CN113173977A - 一种双功能抗原、其制备方法及应用 - Google Patents
一种双功能抗原、其制备方法及应用 Download PDFInfo
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- CN113173977A CN113173977A CN202110518584.1A CN202110518584A CN113173977A CN 113173977 A CN113173977 A CN 113173977A CN 202110518584 A CN202110518584 A CN 202110518584A CN 113173977 A CN113173977 A CN 113173977A
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Abstract
本发明公开了一种双功能抗原、其制备方法及应用。本发明提供的双功能抗原由肺炎球菌荚膜多糖和SARS‑CoV‑2的刺突S蛋白或其片段共价偶联构成;所述双功能抗原中,各血清型的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段的质量比为0.2‑2:1。本发明所述的双功能抗原较其使用的刺突S蛋白或其片段抗原自身,能显著提高针对新冠病毒的中和抗体水平;且同时能产生较高水平的针对肺炎球菌荚膜多糖的特异性抗体。
Description
技术领域
本发明属于生物医药领域,具体涉及一种双功能抗原、其制备方法及应用。
背景技术
致病微生物侵入呼吸道并进行繁殖导致的疾病称呼吸道感染。根据其部位分为上呼吸道感染和下呼吸道感染。前者包括鼻炎、咽炎和喉炎;后者包括气管炎、支气管炎和肺炎。呼吸道感染是由多种微生物包括细菌、病毒、支原体、真菌、寄生虫等引起的感染性疾病。呼吸道感染多由病毒引起,细菌感染常继发于病毒感染之后,形成细菌及病毒共感染从而加重病情。该类型疾病四季、任何年龄均可发病(以婴幼儿及老年人易感),通过含有病毒的飞沫、雾滴或经污染的用具进行传播。
2020年以来,新型冠状病毒感染的常态化进一步使呼吸道感染防控复杂化,做好重点人群尤其是中老年人群中对于肺炎链球菌感染的控制,可能会有利于对新冠疫情及其继发感染的控制。未来,新冠疫苗和肺炎疫苗等的联合也许是最有效的综合防控措施。
肺炎球菌是呼吸道感染最主要的病原体,每年下呼吸道感染导致总体死亡人数在200万以上,其中5岁以下儿童死亡65万,79岁以上老人的死亡超过100万。肺炎链球菌是导致下呼吸道感染最主要的致病原,导致了其中55.4%约151万人的死亡,疾病负担超过了其它病原体总和。
2000年,美国批准了第一个7价肺炎结合疫苗,该疫苗在给婴幼儿提供良好保护的同时,也给未接种者提供间接保护;2010年,血清覆盖率升级版的13价肺炎结合疫苗问世。过去10年的使用效果再次证明肺炎结合疫苗在直接保护和间接保护方面的优势,肺炎结合疫苗过去20年在发达经济体的免疫计划中已经被常规使用,给社会带来的价值已经得到科学界和健康组织的高度认可。
新型冠状病毒属于冠状病毒科冠状病毒属,是有囊膜的正链RNA病毒,在所有RNA病毒中其基因组最大,动物和人类都是冠状病毒的宿主。新型冠状病毒于2020年1月12日被世界卫生组织正式命名。它是以前从未在人体中发现的冠状病毒新毒株,该病毒为第七种可以感染人的冠状病毒。人感染SARS-CoV-2的潜伏期一般为1-14天,感染后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征和肾衰竭,甚至死亡。因此,开发一种安全、有效的针对新型冠状病毒(SARS-CoV-2)的疫苗显得十分紧迫,有着重要的意义。
表面刺突蛋白(S蛋白)是新型冠状病毒的主要中和抗原。新型冠状病毒通过受体结合区(Receptor Biniding Domain,RBD)区域与宿主细胞体的受体血管紧张素转换酶2(Angiotensin Converting Enzyme 2,ACE2)结合而侵入细胞。因此,新型冠状病毒S蛋白的受体结合区被认为是诱导机体产生中和抗体的最主要的抗原靶区域。RBD蛋白作为疫苗抗原能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合,可以提高疫苗的免疫原性和免疫效率。
目前,市面上并无可以同时针对新型冠状病毒和肺炎球菌的疫苗抗原及其形式。
发明内容
为解决现有技术中尚无同时针对新型冠状病毒和肺炎球菌的疫苗抗原的技术问题,本发明提供了一种双功能抗原、其制备方法及应用,所述双功能抗原由肺炎球菌荚膜多糖和SARS-CoV-2的刺突S蛋白或其片段共价偶联构成。本发明所述的双功能抗原较其使用的刺突S蛋白或其片段抗原自身,能显著提高针对新冠病毒的中和抗体水平;且同时能产生较高水平的针对肺炎球菌荚膜多糖的特异性抗体。
为解决上述技术问题,本发明提供的技术方案一为:一种双功能抗原,所述双功能抗原由肺炎球菌荚膜多糖和SARS-CoV-2的刺突S蛋白或其片段共价偶联构成;
所述双功能抗原中,各血清型的所述肺炎球菌荚膜多糖与刺突S蛋白或其片段的质量比为0.2-2:1。
本发明所述双功能抗原的结构示意图可以参见图5。
较佳地,各血清型的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段的质量比为0.7-1.2:1。
和/或,所述肺炎球菌荚膜多糖的血清型选自以下一种或多种:1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F型。所述双功能抗原中,各血清型的所述肺炎球菌荚膜多糖与刺突S蛋白或其片段的质量比优选如下表所示:
较佳地,所述刺突S蛋白包含如SEQ ID NO:1或SEQ ID NO:3所示的氨基酸序列。
优选地:所述刺突S蛋白包含在SEQ ID NO:1上具有以下突变中一种或多种的氨基酸序列:N36D、I40F、D46Y、Y47F、Y47W、V49F、F74W、W118R、N121K、S159N、N183Y、C220S和C220A,所述刺突S蛋白RBD的氨基酸序列优选为SEQ ID NO:2所示的氨基酸序列。
本发明中,SEQ ID NO:1来源于SARS-CoV-2株的S蛋白(GenBank:MN908947.3)的一部分,是SARS-CoV-2Spike蛋白的RBD的(R319-I587)区域;
SEQ ID NO:2来源于SARS-CoV-2株的S蛋白(GenBank:MN908947.3)的一部分,是SARS-CoV-2Spike蛋白的RBD的(R319-I587)区域,其中538位Cysteine取代为Serine/Alanine;
SEQ ID NO:3来源于SARS-CoV-2株的S蛋白(NCBI上的GenBank:MN908947.3)的一部分,是SARS-CoV-2Spike蛋白的RBD的(R319-F541)区域。
较佳地,所述的共价偶联为通过还原胺化反应、碳二亚胺介导的缩合反应、硫烷基化反应、活泼酯法反应或者氰基化反应进行共价偶联。
优选地,所述氰基化反应中所用偶联试剂优选1-氰基-4-(二甲氨基)吡啶四氟硼酸盐(1-Cyano-4-dimethylaminopyridinium tetrafluoroborate,CDAP);和/或,所述氰基化反应中所用偶联试剂优选活化所述多糖上的羟基,同时偶联所述刺突S蛋白或其片段上的赖氨酸残基,形成偶联产物。
为解决上述技术问题,本发明提供的技术方案二为:一种分离的核酸,所述核酸的序列为SEQ ID NO:5所示。
为解决上述技术问题,本发明提供的技术方案三为:一种如技术方案一所述的双功能抗原的制备方法,所述制备方法为:将偶联试剂活化后的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段混合、反应。
较佳地,制备所述刺突S蛋白的宿主细胞包括真核细胞和原核细胞,所述真核细胞优选昆虫细胞Sf9、昆虫细胞系Hi5、CHO细胞、Vero细胞、HEK293细胞、Expi293细胞或酵母细胞,更优选为Expi293细胞;所述原核细胞优选大肠杆菌、棒状杆菌或枯草芽孢杆菌,更优选为大肠杆菌。
发明人意外发现,技术方案二所述的核酸在原核系统中表达效果更好,故在技术方案之三的一个较佳实例中,所述刺突S蛋白的制备包括将如技术方案二所述的核酸转化入大肠杆菌以表达所述刺突S蛋白。
所述刺突S蛋白的纯化方法为本领域常规。
在一所述刺突S蛋白的纯化示例中,所述纯化方法为:将所述真核细胞破碎后过滤除去细胞碎片得上清液,或将所述原核细胞的细菌包涵体变复性后得菌液,通过阳离子交换柱以流穿模式去除所述上清液或菌液中的杂质和聚体,以对所述刺突S蛋白进行粗纯。之后含有所述刺突S蛋白的组分进行精纯:通过pH调节上样阳离子交换柱,进行盐梯度洗脱,将得到的所述刺突S蛋白的洗脱峰合并,再用10kDa MWCO膜截留浓缩。将所得所述刺突S蛋白的样品转换到保存样品的缓冲液中,或用疏水层析柱进一步纯化及浓缩。
在一所述刺突S蛋白的纯化示例中,所述细菌包涵体清洗后用高浓度变性剂(8M尿素或6M盐酸胍)和20mM DTT,于pH7.0-10.0室温溶解后,以0.1-1mg/ml浓度加入复性液中。复性液中含有2-5M尿素,氧化还原剂(谷胱甘肽,半胱氨酸/胱氨酸,半胱胺/胱胺),氧化剂浓度在0.05-0.5mM,还原剂浓度在0.05-2mM,4-10℃复性16-60小时。
在一所述刺突S蛋白的纯化示例中,所述粗纯中的阳离子柱例如Cytiva公司的SPSepharose Bigbeads和SP Sepharose Fast Flow、东曹公司的Toyopearl SP-550C、Toyopearl SP-650M和博格隆公司的SP Bestarose FF等。
在一所述刺突S蛋白的纯化示例中,所述精纯中的阳离子柱例如Cytiva公司的SPSepharose HP和Source 30S、Merck公司的Eshmuon CPS、东曹公司的Toyopearl Sμlfate-650F和博格隆公司的SP Bestarose HP等。
在一所述刺突S蛋白的纯化示例中,所述疏水层析柱的填料例如Cytiva公司的Phenyl Sepharose Fast Flow(high sub)、Phenyl Sepharose Fast Flow(Low Sub)、Butyl Sepharose Fast Flow、博格隆公司的Phenyl Bestarose FF(HS)和ButylBestarose 4FF。
为解决上述技术问题,本发明提供的技术方案四为:一种药物组合物,所述药物组合物包括如技术方案一所述的双功能抗原。
优选地,所述组合物中还含有0.25-1mg/ml的铝佐剂和0.9%的氯化钠。
当所述双功能抗原包含血清型为1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F型的所述肺炎球菌荚膜多糖时,其中1、2、3、4、5、6A、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F型肺炎球菌荚膜多糖的用量相同,均各为6B型肺炎球菌荚膜多糖的一半。
较佳地,所述药物组合物为疫苗,例如联合疫苗和多价疫苗。本领域技术人员可根据受试者的具体确定所述双功能抗原的用量、所述肺炎球菌荚膜多糖的血清型种类及用量和所述刺突S蛋白的用量。
为解决上述技术问题,本发明提供的技术方案五为:一种如技术方案一所述的双功能抗原或如技术方案四所述的药物组合物在制备诊断、治疗或预防肺炎球菌和/或新型冠状病毒的药物中的应用。
为解决上述技术问题,本发明提供的技术方案六为:如技术方案二所述的核酸在制备如技术方案一所述双功能抗原中的应用。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明提供了一种双功能抗原、其制备方法及应用。所述双功能抗原由肺炎球菌荚膜多糖和SARS-CoV-2的刺突S蛋白或其片段共价偶联构成。本发明双特异性抗原,较刺突S蛋白或其片段抗原自身,能显著提高针对新冠病毒的中和抗体水平;且同时能产生较高水平的针对一种或多种肺炎球菌荚膜多糖的特异性抗体(较佳地为24个血清型)肺炎球菌荚膜多糖,有望有效预防新冠病毒和/或肺炎球菌。所述制备方法使用了在原核细胞中可高效表达的刺突S蛋白的核酸序列。所述制备方法成熟、无生物安全风险,能较快、低成本的应用于临床的使用。
附图说明
图1为大肠杆菌表达RBD蛋白(SEQ ID NO:2)的SDS-PAGE图。
图2为纯化后大肠杆菌表达重组RBD蛋白(SEQ ID NO:2)电泳图。
图3为HEK293细胞表达RBD蛋白(SEQ ID NO:3)的SDS-PAGE图。
图4为大肠杆菌表达RBD蛋白(SEQ ID NO:2)的质谱精确分子量图。
图5为多糖与RBD蛋白偶联的化学示意图。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
序列信息:
SEQ ID NO:1,SARS-CoV-2Spike蛋白(GenBank:MN908947.3)的RBD的区域(R319-I587):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI
SEQ ID NO:2,SARS-CoV-2Spike蛋白(GenBank:MN908947.3)的RBD的区域(R319-I587),538位Cysteine取代为Serine/Alanine:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDI
SEQ ID NO:3,SARS-CoV-2Spike蛋白(GenBank:MN908947.3)的RBD的区域(R319-F541):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO:4,为SEQ ID NO:1对应的核苷酸序列:
CGCGTTCAGCCTACAGAATCAATTGTTCGCTTTCCTAACATTACAAACCTTTGTCCTTTCGGAGAGGTCTTCAATGCAACACGCTTTGCTTCAGTTTATGCTTGGAACCGCAAACGCATTTCAAACTGTGTTGCTGATTATTCAGTTCTTTATAACTCAGCTTCATTCTCCACGTTTAAATGTTATGGCGTTTCACCTACAAAGCTGAATGATCTTTGTTTCACTAATGTTTATGCTGATTCATTTGTTATTCGCGGCGATGAAGTTCGCCAGATTGCTCCTGGCCAGACAGGCAAGATAGCGGATTATAACTATAAACTTCCTGATGATTTCACCGGATGTGTTATTGCTTGGAACTCAAACAACCTCGATTCAAAGGTGGGTGGCAACTATAACTATCTTTATCGCCTATTCAGAAAGTCAAACCTTAAACCTTTCGAGCGGGATATTTCAACAGAAATTTATCAGGCTGGCTCAACACCTTGTAACGGCGTTGAAGGCTTTAACTGTTATTTCCCGTTACAGTCTTATGGCTTTCAGCCTACAAACGGCGTTGGCTATCAGCCTTATCGCGTTGTTGTTCTTTCATTTGAACTTCTTCATGCTCCTGCTACAGTTTGTGGCCCTAAGAAGAGCACTAACCTTGTTAAGAATAAGTGTGTTAACTTTAACTTTAACGGCCTTACAGGCACAGGCGTTCTTACAGAATCAAACAAGAAGTTTCTGCCATTTCAGCAGTTTGGCCGCGATATTGCTGATACAACCGACGCCGTCAGAGACCCTCAGACACTTGAAATTCTTGATATTTAA
SEQ ID NO:5,为SEQ ID NO:2对应的核苷酸序列:
CGCGTTCAGCCTACAGAATCAATTGTTCGCTTTCCTAACATTACAAACCTTTGTCCTTTCGGAGAGGTCTTCAATGCAACACGCTTTGCTTCAGTTTATGCTTGGAACCGCAAACGCATTTCAAACTGTGTTGCTGATTATTCAGTTCTTTATAACTCAGCTTCATTCTCCACGTTTAAATGTTATGGCGTTTCACCTACAAAGCTGAATGATCTTTGTTTCACTAATGTTTATGCTGATTCATTTGTTATTCGCGGCGATGAAGTTCGCCAGATTGCTCCTGGCCAGACAGGCAAGATAGCGGATTATAACTATAAACTTCCTGATGATTTCACCGGATGTGTTATTGCTTGGAACTCAAACAACCTCGATTCAAAGGTGGGTGGCAACTATAACTATCTTTATCGCCTATTCAGAAAGTCAAACCTTAAACCTTTCGAGCGGGATATTTCAACAGAAATTTATCAGGCTGGCTCAACACCTTGTAACGGCGTTGAAGGCTTTAACTGTTATTTCCCGTTACAGTCTTATGGCTTTCAGCCTACAAACGGCGTTGGCTATCAGCCTTATCGCGTTGTTGTTCTTTCATTTGAACTTCTTCATGCTCCTGCTACAGTTTGTGGCCCTAAGAAGAGCACTAACCTTGTTAAGAATAAGTCTGTTAACTTTAACTTTAACGGCCTTACAGGCACAGGCGTTCTTACAGAATCAAACAAGAAGTTTCTGCCATTTCAGCAGTTTGGCCGCGATATTGCTGATACAACCGACGCCGTCAGAGACCCTCAGACACTTGAAATTCTTGATATTTAA
