CN105963691A - 一种肺炎链球菌疫苗 - Google Patents
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Abstract
本发明公开了一种肺炎链球菌疫苗及其制备方法,所述肺炎链球菌疫苗是通过S3CS重组抗原或其突变蛋白抗原与佐剂混合制备得到。所述疫苗可以用于免疫,提高机体对于肺炎链球菌的保护,并且能够产生特异性的抗体。其制备方法简单,适合规模化工业生产的需要。
Description
技术领域
本发明涉及疫苗,具体地讲,涉及一种肺炎链球菌疫苗。
背景技术
由肺炎球菌(肺链)所引起的感染是全世界发病率和死亡率的主要起因。肺炎、发热性菌血症和脑膜炎是侵入性肺炎球菌性疾病的最常见表现形式,而呼吸道内的细菌扩散可导致中耳感染、窦炎或复发性支气管炎。与侵入性疾病相比,非侵入性的表现形式通常不那么严重,但更多见。由于抗生素耐药性传染病的扩散,以及肺炎球菌肺炎经常在流感感染之后出现,肺炎球菌疾病在流感期间发作的可能性进一步增加。由肺炎链球菌引发的疾病已成为全球一个重要的公共卫生问题。肺炎球菌已成为全球儿童的头号杀手。
我国肺炎的病死率为16.4%,其中50岁以上中老年人及1岁以下婴幼儿分别高达28.6%和22.0%。我国肺炎球菌在健康儿童中的携带率较高,资料统计显示,在北方地区健康儿童中的携带率为24.2%,南方地区为31.3%。而该病是导致5岁以下儿童死亡的重要原因。主要原因在于婴幼儿免疫***的发育尚不完善,免疫力较弱。并且年龄越小的婴儿,免疫力越弱。
肺炎球菌大约有90种血清型(菌株),我国的统计资料显示肺炎球菌感染菌株前几个血清型依次为:5、6、19、23、14、2、4型。据一项收集了860株肺炎球菌分离株的研究显示,有109株(12.7%)表现为血清群6,在中国肺炎球菌血清型6的红霉素耐药性达100%,其中的6A、6B和6C型分别为62株(56.9%)、38株(34.9%)和9株(8.2%)。
中国生物技术集团成都生物制品研究所生产的23价肺炎球菌疫苗是选取了23种最常见的致病菌(1,2,3,4,5,6B,7F,8,9N,9V,10A,11A,12F,14,15B,17F,18C,19A,19F,20,22F,23F,33F),分别发酵培养并分离提纯肺炎球菌荚膜上的各型多糖,按等比例混合制成疫苗。
细菌的多糖是一种胸腺非依赖性抗原,这种抗原与胸腺依赖性抗原的主要区别在于前者不需要T淋巴细胞的辅助来产生抗体。因此多糖疫苗存在以下问题:(1)在幼小动物或婴幼儿体内只能产生微弱的免疫反应,甚至不产生免疫反应,免疫反应随年龄的增长而增强;(2)产生低亲和力的抗体;(3)只产生短暂的免疫反应,不具备反复接种时的免疫记忆和免疫增强效应;(4)容易产生免疫耐受;(5)普通的佐剂对这种抗原不易起到免疫增强的作用。23价多糖疫苗对于侵入型肺链感染的保护率为50-70%。而且只能用于2岁以上的人群接种,而肺炎的高峰发病年龄为6-12月龄。
在1998年5月7日所公开的W098/18930,题为“肺炎链球菌的抗原与疫苗”(Streptococcus Pneumoniae antigens and vaccine s的PCT专利中描述了许多具抗原性的特定多肽,但是对于这些多肽的生物活性却无相关报告。
因此,目前急需以链球菌抗原当作疫苗的组份,用以预防和/或治疗链球菌感染,这变得越来越重要。
发明内容
本发明提供一种肺炎链球菌疫苗,所述疫苗中的蛋白氨基酸序列如SEQ IDNO:1所示。
本发明的其它方面提供了载体,其包含可操作连接至表达控制区的本发明的多核苷酸,还提供了用所述载体转染的宿主细胞以及制备多肽的方法,该方法包括在适于表达该基因的条件下培养所述宿主细胞。
在另一方面,提供了由本发明多核昔酸所编码的新颖多肽。
根据本发明的一方面,本发明提供一分离的多核苷酸,其所编码的多肽与选自含有SEQ ID NO:1的同一性至少有95%。
根据本发明的一方面,本发明提供一分离的多核苷酸,其所编码的多肽与选自含有SEQ ID NO:1的同一性至少有90%。
