本发明涉及抗呼吸道合胞病毒感染的疫苗,属于医药领域。
背景技术:
1、人呼吸道合胞病毒((respiratory syncytial virus,rsv),是在全球范围内引起5岁以下儿童和老人下呼吸道感染的最重要病原。rsv属于副黏病毒科的肺病毒属(pneumovirus),是一种反义单链rna病毒,只有一种血清型。其主要通过鼻咽黏膜或眼黏膜接触含病毒的分泌物或污染物而传播。科学家从上世纪60年代就开始rsv疫苗研发工作,但当时的rsv灭活疫苗不仅未能预防rsv感染,还导致新发rsv感染的婴儿病情加重。
2、病毒形态为球形,直径为120~300nm,有包膜,基因组为非分节段的单负链rna,主要编码10种蛋白质,即融合蛋白(f)、小疏水蛋白(sh)和黏附蛋白(g)三种跨膜蛋白,两种基质蛋白m1和m2,三种与病毒rna相结合形成核衣壳的蛋白(l、n和p),两种非结构蛋白(ns1和ns2)。病毒包膜上有糖蛋白组成的刺突,无ha、na和hl。
3、融合蛋白(f)是rsv的病毒表面蛋白,主演参与病毒进入细胞的过程,而且拥有高度的序列保守性,因此成为疫苗开发的主要目标蛋白。f蛋白有两个构相,分别为融合前(pre-f)和融合后(post-f)构相。传染性rsv的表面是同时拥有这两种构相的f蛋白,病毒感染细胞后pre-f构相会变构为post-f构相。自1956年首次发现该病毒以来,该病毒的复杂分子结构和安全问题阻碍了疫苗的开发工作。
技术实现思路
1、为解决针对呼吸道合胞病毒感染引起老年人、婴儿和免疫力低下人群的呼吸道感染,尚缺乏有效预防和治疗药物的问题,本发明提供了抗呼吸道合胞病毒感染的腺病毒载体疫苗,该疫苗是以腺病毒为载体,将rsv f蛋白序列优化设计出一系列融合前(pre-f)序列作为抗原基因重组到腺病毒载体上,尤其是基于a型rsv f蛋白设计抗原基因,使腺病毒载体能表达rsv抗原蛋白,帮助宿主抵抗呼吸道合胞病毒感染,具有较好的防治效果。
2、第一方面,本发明提供了预防和/或治疗呼吸道合胞病毒感染的腺病毒载体疫苗,其是通过构建含有表达呼吸道合胞病毒基因的重组腺病毒载体获得;所述表达呼吸道合胞病毒基因的多核苷酸序列选自seq id no.12、seq id no.14、seq id no.16、seq idno.18、seq id no.20、seq id no.22、seq id no.24、seq id no.26、seq id no.28、seq idno.30、seq id no.32、seq id no.34中至少一种。
3、进一步地,上述多核苷酸序列编码的蛋白氨基酸序列选自seq id no.11、seq idno.13、seq id no.15、seq id no.17、seq id no.19、seq id no.21、seq id no.23、seq idno.25、seq id no.27、seq id no.29、seq id no.31、seq id no.33中至少一种。
4、进一步的,所述腺病毒载体疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
5、进一步地,所述的重组腺病毒载体选自如下至少一种:腺病毒、安卡拉痘苗病毒、腺相关病毒。
6、优选地,选自人5型、35型或26型或/和黑猩猩adc68型或adc7型的复制缺陷型腺病毒。
7、更优选地,选自e1、e3联合缺失的人5型复制缺陷型腺病毒。
8、进一步地,所述的腺病毒载体疫苗为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂。
9、优选地,所述的疫苗为鼻喷制剂或肌肉注射制剂。
10、本发明提供了上述腺病毒载体疫苗中腺病毒的制备方法,包括以下步骤:构建含有所述多核苷酸的穿梭质粒载体;然后将构建的穿梭质粒载体与骨架质粒一起转染入宿主细胞,培养宿主细胞;获得复制缺陷重组腺病毒,然后扩大培养、纯化,即可。
11、seq id no.11
12、rsv-a-pref-1(r1)(del104-145aa)
13、mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsaiggyipeaprdgqayvrkdgewvllstfl
14、seq id no.12编码seq id no.11的多核苷酸
15、atggagctgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaacatcaccgaggagttctatcagagcacctgcagcgctgtgagcaaaggctatctgagcgccctgagaaccggctggtacacaagcgtcatcaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctcctgatgcagagcaccccccctacctccgctattgctagcggcgtggccgtgagcaaggtcctgcacctggagggcgaggtgaacaagatcaagagcgccctgctcagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacaagcaaggtgctggacctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccgtgagcacctacatgctgaccaacagcgaactgctgtccctgattaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacaccaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtcaacaagcaagaggggaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgagcgctatcggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctgctgtccaccttcctctga
16、seq id no.13
17、rsv-a-pref-1f(r2)(del104-145aa)
18、mellilkanaittiltavtfcfasgqniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellhnvnagksttnimittiiiviivillsliavglllyckarstpvtlskdqlsginniafsn
19、seq id no.14编码seq id no.13的多核苷酸
20、atggagctgctgatcctgaaggccaacgccatcacaaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaacatcaccgaggagttctatcagagcacctgcagcgccgtgagcaaggggtatctgagcgccctgagaaccggctggtacacctccgtcatcaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcaactgctgatgcaaagcacccctcccacaagcgccatcgcctccggcgtggctgtcagcaaggtcctccatctggagggcgaggtgaacaagatcaagagcgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacctccaaggtgctggatctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtgaatgctggcgtcaccacccccgtgagcacctacatgctgaccaacagcgagctgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcacaaccaacaccaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagcagcgtgattacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtgaataagcaagagggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgcacaacgtgaacgccggcaagagcaccaccaacatcatgatcaccaccatcattatcgtgatcatcgtgattctcctgtccctgatcgccgtcggcctgctcctgtactgcaaggctagaagcacccccgtgaccctgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaactga
21、seq id no.15
22、rsv-a-pref-2(r3)(2个二硫键突变)
23、mellilkanaittiltavtfcfasgqncteefyqstcsavskgylsalrtgwytcvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptnnrarrelprfmnytlnnakktnvtlskkrkrrflgfllgvgsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvctskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsaiggyipeaprdgqayvrkdgewvllstfl
24、seq id no.16编码seq id no.15的多核苷酸
25、atggagctgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaactgcaccgaggagttctatcagagcacctgcagcgccgtcagcaaaggctacctgagcgccctgagaaccggctggtacacctgcgtgattaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagagcaccccccccaccaacaacagagctagaagagagctgcctagattcatgaactacaccctgaacaacgccaagaagaccaacgtgaccctgagcaagaagagaaagagaagattcctgggcttcctgctgggcgtgggcagcgccattgctagcggcgtggccgtgagcaaggtgctgcacctcgagggcgaggtgaacaagatcaagagcgccctgctgtccaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgtgcacaagcaaggtcctggacctgaagaactacatcgacaagcagctgctgcccatcgtcaacaagcaatcctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccgtgagcacctacatgctgaccaacagcgaactcctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacaccaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtgaataaacaagagtgcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgagcgccatcggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctgctgagcaccttcctgtga
26、seq id no.17
27、rsv-a-pref-2f(r4)(2个二硫键突变)
28、mellilkanaittiltavtfcfasgqncteefyqstcsavskgylsalrtgwytcvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptnnrarrelprfmnytlnnakktnvtlskkrkrrflgfllgvgsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvctskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellhnvnagksttnimittiiiviivillsliavglllyckarstpvtlskdqlsginniafsn
29、seq id no.18编码seq id no.17的多核苷酸
30、atggagctgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcaagggctacctgagcgccctgagaaccggctggtacacctgtgtcatcaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagagcaccccccccaccaacaacagagctagaagagagctgcctagattcatgaactacaccctgaacaacgccaagaagaccaacgtgaccctgagcaagaaaagaaagagaagattcctggggttcctgctgggggtgggcagcgccattgctagcggcgtggccgtctccaaggtcctgcacctggagggcgaggtgaacaagatcaagagcgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgtgcacaagcaaggtgctggacctgaagaactacatcgacaagcagctgctgcccatcgtcaacaaacagtcctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtcaatgccggggtgaccacccccgtgagcacctacatgctgaccaacagcgagctgctgagcctcatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgtaccaccaacaccaaagagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagctccgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagcaagagtgcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgcacaacgtgaacgccggcaagagcaccaccaacatcatgatcacaaccatcattatcgtgatcatcgtgattctcctgagcctgatcgccgtgggcctgctcctgtactgcaaggctagaagcacccccgtgaccctgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaactga
31、seq id no.19
32、rsv-a-pref-3(r5)(del104-145aa+1个二硫键突变)
33、mellilkanaittiltavtfcfasgqncteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsaiggyipeaprdgqayvrkdgewvllstfl
34、seq id no.20编码seq id no.19的多核苷酸
35、atggagctgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaactgcaccgaggagttctatcagagcacctgcagcgctgtgagcaaaggctatctgagcgccctgagaaccggctggtacacaagcgtcatcaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctcctgatgcagagcaccccccctacctccgctattgctagcggcgtggccgtgagcaaggtcctgcacctggagggcgaggtgaacaagatcaagagcgccctgctcagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacaagcaaggtgctggacctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccgtgagcacctacatgctgaccaacagcgaactgctgtccctgattaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacaccaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtcaacaagcaagagtgtaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgagcgctatcggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctgctgtccaccttcctctga
36、seq id no.21
37、rsv-a-pref-3f(r6)(del104-145aa+1个二硫键突变)
38、mellilkanaittiltavtfcfasgqncteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellhnvnagksttnimittiiiviivillsliavglllyckarstpvtlskdqlsginniafsn
39、seq id no.22编码seq id no.21的多核苷酸
40、atggagctgctgatcctgaaggccaacgccatcacaaccatcctgaccgccgtgaccttctgcttcgctagcgggcagaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcaaggggtatctgagcgccctgagaaccggctggtacacctccgtcatcaccatcgagctgagcaacatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcaactgctgatgcaaagcacccctcccacaagcgccatcgcctccggcgtggctgtcagcaaggtcctccatctggagggcgaggtgaacaagatcaagagcgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacctccaaggtgctggatctgaagaactacatcgacaagcagctgctgcccatcgtgaacaagcagagctgcagcatcagcaacatcgagaccgtgatcgagtttcagcagaagaacaacagactgctggagatcacaagagagttcagcgtgaatgctggcgtcaccacccccgtgagcacctacatgctgaccaacagcgagctgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcaacaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccctgtacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcacaaccaacaccaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgagacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgagatcaacctgtgcaacgtggacatcttcaaccccaagtacgactgcaagatcatgacaagcaagaccgacgtgagcagcagcgtgattacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcatggacaccgtgagcgtgggcaacaccctgtactacgtgaataagcaagagtgcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaaagagcgacgagctgctgcacaacgtgaacgccggcaagagcaccaccaacatcatgatcaccaccatcattatcgtgatcatcgtgattctcctgtccctgatcgccgtcggcctgctcctgtactgcaaggctagaagcacccccgtgaccctgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaactga
41、seq id no.23
42、rsv-b-pref-1(r7)(del104-145aa)
43、mellihrssaifltlavnalyltssqniteefyqstcsavsrgyfsalrtgwytsvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvltskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellsaiggyipeaprdgqayvrkdgewvllstfl
44、seq id no.24编码seq id no.23的多核苷酸
45、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaacatcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacctccgtgatcacaatcgaactgagcaacattaaggagaccaagtgtaacggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccccgccgctagcgccatcgctagcggcatcgccgtgtccaaagtgctgcacctggagggcgaggtgaacaagatcaagaacgccctgctgagcacaaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacaagcaaggtgctggacctgaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgaactgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaatatcaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagacaaagtgcacagctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagggcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgagctgctcagcgctatcggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctcctgagcaccttcctctga
46、seq id no.25
47、rsv-b-pref-1f(r8)(del104-145aa)
48、mellihrssaifltlavnalyltssqniteefyqstcsavsrgyfsalrtgwytsvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvltskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellhnvntgksttnimittiiiviivvllsliaiglllyckakntpvtlskdqlsginniafsk
49、seq id no.26编码seq id no.25的多核苷酸
50、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaacatcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacaagcgtcatcaccatcgagctgagcaatattaaggagaccaagtgtaatggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccctgccgctagcgccatcgctagcggcatcgccgtgagcaaagtgctgcatctggagggcgaggtgaacaagatcaagaacgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacctccaaggtgctggacctcaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgagctgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacatcaaagagggctccaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagggcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgagctgctgcacaacgtgaacaccggcaagtccaccacaaacatcatgatcaccaccatcattatcgtgatcatcgtggtgctgctgtccctgatcgccatcggcctgctcctgtactgcaaggccaagaacacacctgtgaccctgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaagtga
51、seq id no.27
52、rsv-b-pref-2(r9)(2个二硫键突变)
53、mellihrssaifltlavnalyltssqncteefyqstcsavsrgyfsalrtgwytcvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaannrarreapqymnytinttknlnvsiskkrkrrflgfllgvgsaiasgiavskvlhlegevnkiknallstnkavvslsngvsvctskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellsaiggyipeaprdgqayvrkdgewvllstfl
54、seq id no.28编码seq id no.27的多核苷酸
55、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacctgcgtcatcacaatcgagctgagcaacatcaaagagaccaagtgcaacggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccccgccgccaacaacagagctagaagagaggcccctcagtacatgaactacaccatcaacaccaccaagaacctgaacgtgagcatcagcaagaagagaaagagaagattcctgggcttcctgctgggcgtgggcagcgccatcgctagcggcatcgccgtgtccaaggtcctgcatctcgagggcgaggtgaacaagatcaagaacgccctgctgagcacaaacaaggccgtggtgagcctgagcaacggcgtgagcgtgtgcacaagcaaggtgctggacctcaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgagctgctgagcctcatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacatcaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtcatcacaagcctgggcgccatcgtgagctgctacggcaagaccaaatgtaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagtgcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgaactgctgagcgccattggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctgctgagcaccttcctgtga
56、seq id no.29
57、rsv-b-pref-2f(r10)(2个二硫键突变)
58、mellihrssaifltlavnalyltssqncteefyqstcsavsrgyfsalrtgwytcvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaannrarreapqymnytinttknlnvsiskkrkrrflgfllgvgsaiasgiavskvlhlegevnkiknallstnkavvslsngvsvctskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellhnvntgksttnimittiiiviivvllsliaiglllyckakntpvtlskdqlsginniafsk
59、seq id no.30编码seq id no.29的多核苷酸