SEQ ID NO:6,为SEQ ID NO:3对应的核苷酸序列:
AGAGTGCAACCTACAGAATCAATCGTGAGATTTCCTAACATCACAAACCTTTGCCCTTTCGGAGAAGTGTTCAATGCTACAAGATTTGCATCAGTGTACGCATGGAACAGAAAGAGGATATCAAACTGCGTGGCAGATTACTCAGTGCTTTACAACTCAGCATCATTCTCTACCTTTAAATGCTACGGAGTGTCACCTACAAAGTTAAATGATCTTTGCTTTACAAACGTGTACGCAGATTCATTTGTGATCAGAGGAGATGAAGTGAGACAAATCGCACCTGGACAAACAGGAAAGATAGCTGATTACAACTACAAACTTCCTGATGATTTCACCGGGTGCGTGATCGCATGGAACTCAAACAACTTGGATTCAAAGGTGGGAGGCAATTATAATTATTTATATCGTTTATTTAGGAAGTCCAACCTCAAACCTTTCGAGCGAGATATCTCAACAGAAATCTACCAAGCAGGATCAACACCTTGCAACGGAGTGGAAGGATTTAACTGCTACTTTCCTCTTCAATCATACGGATTTCAACCTACAAACGGAGTGGGATACCAACCTTACAGAGTGGTGGTGCTTTCATTTGAACTTCTTCACGCACCTGCAACAGTGTGCGGACCTAAGAAGAGTACGAACCTTGTGAAGAATAAGTGCGTGAACTTTTAG
实施例1表达SARS-CoV-2重组RBD蛋白的大肠杆菌工程菌的制备
委托苏州金唯智合成分别包含SEQ ID NO:5和SEQ ID NO:6的pET-28a质粒,测序验证正确。
将正确的含SEQ ID NO:5的质粒转化BL21(DE3)感受态,单克隆接种到3mL含100ng/mL卡那霉素的液体LB培养基中,220rpm,30℃振荡过夜培养,作为种子。
将种子按1:500接种到含100ng/mL卡那霉素的TB培养基中,220rpm,37℃振荡培养至OD600=2,加入终浓度0.5mM IPTG,继续培养4h,离心收集菌体用于纯化。
取10OD菌体用1mL裂解液(10mM Tris-HCl,pH8.0)进行重悬,超声破碎裂菌,进行总蛋白、上清、沉淀各组分的SDS-PAGE电泳分析。
实施例2表达SARS-CoV-2重组RBD蛋白(序列为SEQ ID NO:3)的Expi293重组细胞的制备,用QIAGEN质粒提取试剂盒提取测序正确的目的基因的质粒备用
根据转染需要体积准备好新鲜传代至2.0-2.5×10E6个/mL的Expi293细胞悬液(购于ATCC CRL-1573)。
配制转染试剂-质粒复合物,即A液(实施例1中合成的含SEQ ID NO:6的质粒/Opti-MEM=1μg/50μl)和B液(PEI/Opti-MEM=5μl/50μl),A液和B液室温独立放置10分钟。
将B液滴加到A液当中,室温孵育10min后,滴加加入细胞悬液,震荡培养(115rpm,36.8℃,5%CO2),24小时后可以加入0.4mL KT Feed(珠海凯瑞),相同条件继续振荡培养5天-7天,8500rpm离心15min收集细胞上清,交给下游纯化。
按本领域常规步骤进行上清的SDS-PAGE电泳分析,结果见图3。
实施例3 SARS-CoV-2重组RBD蛋白(序列为SEQ ID NO:2)大肠杆菌或细胞表达的纯化及鉴定
将实施例1中的含SEQ ID NO:5的大肠杆菌表达菌体,以1:20(W/V)重悬于pH8.0的Tris缓冲液中,用高压匀浆仪900bar*3次破碎后离心得到包涵体。将包涵体用溶液(20mM乙二醇,8M Urea,50mM DTT pH8.0)溶解变性后加入复性液(20mM Tris,pH8.0,3M尿素,0.05mM胱胺,1mM半胱胺)复性20小时。调节复性上清液pH值到7,10000×g离心1小时,取上清液通过Cytiva SP Sepharose Fast Flow以1-100cm/h流速纯化去除吸附的HCP(宿主细胞残留蛋白,Host cell protein)和多聚体对目标蛋白进行粗纯;收集流穿液目标RBD蛋白峰,并将流穿液调节pH值到5,用Cytiva SP sepharose HP以结合模式做进一步精细纯化,目标蛋白用1M NaCl梯度洗脱,合并含有目标蛋白的洗脱峰,用10kDa MWCO密理博膜包(P2B010A01)浓缩并换液到2x PBS缓冲液中,进行进一步的多糖偶联。
将实施例2中的表达所述抗原的细胞上清液过滤除去细胞碎片。细胞上清液调节pH至7.0,通过Cytiva SP Fast Flow阳离子交换柱以流穿模式去除HCP和多聚体对目的蛋白进行粗纯,流穿液调节pH4.0后上样阳离子交换柱SP sepharose HP进一步精细纯化,用1M NaCl进行梯度洗脱,合并含有目标蛋白的洗脱峰,用10kDa MWCO密理博膜包超滤浓缩并换液到2x PBS缓冲液中进行进一步的多糖偶联。
