CN106039301A - Preparation method of pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine - Google Patents

Preparation method of pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine Download PDF

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CN106039301A
CN106039301A CN201610503169.8A CN201610503169A CN106039301A CN 106039301 A CN106039301 A CN 106039301A CN 201610503169 A CN201610503169 A CN 201610503169A CN 106039301 A CN106039301 A CN 106039301A
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vaccine
pneumococcal
haemophilus influenzae
protein
influenzae type
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刘昊智
吴克
王文灏
袁军
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武汉博沃生物科技有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/05Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/08Clostridium, e.g. Clostridium tetani
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/102Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/70Multivalent vaccine

Abstract

The invention relates to a pneumococcus-type b haemophilus influenzae-diphtheria-pertussis-tetanus combined vaccine. A preparation method is used for preparing a combined vaccine containing a pneumococcus-type b haemophilus influenza combined vaccine and a diphtheria-pertussis-tetanus vaccine; pneumococcus carrier protein and type b haemophilus influenzae carrier protein, type b haemophilus influenzae capsular polysaccharide and pneumococcus capsular saccharide are added; and the mass ratio of the added capsular polysaccharide to the added type b haemophilus influenzae carrier protein ranges from (5: 1) to (1: 5). With adoption of the combined vaccine, five types of antigens including the pneumococcus are immunized at the same time, and pneumococcus protein is used as a virulence factor for inducing a relatively strong immune protection effect; the capsular saccharide is used for supplementing immunization, so that the immune protection effect of the vaccine is enhanced; and meanwhile, self protein of the pneumococcus can be prevented from immune conflicts with other vaccines, such as a diphtheria vaccine and a tetanus vaccine, in vivo.

Description

肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法 Pneumococcal Haemophilus influenzae type -b - DPT vaccine preparation

技术领域 FIELD

[0001] 本发明涉及疫苗生产制备领域,尤其涉及联合疫苗,具体是指一种肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法。 [0001] The present invention relates to a vaccine production, and in particular relates to a combination vaccine, in particular to a pneumococcal Haemophilus influenzae type -b - preparation of DTP combination vaccine.

背景技术 Background technique

[0002] 肺炎链球菌(Streptococcus pneumoniae)简称肺炎球菌(Pneumococcus),寄居于正常人的鼻咽腔中,是细菌性大叶性肺炎、脑膜炎、中耳炎、肺炎、支气管炎的主要病原菌。 [0002] Streptococcus pneumoniae (Streptococcus pneumoniae) referred to pneumococcal (Pneumococcus), dwelling in the normal nasopharyngeal cavity, bacterial lobar pneumonia, meningitis, otitis media, pneumonia, bronchitis major pathogens. 肺炎球菌导致的疾病一直是全球严重的公共卫生问题,在全世界范围内有较高的发病率和病死率,尤其是对2岁以下的儿童和老人。 Pneumococcal disease has been the cause of a serious global public health problem, with high morbidity and mortality worldwide, especially for children under 2 years of age and the elderly. 目前已上市的肺炎球菌荚膜糖疫苗和荚膜糖蛋白质结合疫苗,其设计都基于肺炎球菌荚膜糖,涵盖了导致肺炎球菌性疾病的最常见血清型。 Available now pneumococcal capsular saccharide vaccines and capsular glycoprotein conjugate vaccines, which are based on the design pneumococcal capsular saccharides, covering the lead to pneumococcal disease most common serotypes. 但肺炎球菌荚膜糖为胸腺非依赖性抗原(Thymus independent antigen,TI-Ag),抗体反应主要依赖于其重复单位组成的线性表位,在无T淋巴细胞辅助的情况下直接与B淋巴细胞表面的IgM受体交联,所诱导的抗体主要为IgM和IgG2,缺少较好的补体活化能力,抗体水平不能维持足够长的时间,且不能诱导免疫记忆,无法在2岁以下幼儿中产生免疫保护。 However pneumococcal capsular saccharide is a thymus-independent antigens (Thymus independent antigen, TI-Ag), antibody dependent mainly upon the reaction of the repeating units of the linear epitope, B lymphocytes directly without auxiliary T lymphocytes receptor crosslinking surface IgM, antibody induced are mainly IgM and IgG2, lack the ability to better complement activation, antibody levels can not be maintained sufficiently long time, and can not induce immunological memory, not produce immunity in children under 2 years of age protection. 荚膜糖复杂的结构导致每一个血清型的免疫原性不同,无法针对不同地区的不同人群产生足够有效的免疫应答。 Capsular saccharides complex structures lead to different for each serotype of immunogenicity, can not produce enough effective immune response against different people in different regions. 肺炎球菌结合疫苗包括血清型别多,各型别用于结合的特异性结构不同,导致其每个型别的结合方法相异。 Pneumococcal conjugate vaccine comprising multiple serotype, each typed binding specificity for different structures, each leading to different types other bonding method. 目前肺炎球菌常用的载体蛋白包括白喉类毒素;破伤风类毒素、百日咳类毒素、霍乱类毒素、大肠杆菌LT、大肠杆菌ST、来自铜绿假单胞菌的外毒素A; 外膜复合体c(0MPC)、孔蛋白、转铁蛋白结合蛋白、来自A组或者B组链球菌的C5a肽酶、流感嗜血杆菌蛋白D;卵白蛋白;匙孔血蓝蛋白(KLH);牛血清白蛋白(BSA)或者结核菌素的纯化的蛋白质衍生物(pro);以及部分肺炎球菌自身的高保守性蛋白等。 Pneumococcal currently used carrier proteins include diphtheria toxoid; tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa; C the outer membrane complex ( 0MPC), porins, transferrin binding proteins, of C5a peptidase from group a or group B Streptococcus, and Haemophilus influenzae protein D; ovalbumin; keyhole limpet hemocyanin (KLH); bovine serum albumin (BSA ) or purified protein derivative of tuberculin (Pro); and a portion pneumococcal protein itself highly conserved and the like. 肺炎球菌对荚膜糖的修饰及与载体蛋白的结合要在保证荚膜糖特异基团不丢失、抗原性和免疫原性不受影响的前提下进行,同时为了避免糖链的过度交联和结合物除菌过滤的要求,对荚膜糖及结合物分子的大小应当有一定控制。 Modified binding of pneumococcal capsular saccharide to carrier protein is to ensure that the specific capsular saccharide group is not lost, under the premise of antigenicity and immunogenicity in the unaffected, while in order to avoid excessive crosslinking and sugar chains the conjugate of claim sterile filtration, and the size of the capsular saccharide binding molecules should have some control. 整个工艺过程中需要加入多种化学试剂参与反应,并且荚膜糖蛋白结合疫苗的血清型覆盖率低和非疫苗血清型肺炎球菌感染性疾病的增加使得更多研究者开始关注其他方向的肺炎球菌疫苗开发。 Low serotype coverage throughout the process necessary to add a variety of chemical reagents involved in the reaction, and capsular glycoprotein conjugate vaccine and non-vaccine serotypes increased pneumococcal infections makes more researchers began to focus on other directions of pneumococcal vaccine development.

[0003] 肺炎球菌蛋白疫苗已成为近十年来肺炎疫苗的研发主流,具有种属特异性抗原为基础的疫苗广受研发人员的重视,吸引了大量的目光。 [0003] pneumococcal protein vaccines have become mainstream over the past decade developed pneumonia vaccine, species-specific antigen-based vaccines widely in research and development personnel, attracting a lot of attention. 但由于蛋白本身的结构及理化性质的限制,很多肺炎球菌蛋白无法直接用于人体免疫,需要花费大量的人力和物力进行研究及临床验证。 But due to the structure of the protein itself and the physical and chemical properties, many pneumococcal protein can not be directly used for human immunization, it takes a lot of manpower and material resources to research and clinical validation.

[0004] 流感嗜血杆菌中b型流感嗜血杆菌侵袭力最强的一个血清型,是引起小儿严重感染的重要致病菌,其感染对象主要是5岁以下儿童,尤其是2岁以下的婴幼儿。 [0004] Haemophilus influenzae type b invasive Haemophilus influenzae serotype strongest one, is an important pathogen causing serious infections in children, whose infection was mainly targeted at children under age 5, especially 2 years infants and young children. Hib通过易感者呼吸道传播,定植于鼻咽部,可表现为无症状携带,持续数月,少部分人群则发生Hib侵袭性疾病。 Hib spread through the respiratory tract susceptible, to colonize the nasopharynx, can be expressed as asymptomatic carrier, for several months, a small part of the population of invasive Hib disease occurs. Hib感染最常见的疾病为脑膜炎与肺炎,另外还包括骨髓炎、脓毒性关节炎,会厌炎及败血症等。 Hib infection is the most common disease is meningitis and pneumonia, also including osteomyelitis, septic arthritis, epiglottitis and sepsis. Hib最重要的致病因子是其荚膜多糖PRP,能在Hib感染和克隆过程中提供生存优势,抵抗补体介导的吞噬作用,抑制血清杀菌活性,逃脱鼻粘膜免疫,促进细菌在人群中的传播。 Hib most important pathogenic factor is the capsular polysaccharide PRP, Hib infection and can provide in the cloning process survival advantage against phagocytosis of complement-mediated inhibition of serum bactericidal activity to escape the nasal mucosa immunity, and promote bacteria in the crowd propagation.

[0005] Hib结合疫苗根据PRP长度、载体蛋白不同常见的有四种,其免疫原性各有特点: (1)以白喉类毒素蛋白为载体的结合疫苗(PRP-diphthena toxoid,PRP-D):与Hib多糖疫苗免疫原性相似,具有一定的年龄特点,成人初次接种后就可产生较高水平的抗体,而>15月的儿童虽可以产生较高水平抗体,但加强接种后无长期保持作用。 [0005] Hib conjugate vaccine, there are four common, according to its own characteristics PRP immunogenicity lengths, different carrier proteins: (1) to the carrier protein diphtheria toxoid conjugate vaccine (PRP-diphthena toxoid, PRP-D) : and immunogenicity of the vaccine Hib polysaccharide similar, with the characteristics of a certain age, adult for the first time can produce higher levels of antibodies after vaccination, and> although children under 15 months can produce higher levels of antibodies, but no long-term booster vaccination effect. (2)以B组脑膜炎双球菌胞膜蛋白为载体的结合疫苗(PRP-〇MP):PRP-〇MP对所有年龄组人群均有较好的免疫原性。 (2) to Neisseria meningitis B group membrane protein carrier conjugate vaccine (PRP-〇MP): PRP-〇MP good immunogenicity for all age groups are. 第1剂接种后,大多数人均能产生高水平的抗体。 1 after the first vaccination, the majority produce high levels of antibodies per capita. 再次接种后的抗体效价亦大于PRP-D,Hb-0C,PRP-T,但PRP-0MP的抗体维持的时间短,且抗体峰值水平较Hb-〇C,PRP-T低。 Antibody titer after revaccination also greater than the PRP-D, Hb-0C, PRP-T, but the short PRP-0MP time maintaining an antibody, and the antibody levels than the peak Hb-〇C, PRP-T low. (3)以减毒的白喉类毒素CRM197为载体的结合疫苗(池-〇(:,?1^-〇^197):2月龄婴儿第1剂接种产生的抗体水平较低,但4和6月龄分别再次注射后可诱导出高水平的抗体,1年后抗体仍可维持一定的水平。(4)以破伤风类毒素为载体的结合疫苗(PRP-T):与Hb-〇C类似,在较大儿童及成人,第1剂接种即可显示较好的免疫原性。小婴儿对第1剂接种免疫反应较弱,第2剂及第3剂再次接种后即能产生较高水平的抗体,且抗体水平维持时间较长。目前,国内及国际上主要使用的是PRP-CRM197,PRP-T两种,PRP-T适用于大多数年龄段人群的Hib疫苗接种。 (3) attenuated diphtheria toxoid CRM197 conjugate vaccine as a carrier (cell -〇 (:, 1 ^ 197 ^ -〇):? 2-month-old infants vaccinated lower antibody levels generated by the first one, but 4 and after 6 months of age were injected again to induce high levels of antibodies, one year after the antibody can maintain a certain level (4) to tetanus toxoid as carrier conjugate vaccine (PRP-T):. and Hb-〇C Similarly, in older children and adults, a first vaccination to display good immunogenicity. baby is weak immunization a first reaction, after the second of the three agents can produce high revaccination antibodies, antibody levels and levels maintained for a long time. at present, domestic and international main use of PRP-CRM197, PRP-T two kinds, PRP-T is suitable for most age groups Hib vaccination.

[0006] 由于肺炎球菌多糖-蛋白疫苗所使用的蛋白载体与b型流感嗜血杆菌相同或相似, 而使用同一载体蛋白很有可能在体内引起多糖的免疫干扰,即给予多糖-蛋白质缀合物或将多糖_蛋白质缀合物组合制成多价疫苗可能会出现问题。 [0006] Since the pneumococcal polysaccharide - protein vaccine carrier protein used in the Haemophilus influenzae type b are the same or similar, and use the same carrier protein is likely to cause an immune interference of the polysaccharide in vivo, i.e. administered polysaccharide - protein conjugates _ polysaccharide or protein conjugates formed multivalent vaccine composition may be a problem. 例如,据报导按照标准婴幼儿接种时间表,在一定剂量范围内用(游离)TT和肺炎球菌多糖-TT缀合物疫苗同时进行免疫,并对用破伤风类毒素(TT)作为蛋白载体的流感嗜血菌b型多糖(PRP)疫苗进行了试验。 For example, it is reported according to standard infant vaccination schedule, with (free) TT within a certain dose range and -TT pneumococcal polysaccharide conjugate vaccine immunization simultaneously, and with tetanus toxoid (TT) as the protein carrier Haemophilus influenzae type b polysaccharide (PRP) vaccine were tested. 当肺炎球菌疫苗的剂量增加时,对Hib缀合物疫苗的PRP多糖部分的免疫应答降低,这表明很可能通过使用同一载体蛋白引起了多糖的免疫干扰。 When increasing the dose of pneumococcal vaccine, the immune PRP polysaccharide portion of the Hib conjugate vaccine reduced response, suggesting that it is likely to cause an immune interference of the polysaccharide by using the same carrier protein. 因此为了避免这种免疫干扰的产生,提高免疫应答,便于流行病的防控,研发一种稳定有效的多价联合疫苗是本领域的重点研究方向。 Therefore, to avoid this interference immunity, improve immune response, to facilitate the prevention and control of epidemics, to develop a stable and effective multivalent vaccine is the focus of the joint research in this field.

[0007] 百白破疫苗是我国现行的免疫规划程序规定的针次最多、使用最广泛的疫苗,包括无细胞百白破及全细胞百白破两种联合疫苗,近年来无细胞百白破联合疫苗由于其良好的免疫原性和安全性取代了全细胞百白破联合疫苗而广泛用于儿童的预防接种,已被纳入许多国家的常规免疫计划中。 [0007] DPT vaccine is the most prescribed needle times China's current immunization schedules, the most widely used vaccines, including diphtheria-tetanus-acellular and whole-cell DTP vaccine Two Combining recent years, acellular pertussis due to its favorable combination vaccine immunogenicity and safety of replacing the whole-cell DTP vaccine is widely used in vaccination of children, it has been incorporated into routine immunization programs in many countries. 但百白破疫苗由于其疫苗本身的性质导致其具有多种副作用,难以与其他疫苗进一步联合使用。 But the DPT vaccine due to the nature of its own which has led to a variety of side effects, it is difficult to further combination with other vaccines. 赛诺菲巴斯德公司生产的五联疫苗一度成为免疫学历史上的新高度,其中包括百白破疫苗及脊髓灰质炎病毒及b型流感嗜血杆菌荚膜糖。 Sanofi Pasteur's new pentavalent vaccine became a highly educated in the history of immunization, including polio and DPT vaccine virus and Haemophilus influenzae type b capsular saccharides.

[0008] 肺炎球菌蛋白疫苗已成为近十年来肺炎疫苗的研发主流,具有种属特异性抗原为基础的疫苗广受研发人员的重视,吸引了大量的目光。 [0008] pneumococcal protein vaccines have become mainstream over the past decade developed pneumonia vaccine, species-specific antigen-based vaccines widely in research and development personnel, attracting a lot of attention. 但由于蛋白本身的结构及理化性质的限制,很多肺炎球菌蛋白无法直接用于人体免疫,需要花费大量的人力和物力进行研究及临床验证。 But due to the structure of the protein itself and the physical and chemical properties, many pneumococcal protein can not be directly used for human immunization, it takes a lot of manpower and material resources to research and clinical validation. 蛋白疫苗利用生物工程技术合成蛋白抗原,蛋白抗原由于其选用的均为高度保守的肺炎球菌特异性蛋白,从而消除了不同血清型之间的免疫差异,并且由于不需要其他类型的通用蛋白载体,消除了现有的肺炎球菌疫苗不能与通用载体蛋白同类型的疫苗同时联合使用的障碍;其蛋白抗原本身也可以作为载体蛋白与肺炎球菌荚膜糖进行缀合,因此免疫效果更好,实现了特异性免疫与广泛免疫的联合实现。 Protein vaccines use of biological engineering technology antigen protein synthesis, protein antigens are selected because of their highly conserved pneumococcal specific proteins, thereby eliminating the immunological differences between different serotypes, and since no other type of common protein carrier, removes the barriers existing pneumococcal vaccine is not common carrier protein with the same type of vaccine used in combination at the same time; it can also be a protein antigen itself as a carrier protein conjugated pneumococcal capsular sugars, and therefore better immunity to achieve specific immunity and immune extensive joint implementation.

[0009] 肺炎球菌由于其血清型多,导致其疫苗本身的抗原结构大稳定性差,难以与其他疫苗联合共存使用,并且现有的肺炎球菌荚膜糖蛋白结合疫苗均用白喉或破伤风类毒素作为蛋白载体,这种疫苗的主要成分为血清型特异性的荚膜糖,对未包含在疫苗内的其他血清型肺炎球菌感染无效,即缺乏交叉免疫保护效果;并将与已在儿童常规免疫接种中使用的白喉及破伤风疫苗产生干扰,破坏现有的免疫效果。 [0009] Because of its pneumococcal serotypes, which leads to the stability of the vaccine antigen structure itself is a large difference, it is difficult to coexist with other combined vaccine and conventional pneumococcal conjugate vaccine are capsular saccharide with diphtheria or tetanus toxoid as the carrier protein, the main component of this vaccine is serotype-specific capsular saccharides for other pneumococcal serotypes not contained in the vaccine ineffective infection, namely the lack of cross-protective immunity; and already in routine childhood immunization diphtheria and tetanus vaccination using interference, damage existing immunity. 因此研究一款自身免疫原性够好能够稳定存在并能与b型流感疫苗、百白破疫苗联合起效的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗成为了疫苗研发领域的当务之急。 Therefore, a study of the immunogenicity good enough to be able to own stable and able to influenza type b vaccine, DPT vaccine and the onset of pneumococcal Haemophilus influenzae type -b - DPT vaccine has become a field of vaccine development priority.

发明内容 SUMMARY

[0010] 本发明的目的是克服了上述现有技术的缺点,提供了一种能够实现五种疾病同时预防的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法。 [0010] The object of the present invention is to overcome the disadvantages of the prior art described above, there is provided a five diseases can be achieved while preventing pneumococcal Haemophilus influenzae type -b - DTP vaccine preparation.

[0011] 为了实现上述目的,本发明的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法具有如下构成: [0011] To achieve the above object, the present invention is Streptococcus pneumoniae Haemophilus influenzae type -b - DPT vaccine prepared having the following composition:

[0012] 该肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其主要特点是,制备方法用于制备包括肺炎球菌_b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: [0012] The pneumococcal Haemophilus influenzae type -b - Preparation combination vaccine DPT, whose main characteristic is, the preparation method for preparing comprising pneumococcal _b Haemophilus influenzae type combined vaccine and DPT vaccine the combination vaccine, in particular:

[0013] a.将已纯化备用的肺炎球菌载体蛋白及b型流感嗜血杆菌载体蛋白以质量比1:1 的比例加入制备体系中,其中肺炎球菌载体蛋白为肺炎球菌表达的高保守性并具有免疫原性的蛋白,与具有免疫原性的肺炎球菌荚膜糖缀合,b型流感嗜血杆菌载体蛋白为流感嗜血杆菌的外膜D蛋白HiD; . [0013] a backup of the purified pneumococcal protein carrier Haemophilus influenzae type b and carrier protein mass ratio of 1: 1 ratio was added in the preparation of the system, wherein the carrier protein is pneumococcal highly conserved, and expression of pneumococcal immunogenic protein, the pneumococcal capsular saccharide conjugated to immunogenic, b Haemophilus influenzae type carrier protein is an outer membrane of Haemophilus influenzae protein D HiD;

[0014] b.加入b型流感嗜血杆菌荚膜多糖,加入的荚膜多糖与b型流感嗜血杆菌载体蛋白质量比介于5:1和1:5之间; . [0014] b added Haemophilus influenzae type b capsular polysaccharide, was added and the capsular polysaccharide Haemophilus influenzae type b carrier protein ratio between 5: 1 and 1: 5;

[0015] c.加入肺炎球菌荚膜糖,肺炎球菌血清型包括7种~23种血清型,其中6B型荚膜糖的加入质量是其余荚膜糖加入质量的2倍,肺炎球菌荚膜糖加入质量:b型流感嗜血杆菌荚膜多糖加入质量为1: (1.5~3); [0015] c. Add pneumococcal capsular saccharide, pneumococcal serotypes includes 7 to 23 serotypes, wherein the capsular saccharide 6B added mass of the remaining capsular saccharide added is 2 times the mass of the pneumococcal capsular saccharide Add quality: Haemophilus influenzae type B capsular polysaccharide was added mass of 1: (1.5 to 3);

[0016] d.按照本领域的常规手段制备肺炎球菌-b型流感嗜血杆菌联合疫苗 [0016] d. Combined vaccine prepared according to conventional methods in the art pneumococcal Haemophilus influenzae type -b

[0017] e.百白破疫苗按照百白破现有疫苗的制备方法进行制备。 [0017] e. DPT vaccine was prepared according to the conventional production method DPT vaccines.

[0018] 优选地,肺炎球菌荚膜糖的血清型中包括:1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、 12卩、14、158、17卩、18(:、19卩、19厶、20、22卩、23卩和/或33卩。 [0018] Preferably, the pneumococcal serotypes capsular saccharide comprising: 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12 Jie, Jie 14,158,17 , 18 (:, Jie 19, Si 19, 20, 22 Jie, Jie 23 and / or 33 Jie.

[0019] 优选地,载体蛋白包括:肺炎球菌溶血蛋白及其改性衍生物,更优选地包括改性肺炎球菌溶血蛋白A A146Ply、肺炎球菌溶血蛋白衍生物dPly;肺炎球菌表面蛋白及其改性衍生物,更优选地包括肺炎球菌表面蛋白A、肺炎球菌表面蛋白C;肺炎球具表面黏附蛋白及其改性衍生物,更优选地包括肺炎球菌表面黏附蛋白A、肺炎球菌表面黏附蛋白C;或肺炎球菌三组氨酸蛋白家族融合蛋白,更优选地包括肺炎球菌三组氨酸蛋白PhtA、PhtB、PhtD、PhtE、 PhtDE〇 [0019] Preferably, carrier proteins include: a modified pneumolysin proteins and derivatives thereof, more preferably a modified pneumolysin protein A A146Ply, pneumolysin protein derivative dPly; pneumococcal surface protein and Modification derivative, more preferably a pneumococcal surface protein A, pneumococcal surface protein C; pneumococcal surface adhesion protein with its modified derivatives thereof, more preferably a pneumococcal surface adhesion protein A, pneumococcal surface adhesion protein C; three or pneumococcal histidine fusion protein family protein, more preferably a histidine three pneumococcal proteins PhtA, PhtB, PhtD, PhtE, PhtDE〇

[0020] 作为一种优选的实施方式,肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗包括肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: [0020] As a preferred embodiment, the pneumococcal Haemophilus influenzae type -b - DTP vaccine comprising a pneumococcus -b Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines, in particular:

[0021] a.肺炎球菌-b型流感嗜血杆菌联合疫苗为血清型包括4、6B、9V、14、18C、19F、23F 的7价肺炎球菌-肺炎球菌表面蛋白A(PspA)与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗; . [0021] a pneumococcal -b Haemophilus influenzae type comprising a combined vaccine serotypes 4,6B, 9V, 14,18C, 7-valent pneumococcal conjugate 19F, 23F - A pneumococcal surface protein A (PspA) and type b Hib - Haemophilus influenzae combination vaccine outer membrane protein D;

[0022] b.无细胞百日咳疫苗为百日咳疫苗原液、白喉类疫苗为白喉类毒素和破伤风类疫苗为破伤风类毒素,且百白破疫苗为悬混液。 [0022] b. Acellular pertussis vaccine is liquid pertussis vaccine, diphtheria vaccine for tetanus toxoid and diphtheria toxoid to tetanus toxoid vaccine, and the suspension was DPT mixed solution.

[0023]作为另一种优选地实施方式,肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: [0023] As another preferred embodiment, pneumococcal -b Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines, in particular:

[0024] 肺炎球菌-b型流感嗜血杆菌联合疫苗为血清型包括4、68、利、14、18(:、19?、23?的7 价肺炎球菌-肺炎球菌表面黏附蛋白A(PsaA)与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 [0024] -b Pneumococcal Haemophilus influenzae type combined vaccine serotypes including 4,68, Lee, 14, 18 (:?? 19, 23 of the 7-valent pneumococcal - pneumococcal surface adhesion protein A (PsaA) and Haemophilus influenzae type b - Hib combination vaccine D outer membrane protein.

[0025]作为另一种优选地实施方式,肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: [0025] As another preferred embodiment, pneumococcal -b Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines, in particular:

[0026] 肺炎球菌-b型流感嗜血杆菌联合疫苗包括4、68、利、14、18(:、19?、23?的7价肺炎球菌-改性肺炎球菌溶血蛋白A A146Ply与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 ?? [0026] -b Pneumococcal Haemophilus influenzae type combination vaccine include 4,68, Lee, 14, 18 (: 19, 23, 7-valent pneumococcal - modified pneumolysin and protein A A146Ply influenzae type b Haemophilus - Haemophilus influenzae combination vaccine D outer membrane protein.

[0027]作为另一种优选地实施方式,本申请中所属的肺炎球菌包括血清型为7价~23价, 肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗还可以为: [0027] As another preferred embodiment, the present application pneumococcal serotypes belong includes 7 to 23 monovalent divalent, -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines may also be for:

[0028] 肺炎球菌-b型流感嗜血杆菌联合疫苗包括1、4、5、68、7?、利、14、18(:、19?、23?的10 价肺炎球菌_PspA/PsaA/AA146Ply或其他上述肺炎球菌载体蛋白与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 [0028] -b Pneumococcal Haemophilus influenzae type combination vaccine includes 1,4,5,68,7 ?, Lee, 14, 18 (:?? 19, 23 of the 10-valent pneumococcal _PspA / PsaA / AA146Ply or other carrier protein and above pneumococcal Haemophilus influenzae type b - Hib combination vaccine D outer membrane protein.

[0029]作为另一种优选地实施方式,本申请中所属的肺炎球菌包括血清型为7价~23价, 肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗还可以为: [0029] As another preferred embodiment, the present application pneumococcal serotypes belong includes 7 to 23 monovalent divalent, -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines may also be for:

[0030] 肺炎球菌-b型流感嗜血杆菌联合疫苗包括1、3、4、5、6A、6B、7F、9V、14、18C、19A、 19F、23F的13价肺炎球菌-PspA/PsaA/AA146Ply或其他上述肺炎球菌载体蛋白与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 [0030] -b pneumococcal Haemophilus influenzae type combined vaccine comprising 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 13-valent pneumococcal 23F of -PspA / PsaA / AA146Ply above pneumococcal or other carrier protein with Haemophilus influenzae type b - Hib combination vaccine D outer membrane protein.

[0031]作为另一种优选地实施方式,本申请中所属的肺炎球菌包括血清型为7价~23价, 肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗还可以为: [0031] As another preferred embodiment, the present application pneumococcal serotypes belong includes 7 to 23 monovalent divalent, -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine combination vaccines may also be for:

[0032] 肺炎球菌-b型流感嗜血杆菌联合疫苗包括1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、 12F、14、15B、17F、18C、19F、19厶、20、22卩、23卩和33卩的23价肺炎球菌-?8口厶/?83厶/^厶146卩17 或其他上述肺炎球菌载体蛋白与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 [0032] -b Pneumococcal Haemophilus influenzae type combination vaccine includes 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19F , 19 Si, Jie 20, 22, 23 and 33 Jie Jie 23-valent pneumococcal -?? 8 Si / Si 83 / Si 146 ^ 17 Jie pneumococcal above or other carrier protein of Haemophilus influenzae type b - influenza D combined vaccine Haemophilus outer membrane protein.

[0033] 本发明的另一发明目的在于保护一种由上述肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法所制备得到的联合疫苗。 [0033] Another object of the present invention is protected by the one kind of Streptococcus pneumoniae, Haemophilus influenzae type -b - DTP vaccine preparation prepared combination vaccine obtained.

