACGTTGGCCCTGGTTTTACCGGTAACTTCAGCACTCAAAAAGTGAAGATGCACATTCATAGCACGAACGAGGTTACCCGTATTTACAATGTTATTGGCACCCTCCGTGGTGCTGTCGAGCCGGACCGCTATGTTATCCTGGGTGGCCATCGTGACAGCTGGGTTTTTGGTGGTATCGACCCTCAAAGCGGCGCGGCCGTTGTCCACGAAATCGTGCGTAGCTTCGGTACCCTGAAGAAAGAAGGCTGGCGTCCGCGTCGCACCATTTTGTTCGCCTCCTGGGATGCAGAGGAATTCGGCTTGTTGGGTTCGACCGAATGGGCAGAAGAGAATAGCCGTTTGCTGCAAGAAAGAGGCGTGGCATATATCAATGCGGATTCCAGCATTGAAGGTAACTACACCCTGCGCGTGGATTGCACCCCGCTGATGTATAGCCTTGTTCACAACCTGACGAAGGAGCTGAAGAGTCCGGACGAAGGTTTTGAGGGCAAATCTTTGTACGAGTCCTGGACCAAAAAATCCCCGTCCCCGGAATTCTCCGGTATGCCGCGTATTTCCAAACTGGGCTCTGGCAACGACTTCGAGGTGTTCTTTCAACGTTTAGGCATTGCCAGCGGCCGTGCTAGGTATACCAAAAACTGGGAAACGAATAAATTTAGCGGTTATCCGCTGTACCACAGCGTTTACGAGACATACGAATTGGTAGAGAAGTTCTACGACCCGATGTTTAAATACCACCTGACCGTTGCGCAGGTTCGCGGCGGTATGGTTTTCGAACTCGCGAACAGCATCGTCCTGCCGTTTGACTGTCGTGATTATGCTGTGGTGTTACGCAAATACGCGGACAAAATCTACAGCATCAGCATGAAGCACCCGCAAGAGATGAAGACGTACAGCGTGTCTTTTGACAGCCTGTTCTCCGCGGTGAAGAACTTCACTGAGATCGCGTCAAAATTTTCCGAAAGGCTGCAGGATTTCGACAAGAGCAATCCGATTGTGTTGCGGATGATGAACGATCAGCTGATGTTCTTGGAGCGTGCATTCATCGATCCACTGGGTCTGCCGGATCGTCCGTTTTACCGCCATGTTATTTATGCGCCGTCTTCACATAACAAGTATGCAGGTGAAAGCTTTCCGGGCATCTACGATGCGCTGTTTGACATCGAGTCCAAGGTTGATCCGAGCAAGGCGTGGGGTGAAGTGAAACGTCAGATTTATGTTGCCGCGTTCACCGTGCAGGCAGCTGCGGAAACCCTGTCGGAGGTCGCG[0082]上述各经优化后的编码序列与wt编码序列(SEQIDNo:1)编码的PSMA蛋白是相KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIY