CN112358533B - 重组刺突蛋白及其制备方法和用途 - Google Patents
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Abstract
本发明公开了一种重组刺突蛋白,其氨基酸序列如SEQ ID NO:1或3所示。本发明还公开了编码所述重组刺突蛋白的核酸序列(如SEQ ID NO:2或4所示)、所述重组刺突蛋白的制备方法及用途。所述重组刺突蛋白可用于预防新型冠状病毒感染或由该病毒感染导致的疾病。
Description
技术领域
本发明属于生物医药领域,具体而言,本发明涉及一种重组刺突蛋白、其制备方法和用途。
背景技术
新冠肺炎(COVID-19)是由新型冠状病毒(SARS-CoV-2)感染引起的呼吸道疾病。截止到2020年10月,全球范围内已有超过40,000,000人感染,超过1,100,000人因感染SARS-CoV-2病毒而死亡。新冠肺炎在全球范围内的大流行已经对人类的健康和经济发展带来了巨大的影响。因此,迫切需要寻求一种途径遏制病毒的传播。疫苗是一种有效且经济的预防SARS-CoV-2病毒感染及新冠肺炎的方式。
SARS-CoV-2属于β属B亚群,是一种有包膜的单股正链RNA病毒。该病毒属还包含SARS-CoV和MERS-CoV,它们都是通过病毒颗粒表面的刺突蛋白(Spike,以下简称S蛋白)与宿主细胞表面的血管紧张素转化酶2(ACE2)结合而感染宿主。天然状态下,病毒表面的S蛋白呈现三聚体状态,根据蛋白的结构功能,S蛋白可以被分成两个功能单位:S1和S2蛋白亚基,S1亚基的主要功能是介导与宿主细胞表面受体结合,S2亚基的功能是介导病毒与宿主细胞的融合,主要的中和抗原表位集中在S1亚基的受体结合结构域(Receptor bindingdomain,RBD)上。因此,S蛋白的完整性和结构的正确性确保了疫苗的有效性。但在病毒感染细胞的过程中,处于融合前构象(pre-fusion)的天然S蛋白首先被蛋白酶水解成S1亚基和S2亚基,S1亚基发生解离,S2亚基上的蛋白酶水解位点则暴露出来,导致其进一步被蛋白酶水解,使得位于分子内部的融合肽暴露出来,蛋白构象则转变成融合后构象(post-fusion),进而介导病毒和细胞膜的融合。如果保持S蛋白原有的氨基酸序列进行S蛋白的重组表达,由于表达细胞中蛋白酶的存在,S蛋白很可能被水解而难以保持融合前构象,导致S蛋白表达水平降低,并且不利于其结构的正确性。因此,在以S蛋白作为抗原的SARS-CoV-2病毒疫苗中,需要通过基因工程手段对S蛋白进行改造,一方面保持S蛋白的表达水平,确保疫苗产业化的可行性;另一方面保证S蛋白结构的正确性,确保疫苗的有效性;此外,通过体外重组方式表达、制备的S蛋白,不含SARS-CoV-2病毒的遗传物质,则保证了疫苗的安全性。
发明内容
在本发明的第一方面,提供了一种经基因工程手段改造的重组S蛋白。具体而言,首先将S1/S2酶切位点进行替换,阻止蛋白酶的切割;接着为了进一步阻止分子由融合前构象转变成融合后构象,在关键位点引入连续两个脯氨酸(Pro,P)突变,从而增加其结构刚性。在一个优选的实施方式中,所述重组S蛋白具有SEQ ID NO:1或3所示的氨基酸序列。在一个进一步优选的实施方式中,所述重组刺突蛋白分别由SEQ ID NO:2或4所示的核苷酸序列编码。
在本发明的第二方面,提供了一种编码第一方面的重组S蛋白的基因。在一个优选的实施方式中,所述基因具有SEQ ID NO:2或4所示的核苷酸序列。
在本发明的第三方面,提供了一种工程化细胞,在一个进一步优选的实施方式中,所述细胞基因组中整合有SEQ ID NO:2或4所示的核苷酸序列。在一个进一步优选的实施方式中,所述细胞能够分泌表达重组S蛋白。在一个优选的实施方式中,所述工程化细胞为CHO细胞。在一个优选的实施方式中,所述CHO细胞的培养上清液中重组S蛋白的平均表达量达到200-300mg/L。
在本发明的第四方面,提供了一种通过CHO细胞分泌表达并分离第一方面的重组S蛋白的方法,包括以下步骤:
(1) 将SEQ ID NO:2所示的核苷酸序列克隆入表达载体中;
(2) 将步骤(1)所得的表达载体转染至CHO细胞中;
(3) 通过细胞群的筛选和单克隆筛选,获得稳定表达所述重组S蛋白的细胞株;
(4) 使用步骤(3)所得的细胞株进行表达,获得含所述重组S蛋白的培养上清液;以及
(5) 将步骤(4)所得到的含所述重组S蛋白的培养上清液进行纯化,获得纯化的重组S蛋白。
根据本发明的一个具体实施方案,所述步骤(2)中使用的CHO细胞为CHO-K1Q细胞。
在本发明的第五方面,提供了一种疫苗组合物,所述疫苗组合物包含第一方面的重组S蛋白以及药学上接受的赋形剂。根据本发明的一个具体实施方案,所述药学上接受的赋形剂为佐剂。在一个优选的实施方式中,所述佐剂为氢氧化铝联合CpG ODN复合佐剂。
在本发明的第六方面,提供了第一方面的重组S蛋白在制备疫苗组合物中的用途,所述药物疫苗组合物用于预防新型冠状病毒感染或由所述感染导致的疾病。在一个优选的实施方式中,该疫苗组合物具有较强的免疫原性。
本发明的有益效果是:本发明提供的重组S蛋白具有融合前构象且呈三聚体状态,其在小鼠模型上具有良好的免疫原性,可制备疫苗组合物应用于预防新型冠状病毒。此外,本发明还提供了一种可在CHO细胞中高效表达的编码重组S蛋白的方法,所述方法表达量高,快速简便,可实现大规模生产。
本发明的其它方面根据本文的公开内容,对本领域的技术人员而言是显而易见的。
附图说明:
图1表达载体SNT70-S质粒图谱;
图2纯化后重组S蛋白的变性还原SDS-PAGE分析;
图3纯化后重组S蛋白的SE-HPLC分析;以及
图4重组S蛋白25℃条件下活性稳定性分析。
具体实施方式
以下实施例仅为举例说明本发明的技术方案的目的,而非限制本发明的要求保护的范围。
实施例1 SARS-CoV-2病毒的S蛋白胞外段的克隆构建、表达与纯化
1、S蛋白序列的选择与基因-的合成
根据NCBI数据库和文献检索,选择SARS-CoV-2病毒Wuhan-Hu-1毒株(基因组序列登录号:MN908947)的S蛋白胞外段氨基酸序列(1-1213)作为蛋白优化的基础。为了更好地实现S蛋白的分泌表达,以强分泌性信号肽MEFGLSWLFLVAILKGVQC替换S蛋白本身信号肽MFVFLVLLPLVSS。进一步,为了维持蛋白的稳定性并将其锁定在融合前(pre-fusion)构象,一方面将S蛋白S1/S2蛋白酶切割位点682RRAR685以GGSG取代,同时将关键位点氨基酸986KV987突变成连续的两个脯氨酸(Proline,P)。此外,在其C末端引入T4 fibritin基序(GYIPEAPRDGQAYVRKDGEWVLLSTFL),以进一步稳定S蛋白的三聚化状态。经过设计后的重组S蛋白氨基酸序列如SEQ ID NO:1所示,含有1248个氨基酸,其中N端的19个氨基酸为信号肽,在分泌表达过程中,会被切除,因此所获得的目的S蛋白抗原为1229个氨基酸(SEQ ID NO:3)。
为了利于重组S蛋白在CHO细胞中高效表达,选择CHO细胞偏好的密码子对编码基因进行优化,优化后的核苷酸序列如SEQ ID NO:2所示,并进行人工合成。需要说明的是,优化原则并非简单选择CHO细胞中频率最高的密码子,而是一个较为复杂的优化方案。整体上的优化三个原则:第一,根据密码子的简并性,用CHO细胞中各氨基酸对应的高频密码子替换原有密码子;第二,为避免转录后的mRNA中过高的GC含量对其二级结构造成影响,进而影响翻译效率,在优化过程中尽量将基因的GC%控制在40-60%;第三,避开一些常用的限制性酶切位点。
2、重组S蛋白表达载体的克隆构建
上述合成的基因5’和3’端分别含有HindIII和EcoRI限制性内切酶酶切位点,通过双酶切获得片段并将其克隆至表达载体SNT70中(图1)。载体SNT70携带氨苄霉素抗性基因和谷氨酰胺合成酶筛选标记。使用巨细胞病毒(CMV)启动子/增强子序列来进行目的基因的表达。CMV启动子是目前较为常用的启动真核基因表达的强启动子。经克隆构建获得相应的表达载体SNT70-S,并通过酶切和测序鉴定序列无误。
具体而言,表达质粒SNT70-S的构建可按如下方式进行:
1)用HindIII和EcoRI分别对SNT70质粒和委托苏州金唯智生物科技有限公司(GENEWIZ)基因合成获得的pUC57-S质粒(包含编码重组S蛋白的核苷酸序列)进行双酶切,琼脂糖凝胶分离后采用DNA凝胶回收试剂盒回收,并纯化酶切后的目的片段和酶切后的载体SNT70。
2)连接反应:纯化后的目的片段(约3.8kb)和载体SNT70(约9.4kb)酶切产物采用T4连接酶进行连接反应,构建成表达质粒SNT70-S。
3)将连接反应产物转化到Top10感受态细胞,涂布LB培养基平板,获得单克隆转化菌落。
4)从中挑取10个单克隆菌落进行PCR扩增和测序验证。随后,对测序验证正确的克隆,在LB培养基平板上划线两次,将分离得到的单克隆转接到LB液体培养基中(含100 μg/mL氨苄青霉素),37℃,220 rpm过夜振荡培养,大量抽提质粒DNA,最终得到的质粒命名为SNT70-S。
3、SNT70-S质粒的转染、重组S蛋白的表达和纯化
通过上述2中的方式,大量制备表达质粒,经PvuI酶切线性化后通过电穿孔方式转染至宿主细胞CHO-K1Q。电穿孔转染方法为:取2.4×107个细胞,1000 rpm离心5 min,弃上清;用20 ml CHO CD培养基重悬细胞,1000 rpm离心5 min,弃上清;将1520 μl CHO CD培养基和80 μl质粒分别置于离心后细胞中,重悬混匀;分别取800 μl混合液于2个电转杯中,将电转杯分别置于电转仪中进行电击(电转程序:300 V,900 μF,指数脉冲,电阻(∞));电击后,将2个电转杯中细胞合并转移至含有20 ml预热CHO CD04培养基(含终浓度25 mM L-蛋氨酸亚砜亚胺,MSX)的方瓶中,经行加压恢复和筛选。在本实施例中,共进行了4组转染试验。