按本领域常规步骤进行SDS-PAGE电泳分析,电泳结果见图1-2。
按本领域常规步骤对纯化的SARS-CoV-2重组RBD蛋白进行质谱,结果见图4。
实施例4肺炎球菌荚膜多糖发酵及纯化
按本领域常规的多糖发酵和纯化方法:选取24种血清型(1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F)的肺炎球菌,分别进行发酵培养后,采用脱氧胆酸钠(DOC)灭活细菌。离心或膜过滤分离培养液,收集离心或膜过滤上清;将上清以100KD密理博膜包(P2B100V01)超滤浓缩、缓冲液置换及脱盐,分别收集得到粗制多糖溶液。后采用离子或复合模式交换填料,分别进行精制多糖的柱层析纯化,收集流穿峰,采用超滤对流穿峰进行脱盐,冻干回收精制多糖,用于接下来和SARS-CoV-2重组RBD蛋白进行偶联。
实施例5 SARS-CoV-2重组RBD蛋白的微生物多糖共价偶联及纯化
将实施例1或2中所得到的重组RBD蛋白,通过偶联试剂1-氰基-4-(二甲氨基)吡啶四氟硼酸盐(CDAP)分别活化实施例4中所得的肺炎球菌荚膜多糖,将各自活化的24种血清型的肺炎球菌荚膜多糖与RBD按0.2-2:1的投料质量比(具体比例见下表1)分别混合反应,蛋白序列上赖氨酸的氨基直接共价偶联多糖并不断交联聚合形成多糖-蛋白结合物,反应结束后经0.9%氯化钠溶液以100kDa孔径超滤纯化3遍后获得纯化结合物。采用Lowry法分别测定纯化后24个型别RBD结合物中的RBD蛋白含量,采用地衣酚法测定各型别结合物中的多糖含量。
表1 24个型别肺炎球菌多糖与RBD蛋白偶联物的特性数据
实施例6新型冠状病毒-肺炎球菌联合疫苗的配制
根据地衣酚法测定出的24个型别RBD结合物中的多糖含量,按照每剂0.5mL,含6B型结合多糖4μg,其余23个型别结合多糖各2μg的终含量计算各型别RBD结合物的配制体积并取样进行混合,再加入终浓度0.25mg/mL的磷酸铝佐剂进行混合配制获得联合疫苗抗原。
实施例7小鼠免疫实验
将实施例4得到的抗原按照表1的方法于生理盐水中稀释,然后对4-6周龄的Balb/C雌性小鼠(北京维通利华)进行分组免疫。免疫程序如表2,即通过腹股沟皮下注射的方式,每只小鼠分别在第0天、第14天和第28天,接受3针次疫苗免疫,每次0.1mL的接种体积。第42天(即3针次免后的第14天),对小鼠进行眼眶取血。小鼠血清在静置一段时间待血清析出后,通过3000rpm离心10分钟获得,并于-20℃及以下保存。用于SARS-CoV-2相关总IgG抗体滴度、中和抗体滴度以及24个型别肺炎球菌荚膜多糖特异性IgG抗体含量的测定。
表2动物免疫分组情况
实施例8血清中针对新冠病毒RBD的IgG总抗体滴度测定试验
本试验采用间接ELISA法检测各试验组小鼠血清中的针对新冠病毒RBD的IgG总抗体滴度。
i)抗原包被
分别采用S1蛋白(上海近岸生物科技有限公司)及天然序列的新冠病毒RBD蛋白(上海近岸生物科技有限公司)作为包被抗原,以10μg/ml的蛋白浓度100μl每孔包被酶标板,4℃包被过夜。
ii)血清稀释及孵育
试验组血清(10倍起始)按照2倍的倍比稀释,每孔100μl加入酶标板中,37℃孵育120分钟;
iii)洗板机碱性磷酸酶IgG二抗孵育
每孔加入清洗液200μl清洗酶标板,清洗5次。后加入4000倍稀释的羊抗鼠IgG碱性磷酸酶二抗(Southern Biotech),37℃孵育120分钟;
iiii)洗板及显色
每孔加入清洗液200μl清洗酶标板,清洗5次;洗板结束后,每孔加入100μl显色液。黑暗条件、20-25℃显色60分钟后,加入50μl终止液。
iiiii)读值及结果判定标准
将酶标板放入酶标仪中,OD405nm读值。结果分析时需先计算阴阳性的Cutoff值,计算公式如下。再根据Cutoff值,计算样品的IgG抗体滴度。
Cutoff值=阴性组样品孔OD405平均值+2SD
iiiiii)试验结果分析
原核及真核表达的RBD单体蛋白试验组(试验组1及2),5μg剂量免疫3针次后,在小鼠体内均可诱导出针对SARS-CoV-2刺突蛋白或其片段区域高滴度的IgG抗体水平产生。经细菌多糖共价偶联后的新型冠状病毒-肺炎球菌联合疫苗(试验组4),在5μg剂量下免疫3针次后,在小鼠体内可诱导出针对SARS-CoV-2刺突蛋白或其片段区域区域的高滴度的IgG抗体水平产生,亦可产生较高的中和抗体水平且与试验组1&2可比。结果详见表2。
以上结果说明,无论原核或真核表达的RBD单体蛋白及经细菌多糖偶联后的RBD,均可产生较高的总IgG抗体水平,并且经细菌多糖偶联后的RBD激发小鼠产生的总IgG抗体水平显著优于原核或真核表达的RBD单体蛋白抗原。这提示细菌多糖偶联RBD蛋白作为疫苗在免疫学上的可行性,至于产生的IgG抗体是否具有针对SARS-CoV-2的中和保护活性需要试验进一步证实。
表3试验抗原激发小鼠针对新冠刺突蛋白的总IgG抗体水平
注:表中市售S1蛋白购自上海近岸科技有限公司,货号DRA37。
实施例9血清中针对SARS-CoV-2中和抗体滴度测定试验
本试验采用金斯瑞公司商品化的cPassTM sVNT Kit试剂盒检测各试验组小鼠血清中的新冠病毒中和抗体滴度。cPassTM这项技术允许通过模拟病毒和宿主细胞之间的相互作用过程,快速检测样本中的总中和抗体(NAbs)。为了让病毒感染宿主细胞,病毒受体结合蛋白(RBD)首先需要与宿主细胞的膜受体蛋白ACE2相互作用。病毒与宿主细胞的相互作用和随后的病毒感染导致个体免疫反应的激活,从而产生针对病毒的抗体群。其中一些抗体能与病毒结合,但不一定能阻止病毒感染。其他抗体可以以阻断与ACE2受体相互作用的方式与RBD结合。该结果有助于区分样本中是否含有可能特异性阻断相互作用从而阻断病毒进入宿主细胞的NAbs。