60、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacctgtgtgatcaccatcgagctgagcaacatcaaggagaccaaatgtaacggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccctgccgccaacaacagagctagaagagaggcccctcagtacatgaactacaccatcaacaccaccaagaacctgaacgtgagcatcagcaagaagagaaagagaagattcctgggcttcctgctgggcgtgggcagcgccatcgctagcggcatcgccgtgagcaaggtgctgcacctggagggcgaggtgaacaagatcaagaacgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgtgcacaagcaaagtgctggacctcaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgaactgctcagcctgattaatgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacattaaagaggggagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtgatcacaagcctcggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagtgcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgagctgctgcacaacgtgaacaccggcaagagcaccacaaatatcatgatcaccaccatcattatcgtgatcatcgtggtgctcctgagcctgatcgccatcggcctgctcctgtactgcaaggccaaaaacacccccgtgacactgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaagtga
61、seq id no.31
62、rsv-b-pref-3(r11)(del104-145aa+1个二硫键突变)
63、mellihrssaifltlavnalyltssqncteefyqstcsavsrgyfsalrtgwytsvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvltskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellsaiggyipeaprdgqayvrkdgewvllstfl
64、seq id no.32编码seq id no.31的多核苷酸
65、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacctccgtgatcacaatcgaactgagcaacattaaggagaccaagtgtaacggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccccgccgctagcgccatcgctagcggcatcgccgtgtccaaagtgctgcacctggagggcgaggtgaacaagatcaagaacgccctgctgagcacaaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacaagcaaggtgctggacctgaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgaactgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaatatcaaggagggcagcaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagacaaagtgcacagctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagtgcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgagctgctcagcgctatcggcggctacatccccgaggcccctagagacggccaagcctacgtgagaaaggacggcgagtgggtgctcctgagcaccttcctctga
66、seq id no.33
67、rsv-b-pref-3f(r12)(del104-145aa+1个二硫键突变)
68、mellihrssaifltlavnalyltssqncteefyqstcsavsrgyfsalrtgwytsvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvltskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellhnvntgksttnimittiiiviivvllsliaiglllyckakntpvtlskdqlsginniafsk
69、seq id no.34编码seq id no.33的多核苷酸
70、atggagctgctgatccacagaagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagcagccaaaactgcaccgaggagttctatcagagcacctgcagcgccgtgagcagaggctacttcagcgccctgagaaccggctggtacacaagcgtcatcaccatcgagctgagcaatattaaggagaccaagtgtaatggcaccgacaccaaggtgaagctgatcaagcaagagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagaacacccctgccgctagcgccatcgctagcggcatcgccgtgagcaaagtgctgcatctggagggcgaggtgaacaagatcaagaacgccctgctgagcaccaacaaggccgtggtgagcctgagcaacggcgtgagcgtgctgacctccaaggtgctggacctcaagaactacatcaacaacagactgctgcccatcgtgaatcagcagagctgcagaatcagcaacatcgagaccgtgatcgagtttcagcagatgaacagcagactgctggagatcacaagagagttcagcgtgaacgccggcgtgaccacccccctgagcacctacatgctgaccaacagcgagctgctgagcctgatcaacgacatgcccatcaccaacgatcagaagaagctgatgagcagcaacgtgcagatcgtgagacagcagagctacagcatcatgagcatcatcaaggaagaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacaagccccctgtgcaccaccaacatcaaagagggctccaacatctgcctgacaagaaccgacagaggctggtactgcgacaacgccggcagcgtgagcttcttcccccaagccgacacctgcaaggtgcagagcaacagagtgttctgcgacaccatgaacagcctgaccctgcctagcgaggtgagcctgtgcaacaccgacatcttcaacagcaagtacgactgcaagatcatgacaagcaagaccgacatcagcagcagcgtgatcacaagcctgggcgccatcgtgagctgctacggcaagaccaagtgcaccgctagcaacaagaacagaggcatcatcaagaccttcagcaacggctgcgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaacaccctgtactacgtgaacaagctggagtgcaagaacctgtacgtgaagggcgagcccatcatcaactactacgaccccctggtgttccctagcgacgagttcgacgctagcatcagccaagtgaacgagaagatcaatcagagcctggccttcatcagaagaagcgacgagctgctgcacaacgtgaacaccggcaagtccaccacaaacatcatgatcaccaccatcattatcgtgatcatcgtggtgctgctgtccctgatcgccatcggcctgctcctgtactgcaaggccaagaacacacctgtgaccctgagcaaggatcagctgagcggcatcaacaacatcgccttcagcaagtga