[0034] 优选地,肺炎球菌-b型流感嗜血杆菌联合疫苗为冻干剂型,用于作为肺炎球菌疫苗的冻干制剂的冻干保护剂的为蔗糖。 [0034] Preferably, -b pneumococcal Haemophilus influenzae type combined vaccine is lyophilized dosage form, a lyophilized formulation as lyoprotectant pneumococcal vaccines is sucrose.

[0035] 更优选地,蔗糖在冻干原液中的初始浓度不大于20 % ;蔗糖的初始浓度不小于60%,优选为大于70 %。 [0035] More preferably, the initial concentration of sucrose stock solution is lyophilized in less than 20%; the initial concentration of sucrose is not less than 60%, preferably greater than 70%.

[0036] 优选地,蔗糖在冻干原液中的初始质量百分含量为不大于20 % ;较佳地,为4~ 20% ;作为一种较佳的实施方式,根据所需制备的疫苗的不同为7~10%、8~10%、10~ 15%或12~15%;最佳为7%、8%、10%或12%。 [0036] Preferably, the mass percentage of the initial stock solution is lyophilized in sucrose not more than 20%; preferably, 4 to 20%; as a preferred embodiment, the preparation of the vaccine according to the desired different from 7 to 10%, 8-10%, 10-15%, or 12-15%; most preferably 7%, 8%, 10%, or 12%.

[0037] 优选地,蔗糖选用工业级分析纯或药用级。 [0037] Preferably, the choice of sugar or industrial analytical pharmaceutical grade.

[0038] 进一步优选地,肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗在临用前将百白破悬混液加入肺炎球菌_b型流感嗜血杆菌联合疫苗冻干制剂中,将肺炎球菌-b型流感嗜血杆菌联合疫苗复溶,混合均匀后肌肉注射。 [0038] Further preferably, pneumococcal Haemophilus influenzae type -b - DPT vaccine immediately prior to the DPT mixed suspension was added _b pneumoniae Haemophilus influenzae type combined vaccine lyophilized formulation, the pneumococcal Haemophilus influenzae type -b combined vaccine reconstituted after mixed intramuscular injection.

[0039] 优选地,肺炎球菌多疫苗的血清抗体水平彡0.35yg/mL,b型流感嗜血杆菌的血清抗体水平彡〇• 15yg/mL。 [0039] Preferably, the multi pneumococcal vaccine serum antibody levels San 0.35yg / mL, b Haemophilus influenzae type levels of serum antibody San square • 15yg / mL.

[0040] 更优选地,无细胞百日咳疫苗效价多4.0IU;白喉疫苗效价多30IU;破伤风疫苗效价彡40IU。 [0040] More preferably, acellular pertussis vaccine potency plurality 4.0 IU; diphtheria titers plurality 30IU; tetanus titers San 40IU.

[0041] 与现有技术相比,本发明首创的实现了可同时实现包括肺炎球菌在内的五种抗原的同时免疫的肺炎球菌_b型流感嗜血杆菌-百白破联合疫苗,其中肺炎球菌疫苗的蛋白-荚膜糖结合体可在不同的血清型间起到诱导交叉免疫保护作用;并且蛋白本身的作为毒力因子可诱导更强的免疫保护作用,对荚膜糖在免疫上起补充作用,从而增强了疫苗的免疫保护作用;同时肺炎球菌的自身蛋白可以避免与其他疫苗如白喉疫苗、破伤风疫苗在体内产生免疫冲突,使得本结合疫苗的实现成为了可能。 [0041] Compared with the prior art, the present invention achieves simultaneously the first five antigens can be achieved, including pneumococcal including simultaneous immunization pneumococcal Haemophilus influenzae type _b - DTP vaccine, wherein pneumonia protein conjugate vaccine - capsular saccharide conjugates may function between different serotypes induce cross-protective immunity; and the protein itself as a virulence factor may induce a stronger immune protection, on the capsular saccharide of immunization, complementary role, thereby enhancing the protective effect of the vaccine immunization; while the pneumococcal protein itself can be avoided with other vaccines such as diphtheria, tetanus immune conflict in the body, making the combined vaccine achieved as possible. 本结合疫苗提高了接种者的肺炎球菌感染的免疫记忆应答;肺炎球菌蛋白的加入使得肺炎球菌的免疫需要依赖胸腺免疫,因此使得肺炎球菌的免疫实现了全年龄段及全部血清型的长效、全覆盖式免疫;肺炎球菌荚膜糖与肺炎球菌蛋白偶联之后提高了体内免疫应答的速度及扩展了其免疫的深度和广度,二者之间的协同作用虽然没有单独每种疫苗的效果好,但减少了接种次数,提高了接种效率,减轻了被接种者的身体负担。 This combination vaccine to improve the immune memory pneumococcal infection inoculate's response; adding pneumococcal proteins of the immune pneumococcus need to rely on thymus immunity, thus making the implementation of the pneumococcal immunization of all ages and all serotypes of the long-term, full coverage immunization; after pneumococcal capsular saccharide conjugated to a pneumococcal protein increases the speed of an immune response and extended the depth and breadth of the immune, although good synergy between the two effects is not each individual vaccine , but reduce the number of vaccination, inoculation improved efficiency, reduce the body burden of vaccinated persons. 实验数据表明,本发明中的肺炎球菌及b型流感嗜血杆菌的荚膜糖加入顺序对本联合疫苗的制备成功与否十分重要,其中先加入肺炎球菌荚膜糖的情况下由于其可与两种载体蛋白均发生随机缀合,因此无法实现b型嗜血杆菌疫苗的制备目的,只有在先加入b型流感嗜血杆菌荚膜糖或两种荚膜糖同时加入但b型流感嗜血杆菌远过量于肺炎球菌荚膜糖时,才能够实现两种疫苗的同时实现,加之在使用时可以配合百白破疫苗, 实现了五联疫苗的同时免疫。 Experimental data show that, due to its lower and two in the present invention pneumococcus and Haemophilus influenzae type b capsular saccharide order of addition of the success of the combination vaccine preparation is very important or not, which is added to the case of pneumococcal capsular saccharide random conjugated to a carrier protein species occurred, and therefore can not achieve the purpose of the preparation of Haemophilus type b vaccine, only added prior Haemophilus influenzae type b capsular saccharide or both added simultaneously but capsular saccharide of Haemophilus influenzae type b when far in excess of the pneumococcal capsular saccharides to be able to achieve the realization of the two vaccines at the same time, combined with the use of DPT vaccine can be realized at the same time the immune pentavalent vaccine. 肺炎球菌与b型流感嗜血杆菌疫苗、百白破疫苗的联合从源头上解决了一系列下呼吸道感染性疾病,随着肺炎球菌蛋白荚膜糖结合疫苗的不断发展,该联合疫苗具有广泛的推广和实用价值,对肺炎球菌及相关疫苗的研究有着深远的影响。 Streptococcus pneumoniae and Haemophilus influenzae type b vaccine, DPT vaccine jointly solve a series of lower respiratory tract infections from the source, with the pneumococcal capsular saccharide binding protein evolving vaccine, the combination vaccine has a wide range of promotion and practical value, has a profound impact on research and related pneumococcal vaccine.

具体实施方式 Detailed ways

[0042] 为了能够更清楚地描述本发明的技术内容,下面结合具体实施例来进行进一步的描述。 [0042] In order to more clearly describe the technical contents of the present invention, the following embodiments in conjunction with the specific embodiments to be further described.

[0043] 本发明中由于使用肺炎球菌自身蛋白作为肺炎球菌荚膜糖的载体蛋白,因此解决了肺炎球菌荚膜糖与b型流感嗜血杆菌荚膜多糖无法温度共存的技术问题,故下列实施例中仅对本发明内容涉及改进的部分进行详述,其他未提及步骤或原料来源同现有技术,在此不做赘述。 [0043] In the present invention, the use of pneumococcal protein itself as a pneumococcal capsular saccharide to a carrier protein, thus solving the technical problem pneumococcal capsular saccharide of Haemophilus influenzae type b capsular polysaccharide temperature can not coexist, so the following examples SUMMARY embodiment of the present invention relates to improved only partially be described in detail, other sources of raw materials or steps not mentioned with prior art, which is not repeated herein.

[0044] 实施例1 [0044] Example 1

[0045] 1.制备肺炎球菌荚膜多糖 [0045] 1. Preparation of pneumococcal capsular polysaccharide

[0046] a.取现有的7价肺炎球菌的常见致病血清型4、68、利、14、18(:、19?及23?的肺炎球菌培养; . [0046] a conventional take the 7-valent pneumococcal pathogenic serotypes 4,68, Lee, 14, 18 (:, 19 and 23 of the pneumococcal culture;??

[0047] b.分别提纯以上各种血清型肺炎球菌中抗原性强的荚膜多糖4、9V、14、19F&23F, 寡糖18C及多糖6B; . [0047] b above was separately purified pneumococcal serotypes Strongly antigenic capsular polysaccharide 4,9V, 14,19F & 23F, 18C oligosaccharides and polysaccharides 6B;

[0048] c.肺炎球菌灭活后离心收集上清液,经超滤浓缩,根据各肺炎球菌血清型特性分别加入适量(体积分数为70%的)预冷乙醇,离心收集,得粗制荚膜糖;将粗制荚膜糖溶于乙酸钠溶液中,然后按1:2比例以冷酚混匀,离心去除蛋白,反复酚提5-6次,收集上清,用蒸馏水透析,透析后液体加2mol/L氯化钙溶液,加入乙醇搅拌,离心去除核酸,收集上清,补加乙醇搅拌(终浓度80% ),离心收集沉淀,用乙醇、丙酮洗涤沉淀,脱水干燥后得精制荚膜糖, 置-20 °C保存备用。 [0048] c. After inactivation pneumococcal centrifugation supernatant was collected and concentrated by ultrafiltration, according to each of the pneumococcal serotypes characteristic amount added (volume fraction of 70%) were pre-cooled ethanol, collected by centrifugation, to obtain the crude pods after 2 phenol cold mixing ratio, protein is removed by centrifugation, repeatedly mention phenol 5-6 times, the supernatant was collected, dialyzed against distilled water, dialyzed: crude capsular saccharide was dissolved in sodium acetate solution, and then press 1; membrane glycoprotein liquid added 2mol / L calcium chloride solution, ethanol was added with stirring, the nucleic acid is removed by centrifugation, the supernatant was collected, supplemented with stirring ethanol (80% final concentration), collected by centrifugation, the precipitate was washed with ethanol, acetone, dehydrated and dried to give purified pods membrane glycoprotein, is set at -20 ° C for use.

[0049] 2.制备肺炎球菌表达的高保守性并具有免疫原性蛋白A A146Ply And having a highly conserved immunogenic protein A A146Ply [0049] 2. Preparation of pneumococcal Expression

[0050] 根据GenBank公布的Ply基因(序列登录号:X52474),改性肺炎球菌溶血蛋白A A146Ply的设计引物序列如下: [0050] The Ply gene from GenBank (sequence accession number: X52474), modified pneumolysin protein A A146Ply the primers were designed as follows:

[0051] [0051]

Figure CN106039301AD00091

[0052] 其中划线部分为相应的限制性内切酶酶切位点。 [0052] wherein the underlined part endonuclease restriction sites within the corresponding restriction.

[0053] 根据常规实验方法将A A146Ply基因PCR扩增后,1 %凝胶电泳,回收目标DNA片段, 将目标DNA片段连接入pET-28a质粒中,在大肠杆菌感受态细胞BL21内转化抽提重组DNA质粒,IPTG诱导表达后进行SDS-PAGE凝胶电泳鉴定,确定表达量及表达形式后进行Ni-NTA树脂纯化;采用0.2%福尔马林溶液去除内毒素,即溶血活性,得到去除内毒素后的AA146Ply 蛋白。 [0053] The routine experimentation A A146Ply gene after PCR amplification, 1% gel electrophoresis to recover DNA fragment of the target DNA fragment was ligated into pET-28a plasmid, transformed in E. coli extracts competent cells BL21 the recombinant DNA plasmid, after IPTG induction by SDS-PAGE gel electrophoresis, purified by Ni-NTA resin is determined after expression and expression forms; endotoxin removal using 0.2% formalin solution, hemolytic activity i.e., removed to give the AA146Ply the protein toxin. A A146Ply蛋白为Ply蛋白第146位丙氨酸缺失的改性蛋白,蛋白纯度达到80%以上, 检测证明具有免疫原性且残留的内毒素含量低于0. lEU/yg。 A A146Ply protein is a protein of 146 Ply alanine deleted modified protein, protein purity of more than 80%, and the detection proved immunogenic residual endotoxin content is less than 0. lEU / yg.

[0054] 3.制备b型流感嗜血杆菌荚膜多糖 [0054] 3. Preparation of Haemophilus influenzae type b capsular polysaccharide

[0055] a.采用CY培养基对Hib菌株1842在37 °C下进行发酵,发酵液经甲醛灭活,离心取上清后,利用十六烷基三甲基溴化铵(CTAB)进行沉淀。 [0055] a. CY medium employed for fermentation Hib strain 1842 at 37 ° C, formalin-inactivated broth, supernatant after centrifugation, using cetyl trimethyl ammonium bromide (CTAB) precipitation . 然后用0.5~1M的NaCl对复合物沉淀进行解离,添加冰乙醇至终浓度25% (v/v),沉淀核酸,然后加冰乙醇至终浓度75% (v/v),沉淀得到粗多糖。 NaCl 0.5 ~ 1M then the precipitate dissociated complexes, ice-cold ethanol was added to a final concentration of 25% (v / v), nucleic acid precipitation, and then ice to a final concentration ethanol 75% (v / v), to give a crude precipitate polysaccharide.

[0056] b.上述粗多糖用醋酸钠溶液溶解,冷酚抽提5-6次,最后超滤浓缩去除苯酚残留, 经75 % (v/v)乙醇沉淀得到精糖,置-20 °C保存备用。 [0056] b. The crude polysaccharide with sodium acetate solution was dissolved, cold phenol extraction 5-6 times, concentrated by ultrafiltration and finally removing the residual phenol by 75% (v / v) ethanol precipitation to give refined sugar, is set at -20 ° C spare.

[0057] 4.制备b型流感嗜血杆菌表面蛋白HiD [0057] 4. Preparation of Haemophilus influenzae type b surface protein HiD

[0058]将HiD蛋白克隆至大肠杆菌进行表达并分离纯化,具体包括: [0058] The HiD protein and cloned into E. coli Expression Purification, comprises:

[0059] a.目的基因的优化和重组表达质粒的构建 [0059] a. Optimization of the gene of interest and a recombinant expression plasmid

[0060] 在GenBank中获取HiD基因序列并进行优化,加上Hi S标签后进行全基因合成,将合成的序列经SacI和Ndel双酶切后,定向克隆至同样双酶切的表达载体pET_30a( + )中,转化感受态大肠杆菌BL21Star(DE3),37°C过夜培养,挑取阳性单克隆菌落,扩增培养后提取质粒,用Ndel和SacI双酶切鉴定,并送测序,将测序正确的重组表达质粒命名为pET-30a_HiD。 [0060] Gets HiD gene sequences in GenBank and optimized, with the full gene synthesis Hi S label, the synthesized sequences Ndel digested by SacI and cloned into the same expression vector double digested pET_30a ( +), the transform competent E. coli BL21Star (DE3), 37 ° C overnight culture, monoclonal positive colonies were picked, plasmid amplification incubation after extraction, identification and SacI double digestion with Ndel, and sent to sequencing, sequenced the recombinant plasmid was named pET-30a_HiD. [0061] b.重组蛋白的诱导表达及纯化 [0061] b. Expression induction and purification of recombinant proteins

[0062] 复苏工程菌,以1:100的比例接种2 X LB培养基,在37 °C,237r/min下扩大培养,当菌体值约为12时,加入IPTG至终浓度为lmmol/L,37°C诱导4h,取样进行SDS-PAGE分析离心收集诱导菌体,加入生理盐水重悬洗涤2次,以1:10(g/mL)的比例加入05mol/LNaC15mmol/L 咪唑20mmol/Lro(pH7.4)的缓冲液重悬菌体,超声波破碎菌体,8000 X g离心40min,收集上清,于镍离子层析柱中进行纯化,按说明书操作纯化产物的及分析将载体pET-43. la( + )转化的大肠杆菌BL21Star(DE3)全菌体(对照)和将上步纯化样品经SDS-PAGE分离后电转移至硝酸纤维素膜上,用5%脱脂奶粉摇床轻微振荡封闭2h;加入His鼠源单抗(1:800稀释),4°C 过夜;TBST清洗3次,加入HRP标记的羊抗鼠IgG(l: 2000稀释),室温孵育lh;洗涤3次,DAB显色。 [0062] recovery of engineering bacteria, to 1: 100 ratio of 2 X LB was inoculated medium, expanded and cultured at 37 ° C, 237r / min, when the cell value of about 12, IPTG was added to a final concentration of lmmol / L , 37 ° C induction 4h, samples were taken for SDS-PAGE analysis induced cells were collected by centrifugation, washed with physiological saline twice resuspended, at a ratio of 1:10 (g / mL) was added 05mol / LNaC15mmol / L imidazole 20mmol / Lro ( pH7.4) buffer resuspended cells, cell sonication, 8000 X g centrifugation 40min, the supernatant was collected, and purified on nickel ion chromatography column, by manual operation and analysis of the purified product vector pET-43 . la (+) transformed E. coli BL21 Star (DE3) whole-cell (control) and the samples were separated on further purification by SDS-PAGE electro-transferred to nitrocellulose membranes, blocked with 5% skim milk blocking shaker with gentle shaking 2H; His murine monoclonal antibody was added (1: 800 dilution), 4 ° C overnight; washing three times with TBST, HRP-labeled goat anti-mouse IgG (l: 2000 dilution) and incubated for lh at room temperature; washed three times, DAB significant color.

[0063] 5.多糖-蛋白缀合,制备肺炎球菌-b型流感嗜血杆菌结合疫苗 [0063] The polysaccharide - protein conjugated pneumococcal prepared -b Haemophilus influenzae type conjugate vaccine

[0064] a.将纯化备用的A A146Ply及Hid以质量比1:1的比例加入制备体系中; . [0064] a purified and spare Hid A A146Ply mass ratio of 1: 1 ratio was added in the preparation of the system;

[0065] b.加入b型流感嗜血杆菌荚膜多糖,加入的荚膜多糖与Hid质量比为5:1; . [0065] b added Haemophilus influenzae type b capsular polysaccharide, capsular polysaccharide and Hid added mass ratio of 5: 1;

[0066] c.加入7价肺炎球菌荚膜糖,其中6B型荚膜糖的加入质量是其余荚膜糖加入质量的2倍,肺炎球菌荚膜糖加入质量:b型流感嗜血杆菌荚膜多糖加入质量为1:1.5。 [0066] c was added 7-valent pneumococcal capsular saccharide, wherein the capsular saccharide 6B added mass of the remaining capsular saccharide added is 2 times the mass, added mass pneumococcal capsular saccharide:. B capsular Haemophilus influenzae polysaccharide added mass of 1: 1.5.

[0067]以制备10mL疫苗原液为例,加入荚膜多糖4、9¥、14、19?及23?各40辟,寡糖1802邱及多糖6B 80yg,Hib荚膜多糖60yg;Hid蛋白12yg; AA146Ply蛋白12yg。 [0067] to prepare 10mL of vaccine, for example, adding capsular polysaccharide 4,9 ¥, 14,19 and 23 each provision 40, 1802 Qiu oligosaccharides and polysaccharides 6B 80yg, Hib capsular polysaccharide 60yg;?? Hid protein 12yg; AA146Ply protein 12yg. 经过凝胶层析柱纯化后进行高效液相色谱分析该联合疫苗,结果显示所有血清型均有明显的色谱峰,相邻两峰之间分离度R>1.5,拖尾因子T的范围为0.95~1.05,并且荚膜糖含量达到《中华人民共和国药典》2010版及本申请发明内容中的最低要求含量。 After gel purification by column chromatography the combination vaccine HPLC analysis showed all serotypes had significant peaks, the degree of separation between two adjacent peaks R> 1.5, tailing factor range of 0.95 to T 1.05, and capsular sugar content content content meet the minimum requirements "Chinese Pharmacopoeia" 2010 version of this application and invention.