根据本发明的一方面,本发明提供一分离的多核苷酸,其所编码的多肽与选自含有SEQ ID NO:1的同一性至少有85%。
根据本发明的一方面,本发明提供一分离的多核苷酸,其所编码的多肽与选自含有SEQ ID NO:1的同一性至少有80%。
在具体实施方案中,本发明还涉及嵌合型多肽,其包含偶联蛋白中所定义的一个或数个多肽或其片段、相似物或衍生物。
所述偶联蛋白即为耦联结合的蛋白质为白喉类毒素,破伤风类毒素,载体蛋白CRM197,嗜血流感杆菌表面蛋白HiD,百日咳Prn表面蛋白,百日咳Fha抗原和/或肺炎链球菌表面蛋白PspA。
在一具体实施方案中,根据本发明的多肽或嵌合型多肽具抗原性。
在一具体实施方案中,根据本发明的多肽或嵌合型多肽能够引发个体的免疫反应。
在具体实施方案中,本发明还涉及能够产生对本发明中所定义的多肽或嵌合型多肽具结合特异性的抗体的多肽。
“具结合特异性”的抗体表示抗体能够辨识特定的多肽并与其结合,但对于样本例如天然地包含所选肽的生物样本中的其他分子则基本上不识别并结合。利用酶联免疫吸附法(ELISA),可以测定出将所选的多肽当作抗原时的特异性结合能力。
根据本发明,多肽同时包括多肽与嵌合型多肽。也包括己经与其他化合物融合而己改变生物与药理学性质的多肽,例如:与聚乙二醇融合以增长半衰期;与前导或分泌序列融合以
方便纯化;还有前原(prepro)序列和与原(pro)序列以及(多)糖类融合。
再者,在一些氨基酸区域为多态性的情况下,更有需要改变一个或数个特别的氨基酸,使其更有效地模仿不同链球菌株上不同的抗原表位。
所述的突变为申请人通过近800个突变可能中筛选得到的。
所述的突变位点为在SEQ ID NO:1的基础上进行的点突变。F23V(表示第23位点的F氨基酸替换为V),K42C,P60T,K92S,P122D,E133F,D180S,F189H,K243S,I304G,L320Y,V329P,F332K,I339V,I352M,F361W,K369P,S372K,F380M,L401E。
此外,本发明的多肽可经由末端氨基的酞化(例如,乙酞化或硫基乙酸酞胺化)与末端梭基酞胺化(例如:利用氨或甲胺)予以修饰,提供稳定性与增强疏水性,以便连结或结合至支持物或其他分子上。
依据另一方面,本发明提供了疫苗组合物,其至少包含本发明中的一个或数个链球菌多肤和与之混合的可药用的载体稀释剂或佐剂。适当的佐剂包括油,例如:弗式完全佐剂或不完全佐剂;盐类,例如:AlK(S04)2,AINa(S04)2,A1NH4(S04)a,二氧化硅,高龄土;碳多核昔酸,例如:聚IC(poly IC)与聚AU(polyAU)。较好的佐剂包括奎尔A(QuilA)与铝水凝胶(Alhydrogel)。本发明的疫苗可利用注射、快速输液、鼻咽吸收、皮肤吸收或颊部或口腔等非肠胃方式来施用。可药用的载体也包括破伤风类毒素。
本发明的疫苗组合物是用以治疗或预防链球菌的感染和/或如P.R.Murry(主编),E.J.Baron,M.A.Pfaller,F.C.Tenover和R.H.Yolken等人于Manual ofClinical Microbiology,ASM Press,Washington,D.C.第6版,1995一书中第1482页所述受链球菌感染所造成的疾病与症状(此篇已并入本文做参考)。
在一具体实施方案中,本发明的疫苗组合物被用以治疗与预防脑膜炎、中耳炎、细菌血症与肺炎。在一具体实施方案中,本发明的疫苗组合物被用以治疗与预防链球菌的感染和/或受链球菌,特别是肺炎链球菌、A群链球菌(化脓链球菌)[Grouup Astrepococcus(pyogenes)],B群链球菌(GBS或无乳链球菌)[GrouupBstrepococcus(GBS或agalactiae)]、异常泌乳菌(dysgalactiae)、***菌(uberis)、土壤丝菌(nocardia)以及金黄色葡萄球菌(streptococcus aureus)感染后的疾病与症状。在一具体实施方案中,链球菌的感染是肺炎链球菌。
在一特别的具体化实施例中,疫苗被施用于处于受链球菌感染危险中的个体上,例如婴儿、老年人与免疫力较弱者。
本申请书所使用的“个体”,包括哺乳类。在一具体实施方案中此哺乳类为人类。
疫苗组合物较好的单位剂量约为0.001至100p,g/kg(抗原/体重),再较好的是0.01至1Opg/kg,最好的是0.1至1p,g/kg,免疫接种之间有一至六周的间隔,施用一至三次。
有义的效果:本发明提供了一种具有较好免疫原性的蛋白,该蛋白制备成为的疫苗能够提供较好的肺炎链球菌的免疫保护能力,该蛋白制备简单,成本低廉,并且对人体无副作用,适宜于大面积的推广。
具体实施方式
实施例1蛋白的表达
以肺炎链球菌的DNA为模板,通过设计引物扩增得到SEQ ID NO:1所示的氨基酸序列的基因序列,然后利用QIAgen(Chatworth,CA的QIAquick凝胶萃取试剂盒从琼脂凝胶上将PCR产物回收。与Superlinker载体pSL301(Invitrogen,San Diego,CA)一起通过酶切后,以QIAgen(Chatworth,CA)的QIAquick凝胶萃取试剂盒从琼脂凝胶上回收。将限制酶切过的基因组DNA片段与限制酶切过的pSL301载体粘接。粘接后的产物再根据Simanis的方法(Hanahan,D.DNA Cloning,1985,D.M.Glover(ed),pp.109-13 5)转化至DH5a大肠杆菌中。以QIAgen的试剂盒来纯化含有目的基因的pSL301重组质粒(rpSL301),经过测序鉴定,鉴定重组正确。
将目的基因通过酶切连接构建到pET-32c(+)表达载体(Novagen,Madison,WT)后,转化进入BL21细胞,进行原核表达,通过常规的表达获得了上清表达的蛋白形式,通过过柱纯化,获得目的蛋白,浓度为100mg/mL。
实施例2突变蛋白的获得
突变点的引入
(1)、根据相应的突变位点设计相应的诱变引物,采用PCR定点突变法在所对应的S3CS基因上把野生型对应的氨基酸位点进行突变;所述的突变位点为在SEQ ID NO:1的基础上进行的点突变。F23V(表示第23位点的F氨基酸替换为V),K42C,P60T,K92S,P122D,E133F,D180S,F189H,K243S,I304G,L320Y,V329P,F332K,I339V,I352M,F361W,K369P,S372K,F380M,L401E。
(2)、上述PCR产物经胶回收纯化后,用限制性内切酶进行酶切,与经过相同酶切的质粒pSL301载体片段连接后,转化大肠杆菌DH5α感受态细胞;
(3)、鉴定重组质粒:用酶切、PCR方法对重组质粒进行鉴定,并测序检验,由此得到突变后的核苷酸序列。
将这些突变后的S3CS基因采用实施例1中的表达方法进行相应的表达,通过纯化后,蛋白浓度同样调整为100mg/mL。
实施例3肺炎球菌蛋白疫苗的产生抗体效果评价
12-14g雌性3周龄BALB/c.幼鼠,随机分为23组,每组20只,分别腹腔注射按照实施例1和2蛋白与Al(OH)3作为免疫佐剂配制的结合疫苗,市售13价肺链结合疫苗,空白对照(PBS)。
于第0、2、4周程序注射3次,每只幼鼠0.5ml含(0.5ug蛋白)。每个实验组分别于第三针注射后的7天采血,分离血清,置-20℃保存备用。
对于蛋白抗体滴度检测分别采用间接ELISA法,将蛋白溶解于0.05mol/LpH9.6的碳酸盐缓冲溶液中,以20ug/ml浓度包被酶标板,2%的BSA液封闭。实验时将待测血清100,200,400,800,1600,3200,6400,12800,25600,51200,102400,204800,409600倍稀释后加入酶标板,置37℃反应40min,常规洗涤后加入辣根过氧化物酶标记的羊抗鼠二抗。同时加入不含血清的缓冲液作为阴性对照。用四氨基联苯胺显色,酶标仪450nm波长处读取A值。计算每孔中A值与阴性孔A值得比值,但比值大于2.1时代最大的稀释倍数为血清中的抗体滴度,对20只小鼠的滴度进行几何平均,并以20为底计算对数值,结果见表1。