71、第二方面,本发明提供了预防和/或治疗呼吸道合胞病毒感染的组合物,是含有预防和/或治疗呼吸道合胞病毒感染的重组蛋白疫苗和上述的腺病毒载体疫苗为活性成分的复方制剂。
72、本发明提供了预防和/或治疗呼吸道合胞病毒感染的联合用药物,其含有分别或同时给药的预防和/或治疗呼吸道合胞病毒感染的重组蛋白疫苗和所述的腺病毒载体疫苗。
73、进一步地,所述重组蛋白疫苗含有预防和/或治疗呼吸道合胞病毒感染的重组蛋白,所述重组蛋白的氨基酸序列是与seq id no.1或seq id no.2具有70%以上的同源性且具有相同或相似的生物学活性。
74、优选地,所述重组蛋白的氨基酸序列选自seq id no.1、seq id no.2、seq idno.3、seq id no.4、seq id no.5或seq id no.6中至少一种。
75、进一步地,所述重组蛋白疫苗是通过氨基酸序列选自seq id no.7、seq id no.8中至少一种构建的蛋白前体获得。
76、进一步地,编码上述蛋白前体的核苷酸序列选自seq id no.9或seq id no.10中至少一种。
77、进一步地,所述重组蛋白疫苗中的重组蛋白的制备方法包括如下步骤:培养宿主细胞,使其表达所述的蛋白或前体,然后回收所述的蛋白,即得。
78、进一步地,所述的宿主细胞采用昆虫细胞或哺乳动物细胞。优选地,所述的昆虫细胞选自sf9细胞、sf21细胞、hi5细胞中至少一种。优选地,所述的哺乳动物细胞为cho细胞。进一步优选地,所述的cho细胞表达载体为ptt5或ftp-002。
79、其中,所述宿主细胞含有重组载体,所述重组载体含有编码上述蛋白或蛋白前体的核苷酸。进一步地,所述的重组载体采用昆虫杆状病毒表达载体或哺乳动物细胞表达载体。优选地,所述的昆虫杆状病毒表达载体为pfastbac1。优选地,所述的哺乳动物细胞表达载体为cho细胞表达载体。
80、进一步地,所述重组蛋白疫苗含有所述的重组蛋白和/或所述的蛋白前体,以及药学上可接受的辅料或者辅助性成分。
81、进一步地,所述的辅助性成分为免疫佐剂。优选地,所述的免疫佐剂选自如下至少一种:铝盐、钙盐、植物皂苷、植物多糖、单磷酸脂质a、胞壁酰二肽、胞壁酰三肽、鲨烯水包油乳剂、重组霍乱毒素、gm-csf细胞因子、脂质、阳离子脂质体材料、cpg odn。
82、进一步地,所述的鲨烯水包油乳剂为mf59。进一步地,所述的铝盐选自氢氧化铝、明矾中至少一种。进一步地,所述的钙盐为磷酸三钙。进一步地,所述的植物皂苷为qs-21或iscom。进一步地,所述的植物多糖为黄芪多糖。进一步地,所述的脂质选自如下至少一种:磷脂酰乙醇胺(pe)、磷脂酰胆碱(pc)、胆固醇(chol)、二油酰基磷脂酰乙醇胺(dope)。进一步地,所述的阳离子脂质体材料选自如下至少一种:(2,3-二油氧基丙基)三甲基氯化铵(dotap)、n-[1-(2,3-二油酰氯)丙基]-n,n,n-氯化三甲胺(dotma)、阳离子胆固醇(dc-chol)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(dospa)、溴化三甲基十二烷基铵(dtab)、溴化三甲基十四烷基铵(ttab)、溴化三甲基十六烷基铵(ctab)、溴化二甲基双十八烷基铵(ddab)。
83、进一步地,所述组合物或联合用药物为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂;优选地,所述的疫苗为肌肉注射制剂和鼻喷制剂。
84、第三方面,本发明提供了上述腺病毒载体疫苗、组合物或联合用药物在制备预防和/或治疗呼吸道合胞病毒感染的药物中的用途。
85、其中,所述seq id no.1为(rsv subtype af sequence):
86、qniteefyqstcsavskgylsalrtgwytsvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptnnrarrelprfmnytlnnakktnvtlskkrkrrflgfllgvgsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvltskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqegkslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellhnvnagksttnimittiiiviivillsliavglllyckarstpvtlskdqlsginniafsn
87、其中,所述seq id no.2为(rsv subtype b f sequence):
88、qniteefyqstcsavsrgyfsalrtgwytsvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaannrarreapqymnytinttknlnvsiskkrkrrflgfllgvgsaiasgiavskvlhlegevnkiknallstnkavvslsngvsvltskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklegknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellhnvntgksttnimittiiiviivvllsliaiglllyckakntpvtlskdqlsginniafsk
89、seq id no.3:优化的rsv subtype a fprotein截短序列
90、qncteefyqstcsavskgylsalrtgwytcvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvctskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdell
91、seq id no.4:优化的rsv subtype b f protein截短序列
92、qncteefyqstcsavsrgyfsalrtgwytcvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvctskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdell
93、seq id no.5:其是在优化的rsv subtype af截短序列上添加saig和foldon序列qncteefyqstcsavskgylsalrtgwytcvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvctskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsaiggyipeaprdgqayvrkdgewvllstfl
94、seq id no.6:其是在优化的rsv subtype b f截短序列上添加saig和foldon序列