[0068]需要指出的是,由于肺炎球菌蛋白与肺炎球菌荚膜糖、b性流感嗜血杆菌与Hid是同源的,因此二者之间具有协同作用,但本发明中的蛋白与荚膜糖之间的缀合不仅限于上述氨基还原法,只要是能实现该偶联结果的方法均应包括在本申请的发明范围中。 [0068] It should be noted that, due to the pneumococcal proteins pneumococcal capsular saccharide, b Haemophilus influenzae and Hid is homologous, thus having a synergistic effect between the two, but in the present invention capsular protein conjugation between the amino sugar is not limited to the above-described reduction method, a method can be realized as long as the result of the conjugation should be included within the scope of the invention in the present application.

[0069] 6.制备百白破疫苗 [0069] 6. Preparation of DPT

[0070] 由于本发明中的联合疫苗以肺炎球菌疫苗与b型流感嗜血杆菌疫苗、百白破疫苗减少相互影响,并且可以同时产生免疫为主要改进方向,故与本实施例中可采用现有技术制备百白破疫苗,具体方法在此不作赘述。 [0070] Since the present invention is combined vaccine pneumococcal vaccine and Haemophilus influenzae type b vaccine, DPT vaccine reduced interaction, and may improve the immune main directions simultaneously, it may be employed with the present embodiment is now there DPT vaccine preparation techniques, particularly methods not described herein. 但需要注意的是,本实施例中的百白破疫苗中无细胞百日咳疫苗效价多4.0IU,白喉疫苗效价多30IU且破伤风疫苗效价多40IU,且优选为小水针剂型,其中含无细胞百日咳疫苗效价不低于4.0IU,白喉疫苗效价不低于30IU,破伤风疫苗效价不低于40IU。 Note, however, that the present embodiment DPT vaccine acellular pertussis vaccine potency multi 4.0 IU, diphtheria and tetanus titers plurality 30IU vaccine potency 40IU plurality, and preferably small water injection type, wherein acellular pertussis vaccine containing a titer of not less than 4.0IU, diphtheria titers of not less than 30IU, tetanus titers of not less than 40IU.

[0071] 将上述分别获得的肺炎球菌-b型流感嗜血杆菌联合疫苗和百白破疫苗留用,以《中华人民共和国药典》2010版中要求分别进行免疫原性、阳转率等疫苗效价评估实验,具体实验结果见后述。 [0071] The above obtained respectively -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine retained to "Chinese Pharmacopoeia" 2010 edition claims were immunogenic, seroconversion rate vaccine potency evaluation experiments, specific experimental results will be described later.

[0072] 实施例2 [0072] Example 2

[0073]本实施例与实施例1的区别仅在于:肺炎球菌-b型流感嗜血杆菌联合疫苗为冻干剂型,其中蔗糖作为冻干骨架,其中蔗糖在冻干原液中的初始质量百分含量不大于20%,冻干后得到肺炎球菌_b型流感嗜血杆菌联合疫苗冻干制剂。 [0073] The present embodiment differs from Embodiment 1 only in that: Streptococcus pneumoniae Haemophilus influenzae type -b combination vaccine is lyophilized dosage form, wherein sucrose as a lyophilized skeleton, wherein the initial mass of sucrose during lyophilization dope percent content of not more than 20%, and lyophilized to give _b pneumococcal Haemophilus influenzae type combined vaccine lyophilized preparation. 故本实施例与实施例1的进一步不同在于,百白破疫苗可作为肺炎球菌_b型流感嗜血杆菌联合冻干疫苗的复溶液,即临用前将百白破悬混液加入肺炎球菌_b型流感嗜血杆菌冻干制剂中,复溶,混合均匀后肌肉注射。 Therefore, this further embodiment differs from Example 1 in that the DPT vaccine pneumococcal _b as Haemophilus influenzae type complex solution was lyophilized combined vaccine, i.e., immediately prior to use DPT mixed suspension was added pneumococcal _ Haemophilus influenzae type b lyophilized formulation, the reconstituted mixed uniformly intramuscular injection.

[0074]冻干步骤具体为: [0074] The lyophilization step is specifically:

[0075] 1.配制浓度为70%、经121°C灭菌处理15min的蔗糖母液; [0075] 1. Preparation of 70% over 121 ° C for 15min sterilized sucrose liquor;

[0076] 2.将实施例1所得分离纯化的肺炎球菌_b型流感嗜血杆菌联合疫苗置于无菌容器内,加入步骤1配制成的蔗糖母液,使蔗糖浓度至10%,得混匀制备成含有蔗糖保护剂的病毒原液半成品,再分别以1.0ml/瓶的规格灌装于西林瓶内以进行后续的冻干工艺; [0076] 2. The influenza pneumococcal _b obtained in Example 1 Purification of Haemophilus combined vaccine was placed in sterile containers, the step of adding a sucrose formulated mother liquor to a concentration of 10% sucrose, and mix to give prepared as a semi-finished virus stock containing sucrose protecting agent, respectively, and then 1.0ml / bottle to filling specification resistant bottle for subsequent freeze-drying process;

[0077] 3.将步骤2所得半成品进行预冻,预冻时快速降温至_55°C,维持15h_20h; [0077] 3. Step 2 The resulting semi-finished product pre-frozen, pre-freezing during rapid cooling to _55 ° C, maintained 15h_20h;

[0078] 4.将步骤4所得预冻后半成品进行第一阶段干燥:升温至_45°C~_40°C维持50h, 升温至-40°C~_33°C维持22h; [0078] 4. Step 4 The resulting semi-finished product of the first stage pre-freeze drying: temperature was raised to _45 ° C ~ _40 ° C to maintain 50h, warmed to -40 ° C ~ _33 ° C to maintain 22h;

[0079] 5.将步骤4所得第一阶段干燥后半成品进行第二阶段干燥:设定不同时间内升温至0°C~30°C,不同升温阶段继续维持28h;再制备冻干原液中蔗糖浓度为10%的肺炎球菌-b型流感嗜血杆菌联合疫苗,冻干疫苗的制备方法可参考现有技术制备,在此不做赘述。 [0079] Step 5. 4 obtained after the first stage the second stage of drying semifinished dried: setting different times to warm to 0 ° C ~ 30 ° C, the different heating stages continuation 28H; then lyophilizing liquid sucrose concentration of 10% of pneumococcal -b Haemophilus influenzae type combined vaccine, the vaccine can be lyophilized preparation prepared with reference to the prior art, and is not repeated herein.

[0080] 将上述分别获得的肺炎球菌-b型流感嗜血杆菌联合疫苗和百白破疫苗留用,以《中华人民共和国药典》2010版中要求分别进行免疫原性、阳转率等疫苗效价评估实验,具体实验结果见后述。 [0080] The above were obtained -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine retained to "Chinese Pharmacopoeia" 2010 version were required immunogenicity, seroconversion rate and other vaccine potency evaluation experiments, specific experimental results will be described later.

[0081 ] 实施例3 [0081] Example 3

[0082] 本实施例与实施例1的区别在于,本实施例中的多糖添加顺序为先添加肺炎球菌荚膜糖,再添加b型流感嗜血杆菌荚膜多糖。 [0082] The present embodiment differs from Embodiment 1 in that the embodiment in order to add polysaccharide pneumococcal capsular saccharide added first, then add Haemophilus influenzae type b capsular polysaccharide of the present embodiment. 顺序调换后b型流感嗜血杆菌荚膜多糖无色谱峰,证明了肺炎球菌荚膜糖可以以Hid为载体蛋白进行缀合,肺炎球菌先加入或两种多糖按原比例同时加入会形成竞争关系,影响b型流感嗜血杆菌荚膜多糖的正常缀合过程,因此无法实现本技术方案。 After the order transposing Haemophilus influenzae type b capsular polysaccharide without peaks proved pneumococcal capsular saccharide may be conjugated to a carrier protein is Hid, added first, or both pneumococcal polysaccharide ratio of the original will be added simultaneously in competition Effect normal Haemophilus influenzae type b capsular polysaccharide conjugated to process, and therefore can not present technical solution.

[0083] 实施例4 [0083] Example 4

[0084] 本实施例与实施例3的区别在于,本实施例中的b型流感嗜血杆菌荚膜多糖与肺炎球菌荚膜糖同时加入,Hib:ps质量比为(4~10): 1,即b型流感嗜血杆菌荚膜多糖远过量于肺炎球菌荚膜糖,在同时加入缀合体系中时,竞争关系被质量悬殊弱化。 [0084] This Example is Example 3 except that, in the present embodiment, Haemophilus influenzae type b capsular polysaccharide pneumococcal capsular saccharide added simultaneously, Hib: ps is a mass ratio (4 to 10): 1 that Haemophilus influenzae type b capsular polysaccharide pneumococcal capsular far in excess of sugar, while at the same time adding conjugated system, competition is weakened poor quality. 经过凝胶层析柱纯化后进行高效液相色谱分析该联合疫苗,结果显示所有血清型均有明显的色谱峰,相邻两峰之间分离度R>1.5,拖尾因子T的范围为0.95~1.05,,并且荚膜糖含量达到《中华人民共和国药典》2010版及本申请发明内容中的最低要求含量。 After gel purification by column chromatography the combination vaccine HPLC analysis showed all serotypes had significant peaks, the degree of separation between two adjacent peaks R> 1.5, tailing factor range of 0.95 to T 1.05 ,, and capsular sugar content content content meet the minimum requirements "Chinese Pharmacopoeia" 2010 version of this application and invention.