从数据看出,本发明的蛋白疫苗以及相应的突变体蛋白疫苗对小鼠对肺链多糖的免疫反应显著增强,比市售的疫苗产生抗体的能力也增强。
实施例4免疫小鼠效果评价
1)首次免疫,用PBS稀释2mg的S3CS或其突变的重组蛋白抗原,加入浓度为1mg/mL的Al(OH)3;用5号半型针头,双侧腹股沟、足底及背部皮下对点注射,每只BALB/C小鼠注射量为l00uL,并设置阳性对照组、阴性对照组和空白对照组;
2)第二次免疫,第14天进行第二次免疫,免疫组分同上,注射量蛋白抗原量是首次免疫的1/2,免疫途径同上;
3)第三次免疫,第21天进行第三次免疫,免疫组分同上,注射量蛋白抗原量与第二次免疫相同,免疫途径同上;
在第14天采用致死剂量,尾静脉注射肺炎链球菌菌株WU2活菌进行攻毒实验,每只BALB/C小鼠注射菌液量为1.5*109CFU,观察14天,统计各组小鼠的存活率。结果示于表2。
表2显示;阴性对照组和空白对照组的平均免疫保护率分别为0%,本发明的抗原蛋白或其突变蛋白具有大约100%的保护特性,比现有技术的疫苗效果有提高。
安全性评价
将上述蛋白免疫后的小鼠在攻毒实验结束后,继续培养1月,进行解剖,发现小鼠各个器官正常,没有任何变异。这充分的说明,本发明的疫苗安全可靠。
以上所述仅为本发明的较佳实施例,并非用来限定本发明的实施范围;如果不脱离本发明的精神和范围,对本发明进行修改或者等同替换,均应涵盖在本发明权利要求的保护范围当中。
序列表
〈110〉查文娟
〈120〉一种肺炎链球菌疫苗
〈160〉1
〈210〉1
〈211〉887
〈212〉PRT
〈213〉人工序列
〈400〉S3CS
1 MYTFILMLLD FLKNHDFHFF MLFFVFILIR
WAVIYFHAVR YKSYSCSVSD EKLFSSVIIP
61 VVDEPLNLFE SVLNRISRHK PSEIIVVING
PKNERLLKLC HDFNEKLENN MTPIQCYYTP
121 VPGKRNAIRV GLEHVDSQSD ITVLVDSDTV
WTPRTLSELL KPFVCDKKIG GVTTRQRIFD
181 PERNLVTMFA NLLEEIRAEG SMKAMSVTGK
VGCLPGRTIA FRTEILRECI HEFMNETFMG
241 FHKEVSDDRS LTNLTLKKGY KTVMQDTSVV
YTDAPTSWKK FIRQQLRWAE GSQYNNLKMT
301 PWMIRNAPLM FFIYFTDMIL PMLLISFGVN
IFLLKILNIT TVVYTASWLE IILYVLFGMI
361 FSFGGRNFKA MSRMKWYYVF LIPVFIIVLS
IIMCPIRLLG LMRCSDDLGW GTRNLIEGGG
421 SGGGSGGGSG GGSGGGSMSK YKELAKNTGI
FALANFSSKI LIFLLVPIYT RVLTTTEYGF
481 YDLVYTTIQL FVPILTLNIS EAVMRFLMKD
GVSKKSVFSI AVLDIFIGSI AFALLLLVNN
541 LFSLSDLISQ YSIYIFVIFV FYTLNNFLIQ
FSKGIDKIGV TAISGVISTA VMLAMNVILL
601 VVFDWGLLGF FIANVCGYVI PCIYIVSRLR
LWELFEIKID KKLQWEMVYY ALPLVLNILS
661 WWVNNTSDRY IVTAIVGIQA SAIISVAYKI
PQILSTISAI FIQSWQISAI KIQEDKSDTT
721 FVSNMLLYYN ALLLIIASGI ILFVKPISNI
LFGISFYSAW ELVPFLIISS LFNAISGCIG
781 AIMGAKMDTH NIAKSALVGM IANIILNIVL
TFLMGPQGIT ISTLIASFLI FYMRKDSVKE
841 INSETYRAIY LSWILLVVEA CLLIYMDFII