95、qncteefyqstcsavsrgyfsalrtgwytcvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvctskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellsaiggyipeaprdgqayvrkdgewvllstfl
96、seq id no.7信号肽+优化的rsv subtype af蛋白截短序列+(saig)+foldon序列+thrombin酶切位点+his 6标签+strep ii标签
97、mellilkanaittiltavtfcfasgqncteefyqstcsavskgylsalrtgwytcvitielsnikenkcngtdakvklikqeldkyknavtelqllmqstpptsaiasgvavskvlhlegevnkiksallstnkavvslsngvsvctskvldlknyidkqllpivnkqscsisnietviefqqknnrlleitrefsvnagvttpvstymltnsellslindmpitndqkklmsnnvqivrqqsysimsiikeevlayvvqlplygvidtpcwklhtsplcttntkegsnicltrtdrgwycdnagsvsffpqaetckvqsnrvfcdtmnsltlpseinlcnvdifnpkydckimtsktdvsssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgmdtvsvgntlyyvnkqeckslyvkgepiinfydplvfpsdefdasisqvnekinqslafirksdellsaiggyipeaprdgqayvrkdgewvllstflvprgshhhhhhgswshpqfek
98、seq id no.8信号肽+构建rsv subtype b f蛋白截短序列+(saig)+foldon序列+thrombin酶切位点+his 6标签+strep ii标签
99、mellilkanaittiltavtfcfasgqncteefyqstcsavsrgyfsalrtgwytcvitielsniketkcngtdtkvklikqeldkyknavtelqllmqntpaasaiasgiavskvlhlegevnkiknallstnkavvslsngvsvctskvldlknyinnrllpivnqqscrisnietviefqqmnsrlleitrefsvnagvttplstymltnsellslindmpitndqkklmssnvqivrqqsysimsiikeevlayvvqlpiygvidtpcwklhtsplcttnikegsnicltrtdrgwycdnagsvsffpqadtckvqsnrvfcdtmnsltlpsevslcntdifnskydckimtsktdisssvitslgaivscygktkctasnknrgiiktfsngcdyvsnkgvdtvsvgntlyyvnklecknlyvkgepiinyydplvfpsdefdasisqvnekinqslafirrsdellsaiggyipeaprdgqayvrkdgewvllstflvprgshhhhhhgswshpqfek
100、seq id no.9编码seq id no.7所示的多核苷酸序列,其是基于优化的rsvsubtype af蛋白进行密码子优化得到
101、atggagctgctgatcctgaaggccaacgccatcacaaccatcctgacagccgtgacattctgcttcgcctccggccagaattgcaccgaggagttctaccagtccacatgttccgccgtgagcaagggctacctgtccgccctgagaaccggctggtacacctgtgtgatcaccatcgagctgtccaatatcaaggagaacaagtgcaacggcaccgacgccaaggtgaagctgatcaagcaggagctggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagagcacacctcccacctccgccatcgccagcggcgttgctgtgtccaaggtgctgcacctggagggcgaggtgaacaagatcaagtccgccctgctgtccaccaacaaggccgtggtgtccctgagcaacggcgtgtccgtgtgcaccagcaaggtgctggatctgaagaattacatcgacaagcagctgctgcccatcgtgaataagcagagctgtagcatcagcaacatcgagaccgtgatcgagttccagcagaagaataacaggctgctggagatcaccagggagttctccgtgaacgccggcgtgaccacacccgtgagcacctacatgctgaccaattccgagctgctgtccctgatcaatgatatgcccatcaccaatgatcagaagaagctgatgtccaacaacgtgcagatcgtgaggcagcagtcctacagcatcatgtccatcatcaaggaggaggtgctggcctacgtggtgcagctgcctctgtacggcgtgatcgataccccttgctggaagctgcacaccagccccctgtgcacaacaaacaccaaggagggcagcaacatctgcctgacaaggacagacagaggctggtactgtgataacgccggcagcgtgtccttcttcccccaggccgagacctgcaaggtgcagtccaacagagtgttctgcgataccatgaactccctgaccctgcctagcgagatcaacctgtgcaacgtggatatcttcaatcctaagtacgattgtaagatcatgacctccaagacagacgtgtccagcagcgtgatcacatccctgggcgccatcgtgtcctgttacggcaagaccaagtgtaccgcctccaataagaataggggcatcatcaagacattctccaatggctgcgattacgtgtccaacaagggcatggataccgtgagcgtgggcaatacactgtactacgtgaacaagcaggagtgtaagtccctgtacgtgaagggcgagcctatcatcaacttctacgaccccctggtgttccctagcgacgagttcgacgccagcatctcccaggtgaacgagaagatcaaccagtccctggccttcatcaggaagagcgacgagctgctgagcgccatcggcggctacatccctgaggcccctagggacggccaggcctacgtgaggaaggacggcgagtgggtgctgctgtccacattcctggtgcctagaggctcccaccaccaccaccatcacggcagctggtcccaccctcagttcgagaagtga
102、seq id no.10编码seq id no.8所示的多核苷酸序列。其是基于优化的rsvsubtype b f蛋白进行密码子优化得到