电转后24 h、第7天及随后每2~4天,检测细胞密度和活率,调整细胞密度为0.5×106个细胞/ml,置于37℃,10% CO2培养箱静置培养。当细胞密度大于1×106个细胞/ml,细胞活率大于90%时,细胞群加压筛选完成。随后采用14天流加培养的方法,对加压筛选得到的细胞群进行表达量评估。表达量的评估方法是采用生物膜层干涉技术(BiolayerInterferometry,以下简称BLI),即先以Protein G sensor结合CB6抗体,再以CB6抗体结合目标蛋白,通过建立标准曲来计算待测样品的蛋白浓度。
接下来利用自动化细胞铺板与成像***(VIPS)对加压筛选得到的细胞群进行有限稀释法的单克隆化和筛选,并对所挑选的单克隆在分批补料培养过程中扩大培养,将上述克隆细胞的上清收集,取样进行目的蛋白表达量的检测,并综合考虑克隆的生长情况、活细胞密度、活率、终末乳酸含量以及相关产品质量参数,选择出最优的3个克隆,即为优势细胞株。将上述获得的细胞株,经生物反应器培养表达,即可获得含有重组S蛋白的细胞培养上清,同样对上述上清液取样进行BLI检测。结果显示,上述细胞株的培养上清液中重组S蛋白的平均表达量能够达到200-300 mg/L,满足疫苗生产需求。
为了获得重组S蛋白抗原,优势细胞株的培养上清液经常规的处理方式,包括病毒灭活、阴离子交换层析、阳离子交换层析、凝胶过滤层析和除病毒纳滤等步骤后,即可获得SDS-PAGE纯度超过90%(图2)、并且为三聚体的S蛋白(图3)。
进一步,对重组S蛋白的体外抗体(CB6)结合活性稳定性进行分析,BLI检测结果显示,即使在25℃条件下孵育14天,重组S蛋白依然能够维持80%以上的活性(图4)。
实施例2重组SARS-CoV-2疫苗的免疫原性评价
为了研究本发明所提供的基因及载体所制备的抗原的免疫原性,将所获得的实施例1中所述的重组S蛋白抗原与佐剂吸配制成重组SARS-CoV-2疫苗组合物,并以BALB/c小鼠为动物模型开展了免疫原性研究。具体方法为:以重组S蛋白为抗原,与氢氧化铝联合CpGODN佐剂吸配制成重组SARS-CoV-2疫苗组合物。选择6-8周龄BALB/c小鼠随机分组,其中,实验组(注射疫苗组合物)10只小鼠,对照组(仅注射佐剂)10只小鼠。其中,实验组肌肉注射0.05 ml疫苗(5 μg S + 50 μg Alum + 50 μg CpG);对照组肌肉注射0.05 ml复合佐剂(50μg Alum + 50 μg CpG)。第0和3周免疫,第6周采血并分离血清。随后,采用真病毒中和实验检测血清中的中和抗体滴度。结果显示通过本发明提供的技术方案所获得的重组S蛋白所制备得到的疫苗组合物具有非常好的免疫原性,可作为潜在的重组新型冠状病毒疫苗候选(具体结果如表1所示)。
表1.中和抗体滴度检测结果
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
SEQ ID NO:1:
MEFGLSWLFLVAILKGVQCQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGGSGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPGSGYI PEAPRDGQAYVRKDGEWVLLSTFL
SEQ ID NO:2:
ATGGAGTTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGTCAGTGCGTGAACCTGACCACAAGGACCCAGCTGCCACCTGCCTATACCAACTCTTTCACAAGGGGCGTGTACTATCCTGACAAGGTGTTTCGGTCCAGCGTGCTGCACTCCACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCTATCCACGTGTCCGGCACCAATGGCACAAAGAGATTCGACAATCCTGTGCTGCCCTTCAACGATGGCGTGTACTTCGCCTCCACCGAGAAGAGCAACATCATCCGCGGCTGGATCTTTGGCACCACACTGGACTCTAAGACACAGTCCCTGCTGATCGTGAACAATGCTACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCATAAGAACAATAAGAGCTGGATGGAGTCTGAGTTTCGGGTGTATTCTTCCGCCAACAATTGTACATTTGAGTACGTGTCTCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACAGCAAGCACACCCCCATCAACCTGGTGCGGGACCTGCCTCAGGGCTTCTCTGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCAGGTTTCAGACACTGCTGGCCCTGCATCGGTCCTACCTGACACCCGGCGACAGCTCTTCCGGATGGACCGCTGGAGCTGCTGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCTGTGGATTGCGCCCTGGACCCTCTGAGCGAGACCAAGTGTACACTGAAGTCTTTTACCGTGGAGAAGGGCATCTATCAGACATCTAATTTCAGAGTGCAGCCAACCGAGTCCATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCTACCCGGTTCGCCAGCGTGTACGCTTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATTCTGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCTGATTCGTTCGTGATCAGGGGCGACGAGGTGCGGCAGATCGCTCCTGGACAGACAGGCAAGATCGCTGACTACAATTATAAGCTGCCAGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCTAAAGTGGGCGGCAACTACAATTATCTGTACAGACTGTTTCGCAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCCACAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTGCAGTCTTACGGCTTCCAGCCCACCAACGGCGTGGGCTATCAGCCTTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCTCCAGCTACAGTGTGCGGACCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGAACAGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGAGACATCGCCGATACCACAGACGCTGTGCGCGACCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCCTTCGGCGGCGTGAGCGTGATCACACCAGGAACCAATACATCTAACCAGGTGGCCGTGCTGTATCAGGACGTGAACTGTACCGAGGTGCCTGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGAGGGTGTACTCTACCGGCTCCAACGTGTTTCAGACAAGGGCTGGATGTCTGATCGGAGCTGAGCATGTGAACAATAGCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCTTCCTACCAGACCCAGACAAACAGCCCAGGAGGAAGCGGATCTGTGGCTTCCCAGAGCATCATCGCTTATACCATGTCCCTGGGCGCCGAGAATAGCGTGGCTTACAGCAACAATAGCATCGCTATCCCAACCAACTTCACAATCTCCGTGACCACAGAGATCCTGCCCGTGTCTATGACCAAGACATCCGTGGACTGCACAATGTATATCTGTGGCGATTCCACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCTCGTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCTGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGGACTTTGGCGGCTTCAATTTTTCTCAGATCCTGCCAGATCCCTCCAAGCCCAGCAAGAGATCTTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCTGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCTGCTAGGGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCTCAGTACACATCCGCCCTGCTGGCCGGCACCATCACATCTGGATGGACCTTCGGCGCTGGAGCTGCCCTGCAGATCCCTTTTGCCATGCAGATGGCTTATCGCTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCTATCGGCAAGATCCAGGACTCTCTGAGCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCTCAGGCCCTGAATACCCTGGTGAAGCAGCTGTCCAGCAACTTCGGCGCCATCTCTTCCGTGCTGAATGATATCCTGAGCAGACTGGACCCCCCTGAGGCTGAGGTGCAGATCGACAGACTGATCACAGGCCGCCTGCAGAGCCTGCAGACCTACGTGACACAGCAGCTGATCAGGGCTGCCGAGATCCGGGCTTCTGCCAACCTGGCTGCCACCAAGATGTCTGAGTGCGTGCTGGGCCAGTCCAAGCGCGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCTTTCCCTCAGTCCGCCCCACACGGCGTGGTGTTTCTGCATGTGACCTACGTGCCCGCTCAGGAGAAGAACTTCACCACAGCTCCTGCCATCTGCCACGATGGCAAGGCCCATTTTCCAAGAGAGGGCGTGTTCGTGTCTAACGGCACCCATTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGTCCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCCCTGCAGCCTGAGCTGGACTCTTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCATACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCTGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCTAAGAATCTGAACGAGTCCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCAGGCAGCGGCTATATCCCAGAGGCTCCAAGAGACGGACAGGCTTACGTGCGCAAGGATGGCGAGTGGGTGCTGCTGTCTACCTTCCTGTGATGA
SEQ ID NO:3:
QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGGSGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPGSGYIPEAPRDGQAYVRKDGEWVL LSTFL
SEQ ID NO:4:
CAGTGCGTGAACCTGACCACAAGGACCCAGCTGCCACCTGCCTATACCAACTCTTTCACAAGGGGCGTGTACTATCCTGACAAGGTGTTTCGGTCCAGCGTGCTGCACTCCACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCTATCCACGTGTCCGGCACCAATGGCACAAAGAGATTCGACAATCCTGTGCTGCCCTTCAACGATGGCGTGTACTTCGCCTCCACCGAGAAGAGCAACATCATCCGCGGCTGGATCTTTGGCACCACACTGGACTCTAAGACACAGTCCCTGCTGATCGTGAACAATGCTACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCATAAGAACAATAAGAGCTGGATGGAGTCTGAGTTTCGGGTGTATTCTTCCGCCAACAATTGTACATTTGAGTACGTGTCTCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACAGCAAGCACACCCCCATCAACCTGGTGCGGGACCTGCCTCAGGGCTTCTCTGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCAGGTTTCAGACACTGCTGGCCCTGCATCGGTCCTACCTGACACCCGGCGACAGCTCTTCCGGATGGACCGCTGGAGCTGCTGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCTGTGGATTGCGCCCTGGACCCTCTGAGCGAGACCAAGTGTACACTGAAGTCTTTTACCGTGGAGAAGGGCATCTATCAGACATCTAATTTCAGAGTGCAGCCAACCGAGTCCATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCTACCCGGTTCGCCAGCGTGTACGCTTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATTCTGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCTGATTCGTTCGTGATCAGGGGCGACGAGGTGCGGCAGATCGCTCCTGGACAGACAGGCAAGATCGCTGACTACAATTATAAGCTGCCAGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCTAAAGTGGGCGGCAACTACAATTATCTGTACAGACTGTTTCGCAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCCACAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTGCAGTCTTACGGCTTCCAGCCCACCAACGGCGTGGGCTATCAGCCTTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCTCCAGCTACAGTGTGCGGACCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGAACAGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGAGACATCGCCGATACCACAGACGCTGTGCGCGACCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCCTTCGGCGGCGTGAGCGTGATCACACCAGGAACCAATACATCTAACCAGGTGGCCGTGCTGTATCAGGACGTGAACTGTACCGAGGTGCCTGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGAGGGTGTACTCTACCGGCTCCAACGTGTTTCAGACAAGGGCTGGATGTCTGATCGGAGCTGAGCATGTGAACAATAGCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCTTCCTACCAGACCCAGACAAACAGCCCAGGAGGAAGCGGATCTGTGGCTTCCCAGAGCATCATCGCTTATACCATGTCCCTGGGCGCCGAGAATAGCGTGGCTTACAGCAACAATAGCATCGCTATCCCAACCAACTTCACAATCTCCGTGACCACAGAGATCCTGCCCGTGTCTATGACCAAGACATCCGTGGACTGCACAATGTATATCTGTGGCGATTCCACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCTCGTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCTGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGGACTTTGGCGGCTTCAATTTTTCTCAGATCCTGCCAGATCCCTCCAAGCCCAGCAAGAGATCTTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCTGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCTGCTAGGGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCTCAGTACACATCCGCCCTGCTGGCCGGCACCATCACATCTGGATGGACCTTCGGCGCTGGAGCTGCCCTGCAGATCCCTTTTGCCATGCAGATGGCTTATCGCTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCTATCGGCAAGATCCAGGACTCTCTGAGCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCTCAGGCCCTGAATACCCTGGTGAAGCAGCTGTCCAGCAACTTCGGCGCCATCTCTTCCGTGCTGAATGATATCCTGAGCAGACTGGACCCCCCTGAGGCTGAGGTGCAGATCGACAGACTGATCACAGGCCGCCTGCAGAGCCTGCAGACCTACGTGACACAGCAGCTGATCAGGGCTGCCGAGATCCGGGCTTCTGCCAACCTGGCTGCCACCAAGATGTCTGAGTGCGTGCTGGGCCAGTCCAAGCGCGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCTTTCCCTCAGTCCGCCCCACACGGCGTGGTGTTTCTGCATGTGACCTACGTGCCCGCTCAGGAGAAGAACTTCACCACAGCTCCTGCCATCTGCCACGATGGCAAGGCCCATTTTCCAAGAGAGGGCGTGTTCGTGTCTAACGGCACCCATTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGTCCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCCCTGCAGCCTGAGCTGGACTCTTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCATACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCTGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCTAAGAATCTGAACGAGTCCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCAGGCAGCGGCTATATCCCAGAGGCTCCAAGAGACGGACAGGCTTACGTGCGCAAGGATGGCGAGTGGGTGCTGCTGTCTACCTTCCTGTGATGA
<110> 上海泽润生物科技有限公司
<120> 重组刺突蛋白及其制备方法和用途
<130> CPCH2062641N
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1248
<212> PRT
<213> 人工序列
<400> 1
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro
20 25 30
Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val
35 40 45
Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe
50 55 60
Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn
65 70 75 80
Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val
85 90 95
Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
100 105 110
Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn
115 120 125
Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp
130 135 140
Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu
145 150 155 160
Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr
165 170 175
Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe
180 185 190
Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys
195 200 205
Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln
210 215 220
Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn
225 230 235 240
Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr
245 250 255
Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr
260 265 270
Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn
275 280 285
Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu
290 295 300
Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln
305 310 315 320
Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
325 330 335
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
340 345 350
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
355 360 365
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
370 375 380
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
385 390 395 400
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
405 410 415
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
420 425 430
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
435 440 445
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
450 455 460
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
465 470 475 480
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
485 490 495
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
500 505 510
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
515 520 525
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
530 535 540
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu
545 550 555 560
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
565 570 575
Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp
580 585 590
Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr
595 600 605
Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr
610 615 620
Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg
625 630 635 640
Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu
645 650 655
Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile
660 665 670
Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly
675 680 685
Gly Ser Gly Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser
690 695 700
Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile
705 710 715 720
Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser
725 730 735
Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser
740 745 750
Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln
755 760 765
Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr
770 775 780
Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile
785 790 795 800
Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser
805 810 815
Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val
820 825 830
Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly
835 840 845
Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu
850 855 860
Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr
865 870 875 880
Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala
885 890 895
Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe
900 905 910
Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu
915 920 925
Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu
930 935 940
Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln
945 950 955 960
Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe
965 970 975
Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro
980 985 990
Pro Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln
995 1000 1005
Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu
1010 1015 1020
Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys
1025 1030 1035
Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr
1040 1045 1050
His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1055 1060 1065
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr
1070 1075 1080
Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu
1085 1090 1095
Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1100 1105 1110
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val
1115 1120 1125
Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val
1130 1135 1140
Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1145 1150 1155
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly
1160 1165 1170
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu
1175 1180 1185
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
1190 1195 1200
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
1205 1210 1215
Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala
1220 1225 1230
Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
1235 1240 1245
<210> 2
<211> 3750
<212> DNA
<213> 人工序列
<400> 2
atggagttcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgtcag 60
tgcgtgaacc tgaccacaag gacccagctg ccacctgcct ataccaactc tttcacaagg 120
ggcgtgtact atcctgacaa ggtgtttcgg tccagcgtgc tgcactccac acaggatctg 180
tttctgccat tctttagcaa cgtgacctgg ttccacgcta tccacgtgtc cggcaccaat 240
ggcacaaaga gattcgacaa tcctgtgctg cccttcaacg atggcgtgta cttcgcctcc 300
accgagaaga gcaacatcat ccgcggctgg atctttggca ccacactgga ctctaagaca 360
cagtccctgc tgatcgtgaa caatgctacc aacgtggtca tcaaggtgtg cgagttccag 420
ttttgtaatg atcccttcct gggcgtgtac tatcataaga acaataagag ctggatggag 480
tctgagtttc gggtgtattc ttccgccaac aattgtacat ttgagtacgt gtctcagcct 540
ttcctgatgg acctggaggg caagcagggc aatttcaaga acctgaggga gttcgtgttt 600
aagaatatcg atggctactt caagatctac agcaagcaca cccccatcaa cctggtgcgg 660
gacctgcctc agggcttctc tgccctggag cccctggtgg atctgcctat cggcatcaac 720
atcaccaggt ttcagacact gctggccctg catcggtcct acctgacacc cggcgacagc 780
tcttccggat ggaccgctgg agctgctgcc tactatgtgg gctatctgca gcctaggacc 840
ttcctgctga agtacaacga gaatggcacc atcacagacg ctgtggattg cgccctggac 900
cctctgagcg agaccaagtg tacactgaag tcttttaccg tggagaaggg catctatcag 960
acatctaatt tcagagtgca gccaaccgag tccatcgtgc gctttcctaa tatcacaaac 1020
ctgtgcccat ttggcgaggt gttcaacgct acccggttcg ccagcgtgta cgcttggaat 1080
aggaagcgga tcagcaactg cgtggccgac tattctgtgc tgtacaactc cgcctccttc 1140
tccaccttca agtgctatgg cgtgtccccc acaaagctga atgacctgtg ctttaccaac 1200
gtgtacgctg attcgttcgt gatcaggggc gacgaggtgc ggcagatcgc tcctggacag 1260
acaggcaaga tcgctgacta caattataag ctgccagacg acttcaccgg ctgcgtgatc 1320
gcctggaaca gcaacaatct ggattctaaa gtgggcggca actacaatta tctgtacaga 1380
ctgtttcgca agagcaatct gaagcccttc gagagggaca tctccacaga gatctaccag 1440
gccggcagca ccccttgcaa tggcgtggag ggctttaact gttatttccc actgcagtct 1500
tacggcttcc agcccaccaa cggcgtgggc tatcagcctt accgggtggt ggtgctgtct 1560
tttgagctgc tgcacgctcc agctacagtg tgcggaccca agaagtccac caatctggtg 1620
aagaacaagt gcgtgaactt caacttcaac ggactgaccg gaacaggcgt gctgaccgag 1680
agcaacaaga agttcctgcc atttcagcag ttcggcagag acatcgccga taccacagac 1740
gctgtgcgcg acccacagac cctggagatc ctggatatca caccctgctc cttcggcggc 1800
gtgagcgtga tcacaccagg aaccaataca tctaaccagg tggccgtgct gtatcaggac 1860
gtgaactgta ccgaggtgcc tgtggctatc cacgccgatc agctgacccc aacatggagg 1920
gtgtactcta ccggctccaa cgtgtttcag acaagggctg gatgtctgat cggagctgag 1980