[0085] 将上述分别获得的肺炎球菌-b型流感嗜血杆菌联合疫苗和百白破疫苗留用,以《中华人民共和国药典》2010版中要求分别进行稳定性、免疫原性等疫苗效价评估实验,具体实验结果见后述。 [0085] The above obtained respectively -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine retained to "Chinese Pharmacopoeia" 2010 Edition required stability, immunogenicity, etc. Vaccine potency assessed separately experiments, specific experimental results will be described later.

[0086] 实施例5 [0086] Example 5

[0087] 本实施例与实施例1的区别在于,肺炎球菌载体蛋白不同。 [0087] The present embodiment differs from the embodiment in Example 1 in that the pneumococcal different carrier proteins. 本实施例中使用的蛋白载体为肺炎球菌表面黏附蛋白A(PsaA)。 Protein carrier used in this embodiment is a pneumococcal surface adhesion protein A (PsaA). 其余步骤相同,在此不赘述。 The remaining steps, which is not repeated herein.

[0088] 制备肺炎球菌表达的高保守性并具有免疫原性蛋白肺炎球菌表面黏附蛋白A (PsaA) [0088] Expression of pneumococcal prepared having highly conserved immunogenic protein and pneumococcal surface adhesion protein A (PsaA)

[0089] 根据GenBank公布的PsaA基因(序列登录号:U53509)设计引物序列如下: [0089] The PsaA gene from GenBank (sequence accession number: U53509) primers were designed as follows:

[0090] [0090]

Figure CN106039301AD00111

[0091 ]其中划线部分为相应的限制性内切酶酶切位点。 [0091] wherein the underlined part endonuclease restriction sites within the corresponding restriction.

[0092]根据常规实验方法将PsaA基因PCR扩增后,1 %凝胶电泳,回收目标DNA片段,将目标DNA片段连接入pET-28a质粒中,在大肠杆菌感受态细胞BL21内转化抽提重组DNA质粒, IPTG诱导表达后进行SDS-PAGE凝胶电泳鉴定,确定表达量及表达形式后进行Ni-NTA树脂纯化;得到PsaA蛋白,测试浓度,蛋白纯度达到80 %以上。 [0092] The routine experimentation PsaA gene after PCR amplification, 1% gel electrophoresis, DNA fragment recovered, ligated into the plasmid pET-28a DNA fragment, in the recombinant E. coli transformed extracted competent cells BL21 plasmid DNA, after IPTG induction by SDS-PAGE gel electrophoresis, purified by Ni-NTA resin is determined after expression and expression of the form; obtained PsaA protein concentrations tested, more than 80% protein purity.

[0093]经过凝胶层析柱纯化后进行高效液相色谱分析该联合疫苗,结果显示所有血清型均有明显的色谱峰,相邻两峰之间分离度R> 1.5,拖尾因子T的范围为0.95~1.05,,并且荚膜糖含量达到《中华人民共和国药典》2010版及本申请发明内容中的最低要求含量。 [0093] After gel purification by column chromatography HPLC analysis of the combined vaccine showed all serotypes had significant peaks, the degree of separation between two adjacent peaks R> 1.5, the range of tailing factor T ,, 0.95 to 1.05 and the content of capsular saccharide content meet the minimum content of "Chinese Pharmacopoeia" 2010 edition of the present application and invention.

[0094] 将上述分别获得的肺炎球菌-b型流感嗜血杆菌联合疫苗和百白破疫苗留用,以《中华人民共和国药典》2010版中要求分别进行稳定性、免疫原性等疫苗效价评估实验,具体实验结果见后述。 [0094] The above obtained respectively -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine retained to "Chinese Pharmacopoeia" 2010 Edition required stability, immunogenicity, etc. Vaccine potency assessed separately experiments, specific experimental results will be described later.

[0095] 实施例6 [0095] Example 6

[0096] 本实施例与实施例5的区别仅在于,肺炎球菌-b型流感嗜血杆菌联合疫苗为冻干剂型,其中蔗糖作为冻干骨架,其中蔗糖在冻干原液中的初始质量百分含量不大于20%,冻干后得到肺炎球菌_b型流感嗜血杆菌联合疫苗冻干制剂。 [0096] The present embodiment differs only in Example 5, -b pneumoniae Haemophilus influenzae type combined vaccine is lyophilized dosage form, wherein sucrose as a lyophilized skeleton, wherein the initial mass of sucrose during lyophilization dope percent content of not more than 20%, and lyophilized to give _b pneumococcal Haemophilus influenzae type combined vaccine lyophilized preparation. 故本实施例与实施例1的进一步不同在于,百白破疫苗可作为肺炎球菌-b型流感嗜血杆菌联合冻干疫苗的复溶液,即临用前将百白破悬混液加入肺炎球菌-b型流感嗜血杆菌冻干制剂中,复溶,混合均匀后肌肉注射。 Therefore, a further embodiment of the present embodiment is different from Example 1 in that, as a DPT vaccine pneumococcal -b lyophilized Haemophilus influenzae type combined vaccine complex solution, i.e., immediately prior to use DPT mixed suspension was added pneumococcal - Haemophilus influenzae type b lyophilized formulation, the reconstituted mixed uniformly intramuscular injection. [0097]经过凝胶层析柱纯化后进行高效液相色谱分析该联合疫苗,结果显示所有血清型均有明显的色谱峰,相邻两峰之间分离度R> 1.5,拖尾因子T的范围为0.95~1.05,,并且荚膜糖含量达到《中华人民共和国药典》2010版及本申请发明内容中的最低要求含量。 [0097] After gel purification by column chromatography HPLC analysis of the combined vaccine showed all serotypes had significant peaks, the degree of separation between two adjacent peaks R> 1.5, the range of tailing factor T ,, 0.95 to 1.05 and the content of capsular saccharide content meet the minimum content of "Chinese Pharmacopoeia" 2010 edition of the present application and invention.

[0098] 将上述分别获得的肺炎球菌-b型流感嗜血杆菌联合疫苗和百白破疫苗留用,以《中华人民共和国药典》2010版中要求分别进行稳定性、免疫原性等疫苗效价评估实验,具体实验结果见后述。 [0098] The above obtained respectively -b pneumococcal Haemophilus influenzae type combined vaccine and DPT vaccine retained to "Chinese Pharmacopoeia" 2010 Edition required stability, immunogenicity, etc. Vaccine potency assessed separately experiments, specific experimental results will be described later.

[0099] 对比例1 [0099] Comparative Example 1

[0100] 采用市售七价肺炎球菌荚膜糖-蛋白缀合疫苗(商品名:沛儿)作为对比例,其用量及使用方法同说明书,同上述各实施例所得疫苗一并进行疫苗效价评估实验,具体实验结果见后述。 [0100] using a commercially available heptavalent pneumococcal capsular saccharide - protein conjugate vaccine (trade name: Pei children) as a comparative example, an amount, and to use the same instructions, performed together with the above-described embodiments resulting vaccine vaccine potency evaluation experiments, specific experimental results will be described later.

[0101] 对比例2 [0101] Comparative Example 2

[0102] 采用市售b型流感嗜血杆菌结合疫苗(商品名:安尔宝)作为对比例,其用量及使用方法同说明书,同上述各实施例所得疫苗一并进行疫苗效价评估实验,具体实验结果见后述。 [0102] using a commercially available Haemophilus influenzae type b conjugate vaccine (trade name: Yasuji Po) as a comparative example, an amount, and to use the same instructions, vaccine potency in conjunction with the above-described evaluation experiments vaccine obtained in Example, specific experiment results will be described later.

[0103] 对比例3 [0103] Comparative Example 3

[0104] 采用市售百白破作为对比例(商品名:吸附无细胞百白破联合疫苗),其用量及使用方法同说明书,同上述各实施例所得疫苗一并进行疫苗效价评估实验,具体实验结果见后述。 [0104] The commercially available as Comparative DPT (trade name: adsorbed acellular DTP vaccines), and to use the same amount instructions, vaccine potency together with the above-described embodiments resulting vaccine evaluation experiments, specific experiment results will be described later.

[0105] 疫苗效价评估实验 [0105] Vaccine potency evaluation experiments

[0106] 一、Western-blot分析肺炎球菌蛋白抗原性 [0106] a, Western-blot analysis of pneumococcal protein antigens

[0107] 将抗八4146?17、抗? [0107] The anti eight 4146? 17, the anti? 8 34小鼠血清分别进行胃68七61'11-131〇1:分析,重组表达的八A146Ply蛋白、PsaA蛋白可与小鼠血清发生免疫原性结合,在AA146Ply蛋白在53-kDa附近见目的蛋白条带,PsaA蛋白在37-kDa附近见目的蛋白条带,表明表达的蛋白具有较好的抗原性,都保留了两种蛋白的抗原表位。 834 mouse serum were seven 61'11-131〇1 stomach 68: Analysis, eight A146Ply recombinant proteins, PsaA protein may be combined with the immunogenic mouse serum, see the object in the vicinity of 53-kDa protein in AA146Ply protein bands, see protein PsaA protein band in the vicinity of 37-kDa, indicated expression has good antigenic, epitope have retained both proteins.

[0湖]二、稳定性 [0 Lake] Second, the stability of

[0109] 液体肺炎球菌-b型流感嗜血杆菌联合疫苗:4°C避光保存24个月,蛋白无变性,抗原性良好。 [0109] -b liquid Pneumococcal Haemophilus influenzae type combination vaccine: 4 ° C in the dark for 24 months, no protein denaturation, antigenicity good.

[0110] 冻干制剂肺炎球菌-b型流感嗜血杆菌联合疫苗:-20°C避光保存24个月,蛋白无变性,抗原性良好。 [0110] The lyophilized formulation of pneumococcal -b Haemophilus influenzae type combined vaccine: -20 ° C protected from light for 24 months, no protein denaturation, good antigenicity.