i)血清稀释及孵育
阳性质控血清、阴性质控血清及试验组血清(20倍起始)(阴性质控血清及阳性质控血清均为试剂盒自带质控血清)按照2倍的倍比稀释,与HRP标记的RBD蛋白按照1:1的体积混合,混合液37℃孵育30分钟;
ii)混合液酶标板孵育
阳性质控血清、阴性质控血清及试验组血清混合液,各吸取100μl加入酶标板孔中,37℃孵育15分钟。
iii)洗板及显色
每孔加入260μl清洗液,洗板4次;洗板结束后,每孔加入100μl TMB显色液。黑暗条件、20-25℃显色15分钟后,加入50μl终止液。
iiii)读值及结果判定标准
将酶标板放入酶标仪中,450nm读值。结果分析时需先判定系统阴、阳性血清结果是否成立,判定标准如表4。
表4系统阴、阳性判定标准
质控项目 | OD 450nm | 质控合格要求 |
阴性质控 | >1.0 | 阴性质控成立 |
阳性质控 | <0.3 | 阳性质控成立 |
试验组血清中和抗体滴度通过抑制ACE2结合率计算得到,计算公式如下:
抑制率=(1-试验组样品OD值/阴性质控组OD值)×100%
样品组中和抗体滴度阴阳性的抑制率Cutoff值标准见表5:
表5 SARS-CoV-2中和抗体滴度阴阳性的抑制率Cutoff值标准
iiii)试验结果分析
原核及真核表达的RBD单体蛋白试验组(试验组3及4),10μg剂量免疫2针次后,在小鼠体内均可诱导处针对SARS-CoV-2的中和抗体水平产生,但中和抗体水平均较低;经细菌多糖共价偶联后的RBD蛋白试验组(试验组1),在10μg剂量下免疫2针次后,在小鼠体内均可诱导针对SARS-CoV-2的中和抗体水平产生,且抗体水平较高。且细菌多糖共价偶联后的RBD蛋白试验组(试验组2),在1μg低剂量下免疫2针次后,亦可产生较高的中和抗体水平且与试验组1可比。结果详见表6。
以上结果说明,RBD单体蛋白具有一定的免疫原性,免疫后产生较弱中和抗体水平。经细菌多糖偶联后,RBD蛋白的免疫原性得到显著增强,在较低剂量仍可产生较高的中和抗体水平。这提示细菌多糖偶联RBD蛋白作为疫苗能够显著的提高SARS-CoV-2疫苗的免疫原性,且保护性中和抗体水平明显具有优势。
表6试验抗原激发小鼠中和抗体水平的检测
实施例10血清中针对肺炎球菌荚膜多糖型特异IgG抗体含量测定
本试验采用间接ELISA法检测各试验组小鼠血清中的针对以上24个血清型肺炎球菌荚膜多糖型特异IgG抗体GMC浓度。
i)抗原包被
分别采用24个血清型肺炎球菌荚膜多糖作为包被抗原,以1-20μg/ml的多糖浓度100μl每孔包被酶标板,4℃包被过夜次日封闭。
ii)洗板及封闭
包被的酶标板自2~8℃冰箱中取出,用1×洗液洗板5遍,将板轻轻拍干,100μl/孔加入1%BSA,37℃孵育1小时,再用1×洗液洗板5遍,将板轻轻拍干,37℃孵箱内烘干,取出,保鲜膜封板,放入2-8℃冰箱内备用,有效存放期7天。
iii)血清稀释及孵育
将试验组小鼠血清(即待测样品)与各型别多糖阳性血清分别用稀释液稀释(稀释适当倍数),即得待测样品溶液和阳性血清溶液。试验组血清按照2倍的倍比稀释,每孔100μl加入酶标板中,37℃孵育120分钟;
iiii)洗板机碱性磷酸酶IgG二抗孵育
每孔加入清洗液200μl清洗酶标板,清洗5次。后加入4000倍稀释的羊抗鼠IgG碱性磷酸酶二抗,37℃孵育120分钟;
iiiii)洗板及显色
每孔加入清洗液200μl清洗酶标板,清洗5次;洗板结束后,每孔加入100μl显色液,37℃避光孵育2小时。
iiiiii)读值及结果判定标准
加入终止液50μl后,将酶标板放入酶标仪中,450nm读值。结果分析时软件根据阳性样品结果获得四参数标准曲线,试验组样品值根据标准曲线计算获得血清中针对各个型别肺炎球菌荚膜多糖特异性IgG抗体浓度。
iiiiiii)试验结果分析
原核及真核表达的RBD单体蛋白试验组(试验组1及2),由于未与多糖偶联,免疫小鼠后血清中多糖抗体测定均为阴性。
24价肺炎球菌结合疫苗(试验组3),为24个型别肺炎球菌荚膜多糖与白喉类毒素(DT)偶联,免疫小鼠后血清中可测出高滴度的针对24个型别肺炎球菌荚膜多糖特异性IgG抗体浓度,但无针对SARS-CoV-2的IgG抗体及中和抗体。
新型冠状病毒-肺炎球菌联合疫苗(试验组4),为24个型别肺炎球菌荚膜多糖与新冠病毒重组RBD蛋白偶联,免疫小鼠后血清中既可测出高滴度的针对24个型别肺炎球菌荚膜多糖特异性IgG抗体浓度(与24价肺炎球菌结合疫苗浓度可比),又可测定出针对SARS-CoV-2的IgG抗体及中和抗体。结果详见表7。
表7试验抗原激发小鼠针对肺炎球菌24个型别荚膜多糖的型特异IgG抗体水平
以上结果说明,新型冠状病毒-肺炎球菌联合疫苗作为一种新的联合疫苗形式。免疫机体后能产生针对SARS-CoV-2增强的IgG抗体及中和保护抗体反应,且同时针对24个肺炎球菌血清型多糖产生高滴度的型特异IgG抗体水平。是一种能同时预防肺炎球菌及新型冠状病毒感染的双功能联合疫苗。
SEQUENCE LISTING
<110> 江苏坤力生物制药有限责任公司
<120> 一种双功能抗原、其制备方法及应用
<130> P21011562C
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 269
<212> PRT
<213> Artificial Sequence
<220>
<223> SARS-CoV-2 Spike蛋白(GenBank:
MN908947.3)的RBD的区域(R319-I587)
<400> 1