catgtgaaca atagctatga gtgcgacatc ccaatcggcg ccggcatctg tgcttcctac 2040
cagacccaga caaacagccc aggaggaagc ggatctgtgg cttcccagag catcatcgct 2100
tataccatgt ccctgggcgc cgagaatagc gtggcttaca gcaacaatag catcgctatc 2160
ccaaccaact tcacaatctc cgtgaccaca gagatcctgc ccgtgtctat gaccaagaca 2220
tccgtggact gcacaatgta tatctgtggc gattccaccg agtgcagcaa cctgctgctg 2280
cagtacggct cgttttgtac ccagctgaat agagccctga caggcatcgc tgtggagcag 2340
gataagaaca cacaggaggt gttcgcccag gtgaagcaga tctacaagac cccacccatc 2400
aaggactttg gcggcttcaa tttttctcag atcctgccag atccctccaa gcccagcaag 2460
agatctttta tcgaggacct gctgttcaac aaggtgaccc tggctgatgc cggcttcatc 2520
aagcagtatg gcgattgcct gggcgacatc gctgctaggg acctgatctg tgcccagaag 2580
tttaatggcc tgaccgtgct gcctccactg ctgacagatg agatgatcgc tcagtacaca 2640
tccgccctgc tggccggcac catcacatct ggatggacct tcggcgctgg agctgccctg 2700
cagatccctt ttgccatgca gatggcttat cgcttcaacg gcatcggcgt gacccagaat 2760
gtgctgtacg agaaccagaa gctgatcgcc aatcagttta actccgctat cggcaagatc 2820
caggactctc tgagctctac agcctccgcc ctgggcaagc tgcaggatgt ggtgaatcag 2880
aacgctcagg ccctgaatac cctggtgaag cagctgtcca gcaacttcgg cgccatctct 2940
tccgtgctga atgatatcct gagcagactg gacccccctg aggctgaggt gcagatcgac 3000
agactgatca caggccgcct gcagagcctg cagacctacg tgacacagca gctgatcagg 3060
gctgccgaga tccgggcttc tgccaacctg gctgccacca agatgtctga gtgcgtgctg 3120
ggccagtcca agcgcgtgga cttttgtggc aagggctatc acctgatgtc tttccctcag 3180
tccgccccac acggcgtggt gtttctgcat gtgacctacg tgcccgctca ggagaagaac 3240
ttcaccacag ctcctgccat ctgccacgat ggcaaggccc attttccaag agagggcgtg 3300
ttcgtgtcta acggcaccca ttggtttgtg acacagcgca atttctacga gccccagatc 3360
atcaccacag acaatacctt cgtgtccggc aactgtgacg tggtcatcgg catcgtgaac 3420
aataccgtgt atgatcccct gcagcctgag ctggactctt ttaaggagga gctggataag 3480
tacttcaaga atcatacctc ccctgacgtg gatctgggcg acatcagcgg catcaatgcc 3540
tctgtggtga acatccagaa ggagatcgac aggctgaacg aggtggctaa gaatctgaac 3600
gagtccctga tcgatctgca ggagctgggc aagtatgagc agtacatcaa gtggccaggc 3660
agcggctata tcccagaggc tccaagagac ggacaggctt acgtgcgcaa ggatggcgag 3720
tgggtgctgc tgtctacctt cctgtgatga 3750
<210> 3
<211> 1229
<212> PRT
<213> 人工序列
<400> 3
Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr
1 5 10 15
Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser
20 25 30
Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn
35 40 45
Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
50 55 60
Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala
65 70 75 80
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
85 90 95
Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn
100 105 110
Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu
115 120 125
Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe
130 135 140
Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln
145 150 155 160
Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu
165 170 175
Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
180 185 190
Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
195 200 205
Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg
210 215 220
Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp
225 230 235 240
Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr
245 250 255
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile
260 265 270
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
275 280 285
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
290 295 300
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
305 310 315 320
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
325 330 335
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
340 345 350
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
355 360 365
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
370 375 380
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
385 390 395 400
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
405 410 415
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
420 425 430
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
435 440 445
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
450 455 460
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
465 470 475 480
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
485 490 495
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
500 505 510