[0111] 三、免疫原性试验 [0111] Third, the immunogenicity test

[0112] 将上述制备的结合疫苗以人用剂量稀释10倍免疫14~18g的NIH小鼠,每组10只, 每一试验疫苗用两组,皮下注射结合疫苗,阴性对照组注射0.5ml无菌生理盐水。 [0112] The combined vaccine prepared above was diluted 10-fold in human NIH mice immunized with doses of 14 ~ 18g, 10 in each group, the two groups each test vaccine, conjugate vaccines were injected subcutaneously, no negative control group was injected 0.5ml bacteria saline. 0、14天2针腹腔免疫,每次〇. 5ml,第28天采血,采用ELISA方法检测血清抗上述所有荚膜多糖的抗体滴度,检测结果见表1,阳转率结果见表2。 2 Days 0,14 immunized intraperitoneally needle, each square. 5ml, bled on day 28 by ELISA serum antibody titers against all the above capsular polysaccharide, the detection results shown in Table 1, seroconversion rates (Table 2).

[0113] 表1 [0113] TABLE 1

[0114] [0114]

Figure CN106039301AD00131

[0115] 表2 [0115] TABLE 2

[0116] [0116]

Figure CN106039301AD00141

[0117]在此说明书中,本发明已参照其特定的实施例作了描述。 [0117] In this specification, the present invention has been described with reference to specific embodiments thereof. 但是,很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。 However, it is still apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. 因此,说明书应被认为是说明性的而非限制性的。 Accordingly, the specification is to be considered as illustrative and not restrictive.

Claims (10)

1. 一种肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,制备方法用于制备包括肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,肺炎球菌-b型流感嗜血杆菌联合疫苗的制备方法包括: a. 将已纯化备用的肺炎球菌载体蛋白及b型流感嗜血杆菌载体蛋白以质量比1:1的比例加入制备体系中; b. 加入b型流感嗜血杆菌荚膜多糖,加入的荚膜多糖与b型流感嗜血杆菌载体蛋白质量比介于5:1和1:5之间; c. 加入肺炎球菌荚膜糖,其中6B型荚膜糖的加入质量是其余荚膜糖加入质量的2倍,肺炎球菌荚膜糖加入质量:b型流感嗜血杆菌荚膜多糖加入质量为1: (1.5~3); 其中, 肺炎球菌血清型包括7种~23种血清型,b型流感嗜血杆菌载体蛋白为流感嗜血杆菌的外膜D蛋白,肺炎球菌载体蛋白为肺炎球菌表达的高保守性并具有免疫原性的蛋白,与具有免 A pneumococcal Haemophilus influenzae type -b - DTP vaccine preparation, wherein the preparation comprises a process for the preparation of pneumococcal -b Haemophilus influenzae type combined vaccine and DTP vaccine the method of preparing a combination vaccine, pneumococcal -b Haemophilus influenzae type combination vaccine comprising:. a spare the purified pneumococcal protein carrier Haemophilus influenzae type b and carrier protein mass ratio of 1: 1 ratio system prepared by adding ; b. Add Haemophilus influenzae type b capsular polysaccharide, was added and the capsular polysaccharide Haemophilus influenzae type b carrier protein ratio between 5: 1 and 1: 5 between; C was added pneumococcal capsular sugar, wherein the mass added capsular saccharide 6B capsular saccharide is added to the remaining mass of 2 times the mass of pneumococcal capsular saccharide added: Haemophilus influenzae type B capsular polysaccharide was added mass of 1: (1.5 to 3); wherein pneumococcal serotypes includes 7 to 23 serotypes, b Haemophilus influenzae type carrier protein is the outer membrane of Haemophilus influenzae protein D, pneumococcal carrier protein is highly conserved and has an immunogen expression pneumococcus of protein, and has a free 疫原性的肺炎球菌荚膜糖缀合。 Immunogenic pneumococcal capsular saccharide conjugated.
2. 根据权利要求1所述的肺炎球菌_b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,肺炎球菌荚膜糖的血清型中包括:1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、12F、14、 158、17卩、18(:、19卩、19厶、20、22卩、23卩和/或33卩。 The pneumococcal according to claim 1 Haemophilus influenzae type _b - DTP vaccine preparation, wherein the pneumococcal capsular saccharide serotypes included: 1,2,3,4 , 5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 158,17 Jie, 18 (:, Jie 19, Si 19, 20, 22 Jie, Jie 23 and / or 33 Jie.
3. 根据权利要求1所述的肺炎球菌_b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,载体蛋白包括:肺炎球菌溶血蛋白及其改性衍生物、肺炎球菌表面蛋白及其改性衍生物、肺炎球具表面黏附蛋白及其改性衍生物或肺炎球菌三组氨酸蛋白家族融合蛋白。 The pneumococcal according to claim 1 Haemophilus influenzae type _b - DTP vaccine preparation, wherein the carrier protein comprises: pneumolysin proteins and their modified derivatives, pneumococcal surface protein and modified derivatives thereof, pneumococcal surface adhesion protein having their modified derivatives or pneumococcal protein family three histidine fusion protein.
4. 根据权利要求3所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,肺炎球菌蛋白具体为:改性肺炎球菌溶血蛋白△ A146Ply、肺炎球菌溶血蛋白衍生物dPly、肺炎球菌表面蛋白A、肺炎球菌表面黏附蛋白A或肺炎球菌三组氨酸蛋白PhtA、 PhtB、PhtD、PhtE、PhtDE。 The pneumococcal according to claim 3 H. influenzae type -b - DTP vaccine preparation, wherein the pneumococcal protein is specifically: the modified pneumolysin protein △ A146Ply, pneumolysin protein derivative dPly, pneumococcal surface protein A, pneumococcal surface adhesion protein A or protein histidine three pneumococcal PhtA, PhtB, PhtD, PhtE, PhtDE.
5. 根据权利要求1~3任一项所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,肺炎球菌_b型流感嗜血杆菌-百白破联合疫苗包括肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: a. 肺炎球菌-b型流感嗜血杆菌联合疫苗为7价肺炎球菌-肺炎球菌表面蛋白A与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗; b. 无细胞百日咳疫苗为百日咳疫苗原液、白喉类疫苗为白喉类毒素和破伤风类疫苗为破伤风类毒素,且百白破疫苗为悬混液。 The pneumococcal one of the Haemophilus influenzae type -b according to any of claims 1 to 3, - DTP vaccine preparation, comprising, pneumococcal Haemophilus influenzae type _b - DTP -b combination vaccines comprising a pneumococcal Haemophilus influenzae type combined vaccine and combined vaccine DPT vaccine, specifically: a pneumococcal -b Haemophilus influenzae type joint 7-valent pneumococcal conjugate vaccine - pneumococcal surface protein a. and Haemophilus influenzae type b - Hib combination vaccine D outer membrane protein; b acellular pertussis vaccine for whooping cough vaccine stock, diphtheria toxoid vaccine for diphtheria and tetanus vaccine for tetanus toxoid, and. DPT mixed solution is suspended.
6. 根据权利要求5所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: 肺炎球菌-b型流感嗜血杆菌联合疫苗为7价肺炎球菌-肺炎球菌表面黏附蛋白A与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 The pneumococcal claimed in claim 5, wherein the Haemophilus influenzae type -b - DTP vaccine preparation, wherein -b pneumoniae Haemophilus influenzae type combined vaccine and DTP combined vaccine vaccine, specifically: Streptococcus pneumoniae Haemophilus influenzae type -b combined vaccine is heptavalent pneumococcal - pneumococcal surface adhesion protein a and Haemophilus influenzae type b - Haemophilus influenzae outer membrane protein D combination vaccine.
7. 根据权利要求5所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗的制备方法,其特征在于,肺炎球菌-b型流感嗜血杆菌联合疫苗及百白破疫苗的联合疫苗,具体为: 肺炎球菌-b型流感嗜血杆菌联合疫苗为7价肺炎球菌-改性肺炎球菌溶血蛋白△ A146Ply与b型流感嗜血杆菌-流感嗜血杆菌外膜D蛋白的联合疫苗。 The pneumococcal claimed in claim 5, wherein the Haemophilus influenzae type -b - DTP vaccine preparation, wherein -b pneumoniae Haemophilus influenzae type combined vaccine and DTP combined vaccine vaccine, specifically: Streptococcus pneumoniae Haemophilus influenzae type -b combined vaccine is heptavalent pneumococcal - modified pneumolysin protein △ A146Ply and Haemophilus influenzae type b - Haemophilus influenzae outer membrane protein D combination vaccine.
8. -种根据权利要求1所述的肺炎球菌_b型流感嗜血杆菌-百白破联合疫苗的制备方法所制备得到的联合疫苗,其特征在于:联合疫苗由权利要求1~7任一项制备方法制得。 8. - The pneumococcal types 1 _B according to claim Haemophilus influenzae type - Preparation combination vaccine DPT vaccine is prepared, characterized in that: a combination vaccine according to any one of claims 1-7 item preparation methods.
9. 根据权利要求8所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗,其特征在于: 肺炎球菌-b型流感嗜血杆菌联合疫苗为冻干剂型,用于作为肺炎球菌疫苗的冻干制剂的冻干保护剂的为蔗糖。 According to claim. 8 pneumococcal the Haemophilus influenzae type -b - DTP vaccine, which is characterized in that: Streptococcus pneumoniae Haemophilus influenzae type -b combination vaccine is lyophilized dosage form, for a pneumococcal lyoprotectant lyophilized vaccine formulation is sucrose.
10. 根据权利要求9所述的肺炎球菌-b型流感嗜血杆菌-百白破联合疫苗,其特征在于: 蔗糖在冻干原液中的初始浓度不大于20%。 10. The pneumococcal according to claim 9, wherein the Haemophilus influenzae type -b - DTP vaccine, which is characterized in that: the initial concentration of sucrose stock solution is lyophilized in less than 20%.
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Publication number Priority date Publication date Assignee Title
CN101112619A (en) * 2001-04-03 2008-01-30 葛兰素史密丝克莱恩生物有限公司 Vaccine composition
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