GALIAMVINL FLLKDVI
Claims (3)
1.一种肺炎链球菌疫苗,其含有抗原蛋白与佐剂。
2.如权利要求1所述的疫苗,其中抗原蛋白为SEQ
ID NO:1所示的序列,或其在SEQ ID NO:1的基础上进行的点突变,具体的突变位点为F23V,K42C,P60T,K92S,P122D,E133F,D180S,F189H,K243S,I304G,L320Y,V329P,F332K,I339V,I352M,F361W,K369P,S372K,F380M或L401E。
3. 一种预防肺炎的方法,包括使用权利要求1-2任一项所示的疫苗。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101024079A (zh) * | 2006-02-17 | 2007-08-29 | 福州昌晖生物工程有限公司 | 肺炎链球菌多糖-外膜蛋白结合疫苗及制备方法 |
CN101977927A (zh) * | 2008-03-17 | 2011-02-16 | 英特塞尔股份公司 | 针对肺炎链球菌保护的肽以及与其有关的组合物、方法和用途 |
CN103830723A (zh) * | 2012-11-26 | 2014-06-04 | 天士力制药集团股份有限公司 | 一种肺炎链球菌荚膜多糖蛋白结合疫苗的制备方法 |
CN103893751A (zh) * | 2014-03-26 | 2014-07-02 | 天津康希诺生物技术有限公司 | 一种肺炎球菌多糖蛋白缀合疫苗及其制备方法 |
CN104844712A (zh) * | 2015-04-03 | 2015-08-19 | 长春百克生物科技股份公司 | 肺炎链球菌蛋白抗原及其制备方法和应用 |
CN105008539A (zh) * | 2012-11-07 | 2015-10-28 | 格林考瓦因有限公司 | 通过酶缀合在大肠杆菌中生产重组疫苗 |
-
2016
- 2016-07-03 CN CN201610524959.4A patent/CN105963691B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101024079A (zh) * | 2006-02-17 | 2007-08-29 | 福州昌晖生物工程有限公司 | 肺炎链球菌多糖-外膜蛋白结合疫苗及制备方法 |
CN101977927A (zh) * | 2008-03-17 | 2011-02-16 | 英特塞尔股份公司 | 针对肺炎链球菌保护的肽以及与其有关的组合物、方法和用途 |
CN105008539A (zh) * | 2012-11-07 | 2015-10-28 | 格林考瓦因有限公司 | 通过酶缀合在大肠杆菌中生产重组疫苗 |
CN103830723A (zh) * | 2012-11-26 | 2014-06-04 | 天士力制药集团股份有限公司 | 一种肺炎链球菌荚膜多糖蛋白结合疫苗的制备方法 |
CN103893751A (zh) * | 2014-03-26 | 2014-07-02 | 天津康希诺生物技术有限公司 | 一种肺炎球菌多糖蛋白缀合疫苗及其制备方法 |
CN104844712A (zh) * | 2015-04-03 | 2015-08-19 | 长春百克生物科技股份公司 | 肺炎链球菌蛋白抗原及其制备方法和应用 |
Non-Patent Citations (3)
Title |
---|
DENAPAITE,D.ET AL.: "ACCESSION NO.AEP95727.1", 《GENBANK》 * |
刘孟涓 等: "46株肺炎链球菌的血清型别和毒力因子基因的保守性分析", 《临床检验杂志》 * |
曹炬 等: "肺炎链球菌蛋白质疫苗研究进展", 《国际检验医学杂志》 * |
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