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
530 535 540
Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr
545 550 555 560
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
565 570 575
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser
580 585 590
Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro
595 600 605
Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser
610 615 620
Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala
625 630 635 640
Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly
645 650 655
Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Gly Ser Gly
660 665 670
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
675 680 685
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
690 695 700
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
705 710 715 720
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
725 730 735
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
740 745 750
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
755 760 765
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
770 775 780
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
785 790 795 800
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
805 810 815
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
820 825 830
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
835 840 845
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
850 855 860
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
865 870 875 880
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
885 890 895
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
900 905 910
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
915 920 925
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
930 935 940
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
945 950 955 960
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala
965 970 975
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
980 985 990
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
995 1000 1005
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln
1010 1015 1020
Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser
1025 1030 1035
Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr
1040 1045 1050
Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile
1055 1060 1065
Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val
1070 1075 1080
Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu
1085 1090 1095
Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys
1100 1105 1110
Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu
1115 1120 1125
Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe
1130 1135 1140
Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1145 1150 1155
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu
1160 1165 1170
Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln
1175 1180 1185
Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Gly Ser Gly
1190 1195 1200
Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys
1205 1210 1215
Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
1220 1225
<210> 4
<211> 3693
<212> DNA
<213> 人工序列
<400> 4
cagtgcgtga acctgaccac aaggacccag ctgccacctg cctataccaa ctctttcaca 60
aggggcgtgt actatcctga caaggtgttt cggtccagcg tgctgcactc cacacaggat 120
ctgtttctgc cattctttag caacgtgacc tggttccacg ctatccacgt gtccggcacc 180
aatggcacaa agagattcga caatcctgtg ctgcccttca acgatggcgt gtacttcgcc 240
tccaccgaga agagcaacat catccgcggc tggatctttg gcaccacact ggactctaag 300
acacagtccc tgctgatcgt gaacaatgct accaacgtgg tcatcaaggt gtgcgagttc 360
cagttttgta atgatccctt cctgggcgtg tactatcata agaacaataa gagctggatg 420
gagtctgagt ttcgggtgta ttcttccgcc aacaattgta catttgagta cgtgtctcag 480
cctttcctga tggacctgga gggcaagcag ggcaatttca agaacctgag ggagttcgtg 540
tttaagaata tcgatggcta cttcaagatc tacagcaagc acacccccat caacctggtg 600
cgggacctgc ctcagggctt ctctgccctg gagcccctgg tggatctgcc tatcggcatc 660
aacatcacca ggtttcagac actgctggcc ctgcatcggt cctacctgac acccggcgac 720
agctcttccg gatggaccgc tggagctgct gcctactatg tgggctatct gcagcctagg 780
accttcctgc tgaagtacaa cgagaatggc accatcacag acgctgtgga ttgcgccctg 840
gaccctctga gcgagaccaa gtgtacactg aagtctttta ccgtggagaa gggcatctat 900
cagacatcta atttcagagt gcagccaacc gagtccatcg tgcgctttcc taatatcaca 960