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
260 265
<210> 2
<211> 269
<212> PRT
<213> Artificial Sequence
<220>
<223> SARS-CoV-2 Spike蛋白(GenBank:
MN908947.3)的RBD的区域(R319-I587),538位Cysteine取代为Serine/A
lanine
<400> 2
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
260 265
<210> 3
<211> 223
<212> PRT
<213> Artificial Sequence
<220>
<223> SARS-CoV-2 Spike蛋白(GenBank:
MN908947.3)的RBD的区域(R319-F541)
<400> 3
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 4
<211> 810
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO: 1对应的核苷酸序列
<400> 4
cgcgttcagc ctacagaatc aattgttcgc tttcctaaca ttacaaacct ttgtcctttc 60
ggagaggtct tcaatgcaac acgctttgct tcagtttatg cttggaaccg caaacgcatt 120
tcaaactgtg ttgctgatta ttcagttctt tataactcag cttcattctc cacgtttaaa 180
tgttatggcg tttcacctac aaagctgaat gatctttgtt tcactaatgt ttatgctgat 240
tcatttgtta ttcgcggcga tgaagttcgc cagattgctc ctggccagac aggcaagata 300
gcggattata actataaact tcctgatgat ttcaccggat gtgttattgc ttggaactca 360
aacaacctcg attcaaaggt gggtggcaac tataactatc tttatcgcct attcagaaag 420
tcaaacctta aacctttcga gcgggatatt tcaacagaaa tttatcaggc tggctcaaca 480
ccttgtaacg gcgttgaagg ctttaactgt tatttcccgt tacagtctta tggctttcag 540
cctacaaacg gcgttggcta tcagccttat cgcgttgttg ttctttcatt tgaacttctt 600
catgctcctg ctacagtttg tggccctaag aagagcacta accttgttaa gaataagtgt 660
gttaacttta actttaacgg ccttacaggc acaggcgttc ttacagaatc aaacaagaag 720
tttctgccat ttcagcagtt tggccgcgat attgctgata caaccgacgc cgtcagagac 780
cctcagacac ttgaaattct tgatatttaa 810
<210> 5
<211> 810
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO: 2对应的核苷酸序列
<400> 5
cgcgttcagc ctacagaatc aattgttcgc tttcctaaca ttacaaacct ttgtcctttc 60
ggagaggtct tcaatgcaac acgctttgct tcagtttatg cttggaaccg caaacgcatt 120
tcaaactgtg ttgctgatta ttcagttctt tataactcag cttcattctc cacgtttaaa 180
tgttatggcg tttcacctac aaagctgaat gatctttgtt tcactaatgt ttatgctgat 240
tcatttgtta ttcgcggcga tgaagttcgc cagattgctc ctggccagac aggcaagata 300
gcggattata actataaact tcctgatgat ttcaccggat gtgttattgc ttggaactca 360
aacaacctcg attcaaaggt gggtggcaac tataactatc tttatcgcct attcagaaag 420
tcaaacctta aacctttcga gcgggatatt tcaacagaaa tttatcaggc tggctcaaca 480
ccttgtaacg gcgttgaagg ctttaactgt tatttcccgt tacagtctta tggctttcag 540
cctacaaacg gcgttggcta tcagccttat cgcgttgttg ttctttcatt tgaacttctt 600
catgctcctg ctacagtttg tggccctaag aagagcacta accttgttaa gaataagtct 660
gttaacttta actttaacgg ccttacaggc acaggcgttc ttacagaatc aaacaagaag 720