103、atggagctgctgatccacaggagcagcgccatcttcctgaccctggccgtgaacgccctgtacctgacaagctcccagaactgcaccgaggagttctaccagagcacctgtagcgccgtgtccaggggctacttcagcgccctgaggaccggctggtacacctgcgtgatcaccatcgagctgtccaatatcaaggagaccaagtgtaacggcaccgataccaaggtgaagctgatcaagcaggagctggataagtacaagaatgccgtgaccgagctgcagctgctgatgcagaatacacctgccgcctccgccatcgccagcggaatcgctgtgtccaaggtgctgcacctggagggcgaggtgaataagatcaagaatgccctgctgtccacaaacaaggccgtggtgagcctgagcaacggcgtgagcgtgtgtacatccaaggtgctcgacctgaagaactacatcaacaatagactgctgcctatcgtgaaccagcagagctgtaggatcagcaatatcgagacagtgatcgagttccagcagatgaacagcaggctgctggagatcacaagagagttctccgtgaatgccggcgtgacaacacctctgtccacctacatgctgacaaattccgagctgctgtccctgatcaatgatatgcctatcacaaacgatcagaagaagctgatgagctccaatgtgcagatcgtgaggcagcagagctactccatcatgtccatcatcaaggaggaggtgctggcctacgtggtgcagctgcccatctacggcgtgatcgacaccccctgctggaagctgcacacatcccctctgtgcaccaccaatatcaaggaaggctccaatatctgtctgaccaggaccgataggggctggtactgcgataatgccggctccgtgagcttcttccctcaggccgacacctgtaaggtgcagagcaatagagtgttctgtgataccatgaactccctgaccctgccctccgaggtgagcctgtgtaatacagatatcttcaatagcaagtacgattgcaagatcatgaccagcaagaccgatatctccagcagcgtgatcacctccctgggcgccatcgtgagctgctacggcaagaccaagtgtaccgcctccaacaagaacagaggcatcatcaagaccttcagcaatggctgtgactacgtgagcaacaagggcgtggacaccgtgagcgtgggcaatacactgtactacgtgaacaagctggagtgtaagaacctgtacgtgaagggcgagcctatcatcaattactacgaccctctggtgttcccctccgatgagttcgacgccagcatctcccaggtgaacgagaagatcaatcagtccctggccttcatcagaagatccgatgagctgctgagcgccatcggcggctacatccccgaggcccctagagatggccaggcctacgtgagaaaggatggcgagtgggtgctgctgtccaccttcctggtgcccaggggcagccaccaccaccaccatcacggctcctggtcccacccccagttcgagaagtga。
104、有益效果:本发明基于呼吸道合胞病毒f蛋白序列经过优化设计了一系列抗呼吸道合胞病毒感染的腺病毒载体疫苗。动物实验证明,携带密码子优化的a型rsv-pref(rsv-a-pref)基因的重组腺病毒作为腺病毒载体疫苗,免疫动物后能同时诱导产生针对融合前(pre-f)和融合后(postf)糖蛋白的血清抗体,且在支气管中,能诱导产生高效针对融合前(pre-f)和融合后(postf)糖蛋白的抗体,可见,该腺病毒载体疫苗针对rsv病毒具有较强的体液免疫保护能力。
105、其中,含有优化的a型rsv-pref(rsv-a-pref)r2基因(氨基酸序列如seq id no.13所示)的重组腺病毒作为疫苗,免疫动物后,表现出更强的体液免疫保护能力及安全性,可以大规模制备,帮助宿主抵抗呼吸道合胞病毒感染,具有广阔应用前景。
1.预防和/或治疗呼吸道合胞病毒感染的腺病毒载体疫苗,其特征是:其是通过构建含有表达呼吸道合胞病毒基因的重组腺病毒载体获得;所述表达呼吸道合胞病毒基因的多核苷酸序列选自seq id no.12、seq id no.14、seq id no.16、seq id no.18、seq id no.20、seq id no.22、seq id no.24、seq id no.26、seq id no.28、seq id no.30、seq idno.32、seq id no.34中至少一种。
2.根据权利要求1所述的腺病毒载体疫苗,其特征是:多核苷酸序列编码的蛋白氨基酸序列选自seq id no.11、seq id no.13、seq id no.15、seq id no.17、seq id no.19、seqid no.21、seq id no.23、seq id no.25、seq id no.27、seq id no.29、seq id no.31、seqid no.33中至少一种。
3.根据权利要求1或2所述的腺病毒载体疫苗,其特征是:所述腺病毒载体疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
4.根据权利要求1或2所述的腺病毒载体疫苗,其特征是:所述腺病毒载体选自如下至少一种:腺病毒、安卡拉痘苗病毒、腺相关病毒;优选地,选自人5型、35型或26型或/和黑猩猩adc68型或adc7型的复制缺陷型腺病毒;更优选地,选自e1、e3联合缺失的人5型复制缺陷型腺病毒。
5.根据权利要求1-4任一项所述的腺病毒载体疫苗,其特征是:所述的疫苗为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂;优选地,所述的疫苗为鼻喷制剂或肌肉注射制剂。
6.权利要求1-5任一项所述腺病毒载体疫苗中的腺病毒的制备方法,其特征是:包括以下步骤:构建含有所述多核苷酸的穿梭质粒载体;然后将构建的穿梭质粒载体与骨架质粒一起转染入宿主细胞,培养宿主细胞;获得复制缺陷重组腺病毒,然后扩大培养、纯化,即可。
7.预防和/或治疗呼吸道合胞病毒感染的组合物,其特征是:是含有预防和/或治疗呼吸道合胞病毒感染的重组蛋白疫苗和权利要求1-5任一项所述的腺病毒载体疫苗为活性成分的复方制剂。
8.预防和/或治疗呼吸道合胞病毒感染的联合用药物,其特征是:其含有分别或同时给药的预防和/或治疗呼吸道合胞病毒感染的重组蛋白疫苗和权利要求1-5任一项所述的腺病毒载体疫苗。
9.根据权利要求7所述的组合物或权利要求8所述的联合用药物,其特征是:所述重组蛋白疫苗含有预防和/或治疗呼吸道合胞病毒感染的重组蛋白,所述重组蛋白的氨基酸序列是与seq id no.1或seq id no.2具有70%以上的同源性且具有相同或相似的生物学活性;优选地,所述重组蛋白的氨基酸序列选自seq id no.1、seq id no.2、seq id no.3、seqidno.4、seq id no.5或seq id no.6中至少一种。
10.根据权利要求9所述的组合物或联合用药物,其特征是:所述重组蛋白疫苗含有蛋白前体,所述蛋白前体是通过氨基酸序列选自seq id no.7、seq id no.8中至少一种构建的;优选地,编码所述蛋白前体的核苷酸序列选自seq id no.9或seq id no.10中至少一种。
11.根据权利要求7、9-10任一项所述的组合物或权利要求8-10任一项所述的联合用药物,其特征是:所述组合物或联合用药物为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂;优选地,所述的疫苗为鼻喷制剂。
12.权利要求1-5任一项所述的腺病毒载体疫苗、权利要求7、9-11所述的组合物、或权利要求8-11任一项所述的联合用药物在制备预防和/或治疗呼吸道合胞病毒感染的药物上的用途。