aacctgtgcc catttggcga ggtgttcaac gctacccggt tcgccagcgt gtacgcttgg 1020
aataggaagc ggatcagcaa ctgcgtggcc gactattctg tgctgtacaa ctccgcctcc 1080
ttctccacct tcaagtgcta tggcgtgtcc cccacaaagc tgaatgacct gtgctttacc 1140
aacgtgtacg ctgattcgtt cgtgatcagg ggcgacgagg tgcggcagat cgctcctgga 1200
cagacaggca agatcgctga ctacaattat aagctgccag acgacttcac cggctgcgtg 1260
atcgcctgga acagcaacaa tctggattct aaagtgggcg gcaactacaa ttatctgtac 1320
agactgtttc gcaagagcaa tctgaagccc ttcgagaggg acatctccac agagatctac 1380
caggccggca gcaccccttg caatggcgtg gagggcttta actgttattt cccactgcag 1440
tcttacggct tccagcccac caacggcgtg ggctatcagc cttaccgggt ggtggtgctg 1500
tcttttgagc tgctgcacgc tccagctaca gtgtgcggac ccaagaagtc caccaatctg 1560
gtgaagaaca agtgcgtgaa cttcaacttc aacggactga ccggaacagg cgtgctgacc 1620
gagagcaaca agaagttcct gccatttcag cagttcggca gagacatcgc cgataccaca 1680
gacgctgtgc gcgacccaca gaccctggag atcctggata tcacaccctg ctccttcggc 1740
ggcgtgagcg tgatcacacc aggaaccaat acatctaacc aggtggccgt gctgtatcag 1800
gacgtgaact gtaccgaggt gcctgtggct atccacgccg atcagctgac cccaacatgg 1860
agggtgtact ctaccggctc caacgtgttt cagacaaggg ctggatgtct gatcggagct 1920
gagcatgtga acaatagcta tgagtgcgac atcccaatcg gcgccggcat ctgtgcttcc 1980
taccagaccc agacaaacag cccaggagga agcggatctg tggcttccca gagcatcatc 2040
gcttatacca tgtccctggg cgccgagaat agcgtggctt acagcaacaa tagcatcgct 2100
atcccaacca acttcacaat ctccgtgacc acagagatcc tgcccgtgtc tatgaccaag 2160
acatccgtgg actgcacaat gtatatctgt ggcgattcca ccgagtgcag caacctgctg 2220
ctgcagtacg gctcgttttg tacccagctg aatagagccc tgacaggcat cgctgtggag 2280
caggataaga acacacagga ggtgttcgcc caggtgaagc agatctacaa gaccccaccc 2340
atcaaggact ttggcggctt caatttttct cagatcctgc cagatccctc caagcccagc 2400
aagagatctt ttatcgagga cctgctgttc aacaaggtga ccctggctga tgccggcttc 2460
atcaagcagt atggcgattg cctgggcgac atcgctgcta gggacctgat ctgtgcccag 2520
aagtttaatg gcctgaccgt gctgcctcca ctgctgacag atgagatgat cgctcagtac 2580
acatccgccc tgctggccgg caccatcaca tctggatgga ccttcggcgc tggagctgcc 2640
ctgcagatcc cttttgccat gcagatggct tatcgcttca acggcatcgg cgtgacccag 2700
aatgtgctgt acgagaacca gaagctgatc gccaatcagt ttaactccgc tatcggcaag 2760
atccaggact ctctgagctc tacagcctcc gccctgggca agctgcagga tgtggtgaat 2820
cagaacgctc aggccctgaa taccctggtg aagcagctgt ccagcaactt cggcgccatc 2880
tcttccgtgc tgaatgatat cctgagcaga ctggaccccc ctgaggctga ggtgcagatc 2940
gacagactga tcacaggccg cctgcagagc ctgcagacct acgtgacaca gcagctgatc 3000
agggctgccg agatccgggc ttctgccaac ctggctgcca ccaagatgtc tgagtgcgtg 3060
ctgggccagt ccaagcgcgt ggacttttgt ggcaagggct atcacctgat gtctttccct 3120
cagtccgccc cacacggcgt ggtgtttctg catgtgacct acgtgcccgc tcaggagaag 3180
aacttcacca cagctcctgc catctgccac gatggcaagg cccattttcc aagagagggc 3240
gtgttcgtgt ctaacggcac ccattggttt gtgacacagc gcaatttcta cgagccccag 3300
atcatcacca cagacaatac cttcgtgtcc ggcaactgtg acgtggtcat cggcatcgtg 3360
aacaataccg tgtatgatcc cctgcagcct gagctggact cttttaagga ggagctggat 3420
aagtacttca agaatcatac ctcccctgac gtggatctgg gcgacatcag cggcatcaat 3480
gcctctgtgg tgaacatcca gaaggagatc gacaggctga acgaggtggc taagaatctg 3540
aacgagtccc tgatcgatct gcaggagctg ggcaagtatg agcagtacat caagtggcca 3600
ggcagcggct atatcccaga ggctccaaga gacggacagg cttacgtgcg caaggatggc 3660
gagtgggtgc tgctgtctac cttcctgtga tga 3693
Claims (9)
1.一种重组刺突蛋白,其特征在于,所述重组刺突蛋白由SEQ ID NO:1或3所示的氨基酸序列组成。
2.一种编码重组刺突蛋白的基因,其特征在于,所述编码重组刺突蛋白的基因由SEQID NO:2或4所示的核苷酸序列组成。
3.一种工程化的细胞,其特征在于,所述细胞基因组整合有SEQ ID NO:2或4所示的核苷酸序列。
4.如权利要求3所述的细胞,其特征在于,所述细胞可以分泌表达重组刺突蛋白。
5.如权利要求3所述的细胞,其特征在于,所述细胞为CHO细胞。
6.一种通过CHO细胞分泌表达并制备具有SEQ ID NO:3所示的氨基酸序列的重组刺突蛋白的方法,包括下述步骤:
(1)将SEQ ID NO:2所示的核苷酸序列克隆入表达载体中;
(2)将步骤(1)所得的表达载体转染至CHO细胞中;
(3)通过细胞群的筛选和单克隆筛选,获得稳定表达所述重组刺突蛋白的细胞株;
(4)使用步骤(3)所得的细胞株进行表达,获得含所述重组刺突蛋白的培养上清液;以及
(5)将步骤(4)所得到的含所述重组刺突蛋白的培养上清液进行纯化,获得纯化的重组刺突蛋白。
7.一种疫苗组合物,其特征在于,所述疫苗组合物包含权利要求1所述的重组刺突蛋白以及药学上接受的赋形剂。
8.如权利要求7所述的疫苗组合物,其特征在于,所述赋形剂为佐剂。
9.权利要求1所述的重组刺突蛋白在制备疫苗组合物中的用途,所述疫苗组合物用于预防新型冠状病毒感染或由所述感染导致的疾病。
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CN113402591A (zh) * | 2021-06-30 | 2021-09-17 | 李丁 | 基于棘突蛋白基因修饰干细胞的新型冠状病毒疫苗、其制备方法及应用 |
WO2023001259A1 (zh) * | 2021-07-23 | 2023-01-26 | 神州细胞工程有限公司 | 一种可诱导广谱中和活性重组多价新冠病毒三聚体蛋白疫苗的制备及应用 |
WO2023020623A1 (zh) * | 2021-08-20 | 2023-02-23 | 百奥泰生物制药股份有限公司 | 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用 |
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