tttctgccat ttcagcagtt tggccgcgat attgctgata caaccgacgc cgtcagagac 780
cctcagacac ttgaaattct tgatatttaa 810
<210> 6
<211> 672
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO: 3对应的核苷酸序列
<400> 6
agagtgcaac ctacagaatc aatcgtgaga tttcctaaca tcacaaacct ttgccctttc 60
ggagaagtgt tcaatgctac aagatttgca tcagtgtacg catggaacag aaagaggata 120
tcaaactgcg tggcagatta ctcagtgctt tacaactcag catcattctc tacctttaaa 180
tgctacggag tgtcacctac aaagttaaat gatctttgct ttacaaacgt gtacgcagat 240
tcatttgtga tcagaggaga tgaagtgaga caaatcgcac ctggacaaac aggaaagata 300
gctgattaca actacaaact tcctgatgat ttcaccgggt gcgtgatcgc atggaactca 360
aacaacttgg attcaaaggt gggaggcaat tataattatt tatatcgttt atttaggaag 420
tccaacctca aacctttcga gcgagatatc tcaacagaaa tctaccaagc aggatcaaca 480
ccttgcaacg gagtggaagg atttaactgc tactttcctc ttcaatcata cggatttcaa 540
cctacaaacg gagtgggata ccaaccttac agagtggtgg tgctttcatt tgaacttctt 600
cacgcacctg caacagtgtg cggacctaag aagagtacga accttgtgaa gaataagtgc 660
gtgaactttt ag 672
Claims (10)
1.一种双功能抗原,所述双功能抗原由肺炎球菌荚膜多糖和SARS-CoV-2的刺突S蛋白或其片段共价偶联构成;
所述双功能抗原中,各血清型的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段的质量比为0.2-2:1。
2.如权利要求1所述的双功能抗原,其特征在于,所述双功能抗原中,各血清型的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段的质量比为0.7-1.2:1;
和/或,所述肺炎球菌荚膜多糖的血清型选自以下一种或多种:1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F型。
3.如权利要求1所述的双功能抗原,其特征在于,所述刺突S蛋白包含如SEQ ID NO:1或SEQ ID NO:3所示的氨基酸序列;
优选地:
所述刺突S蛋白包含在SEQ ID NO:1上具有以下突变中的一种或多种的氨基酸序列:N36D、I40F、D46Y、Y47F、Y47W、V49F、F74W、W118R、N121K、S159N、N183Y、C220S和C220A,所述刺突S蛋白的氨基酸序列优选为SEQ ID NO:2所示的氨基酸序列。
4.如权利要求1所述的双功能抗原,其特征在于,所述的共价偶联为通过还原胺化反应、碳二亚胺介导的缩合反应、硫烷基化反应、活泼酯法反应或者氰基化反应进行共价偶联;
优选地,所述氰基化反应中所用偶联试剂为1-氰基-4-(二甲氨基)吡啶四氟硼酸盐;和/或,所述氰基化反应中所用偶联试剂活化所述多糖上的羟基,同时偶联所述刺突S蛋白或其片段上的赖氨酸残基,形成偶联产物。
5.一种分离的核酸,所述核酸的序列为SEQ ID NO:5所示。
6.一种如权利要求1-4任一项所述的双功能抗原的制备方法,其特征在于,所述制备方法为:将经偶联试剂活化后的所述肺炎球菌荚膜多糖与所述刺突S蛋白或其片段混合、反应。
7.如权利要求6所述的制备方法,其特征在于,制备所述刺突S蛋白的宿主细胞包括真核细胞和原核细胞,所述真核细胞优选昆虫细胞Sf9、昆虫细胞系Hi5、CHO细胞、Vero细胞、HEK293细胞、Expi293细胞或酵母细胞,更优选为Expi293细胞;所述原核细胞优选大肠杆菌、棒状杆菌或枯草芽孢杆菌,更优选为大肠杆菌;
优选地,所述刺突S蛋白的制备包括将如权利要求5所述的核酸转化入大肠杆菌以表达所述刺突S蛋白。
8.一种药物组合物,其特征在于,所述药物组合物包括如权利要求1-4任一项所述的双功能抗原;
优选地,所述药物组合物中还含有0.25-1mg/ml的铝佐剂和0.9%的氯化钠。
9.一种如权利要求1-4任一项所述的双功能抗原或如权利要求8所述的药物组合物在制备诊断、治疗或预防肺炎球菌和/或新型冠状病毒的药物中的应用,所述药物组合物优选为疫苗。
10.如权利要求5所述的核酸在制备如权利要求1-4任一项所述的双功能抗原中的应用。
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CN113755421A (zh) * | 2021-09-28 | 2021-12-07 | 萝芊细胞因子有限公司 | 一种用于covid-19的口服性疫苗及抗体加强剂 |
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