WO2019223749A1 - 鲍曼不动杆菌免疫原性蛋白及其组合物和应用 - Google Patents
鲍曼不动杆菌免疫原性蛋白及其组合物和应用 Download PDFInfo
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- acinetobacter baumannii
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
- C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
- A61K39/1045—Moraxella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1218—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Acinetobacter
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
Definitions
- the invention belongs to the field of biotechnology, and relates to Acinetobacter baumannii immunogenic protein and its composition and application, in particular to an immunogenic fusion protein containing Ata baumannii Ata protein component, and a vaccine containing the protein Composition and its use in a method for immunizing an animal or human against A. baumannii infection.
- Acinetobacter baumannii is a gram-negative bacterium widely existing in nature. It belongs to an opportunistic bacterium and is easily infected by people with low immune function. One of the clinical pathogens. Because of its rapid proliferation and strong adhesion, it is widely distributed in the hospital environment, and with the large amount of antibiotics used, more and more drug-resistant strains have appeared. Posed a serious threat. At present, antibiotics are mainly used in clinical practice to fight A. baumannii infection. However, the emergence of multidrug-resistant strains makes doctors have to use drugs in combination. However, there are many drawbacks to the combined use of drugs, so the development of related vaccines is urgent to prevent the outbreak of Acinetobacter baumannii. Unfortunately, there is currently no vaccine available for Acinetobacter baumannii.
- the present invention aims to provide an immunogenic fusion protein containing an Ata baumannii Ata protein fragment, a vaccine composition containing the protein, and its application in a method for immunizing a mammal against A. baumannii infection .
- the inventors have discovered that the component of the surface protein Ata (Acinetobacter trimeric autotransporter) of Acinetobacter baumannii is fused with an adjuvant protein to form a fusion protein.
- the fusion protein has immunogenicity against Acinetobacter baumannii,
- the vaccine can be used for preparing a vaccine for preventing Acinetobacter baumannii infection and using the vaccine. Based on the above findings, the present inventors have completed the present invention.
- an immunogenic fusion protein which comprises an Ata protein component of Acinetobacter baumannii, which comprises the amino acid sequence of the N-terminal ⁇ -helix portion of the Ata protein Baumannia Ata protein transport functional region. Fragments; and adjuvant proteins or protein fragments with adjuvant function.
- Another aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned fusion protein.
- Another aspect of the present invention provides a biological material, the biological material is selected from:
- Another aspect of the present invention provides a method for making an animal or a human resistant to Acinetobacter baumannii infection, comprising the steps of immunizing the protein composition or the fusion protein or the nucleic acid molecule or the above to an animal or human in need. Biomaterials to achieve resistance to A. baumannii infection.
- Another aspect of the present invention provides a method for producing A. baumannii antibodies, comprising the steps of: effectively immunizing the above-mentioned protein composition or the above-mentioned fusion protein or the above-mentioned nucleic acid molecule or the above-mentioned biological material to an animal or a human to obtain the Baumann Acinetobacter antibodies.
- the immunization may be intraperitoneal immunization or subcutaneous immunization.
- Another aspect of the present invention provides a product comprising the above-mentioned protein or the above-mentioned protein composition or the above-mentioned fusion protein or the above-mentioned nucleic acid molecule or the above-mentioned biological material or the above-mentioned antibody.
- Another aspect of the present invention provides the use of the fusion protein of the present invention or a nucleic acid molecule encoding the fusion protein of the present invention in the preparation of a vaccine composition for combating A. baumannii infection.
- FIG. 1 is a schematic diagram of 39 amino acids in the N-terminal ⁇ -helix portion of the protein Ata transport functional region.
- Figure 2 shows the results of SDS-PAGE analysis of the purified fusion protein CTB-Ata.
- Figure 3 shows the results of antibody titers against Acinetobacter baumannii in the serum of mice in each group by an indirect ELISA method.
- Figure 4 shows the experimental results of mice challenged with Acinetobacter baumannii after the fusion protein CTB-Ata was immunized.
- protein refers to the molecular chain of amino acids and includes peptides, oligopeptides and polypeptides. If desired, the protein can be modified in vivo or in vitro by, for example, glycosylation, amidation, carboxylation or phosphorylation.
- the protein or peptide can be natural or synthetic.
- fusion protein as used in the present invention means a protein formed by two or more polypeptides covalently linked to each other directly or indirectly through a certain method.
- immunoprotective means the ability (partially or fully) of serum antibodies and / or cytotoxic T cell responses induced during immunization to protect against diseases caused by, for example, Acinetobacter baumannii.
- homology means the homology of the nucleotide sequence of DNA and the amino acid sequence of the protein, the former can be determined by a DNA sequencing method, and the latter can be determined by a method such as mass spectrometry or Edman degradation method.
- an immunogenic protein composition comprising 39 amino acids at the N-terminal ⁇ -helix portion of the Ata protein transfer functional region of Acinetobacter baumannii and an adjuvant protein or a protein having an adjuvant function Fragment.
- an immunogenic fusion protein including a component part of Acinetobacter baumannii Ata protein, the component part comprising an N-terminal ⁇ -helix of the Ata baumannii Ata protein transport functional region Part of the 39 amino acids, namely amino acid residues 127-165 of Sequence 2; and adjuvant proteins or protein fragments with adjuvant function.
- the adjuvant protein is specifically the cholera toxin B subunit, ie, the CTB protein.
- the fusion protein is any one of the following a) -e):
- amino acid sequence consists of the amino acid residues shown in sequence 2 in the sequence listing;
- a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in any of a) to d).
- nucleic acid molecule encoding the above-mentioned fusion protein
- nucleic acid molecule is a nucleic acid molecule shown in any one of the following 1) -4):
- a biological material is provided, the biological material is selected from:
- a method for making an animal or a human resistant to Acinetobacter baumannii comprises the step of immunizing an animal or a human with a protein or a protein composition or a fusion protein according to any one of the above schemes or Nucleic acid molecules or biological materials to achieve resistance to A. baumannii infection.
- a method for producing Acinetobacter baumannii antibodies comprising the steps of: immunizing an animal or a human with a protein or protein composition or a fusion protein or a nucleic acid molecule or a biological material according to any of the above schemes To obtain A. baumannii antibodies.
- Antibodies prepared by the above methods are also within the scope of the present invention.
- a product comprising a protein or a protein composition or a fusion protein or a nucleic acid molecule or a biological material or an antibody according to any one of the above schemes.
- the product of the present invention has at least one of the following functions of ac: a), promoting the production of antibodies against Acinetobacter baumannii in animals or humans; b) preventing and / or treating diseases caused by A. baumannii C) Resistant to Acinetobacter baumannii infection or infection.
- the product is a pharmaceutical or health supplement or a test kit or vaccine.
- the animal is a mouse.
- the fusion protein CTB-Ata includes the amino acid sequence of CTB and the surface protein Ata (Acinetobacter trimeric autotransporter) of Acinetobacter baumannii. It has 39 amino acids in the N-terminal ⁇ -helix of the functional domain.
- Sequences 20-123 are the fusion-expressed CTB protein, 127-165 are the ⁇ -helical portion of the Ata protein, and 201-206 are His tags.
- the nucleotide sequence of the fusion protein encoding gene CTB-Ata is Sequence 1 in the Sequence Listing, where the 103-131 position is the tac promoter, the 235-543 position is the CTB protein encoding gene, and the 556-672 position is Ata The ⁇ -helix part of the protein encodes a gene, and the 778-795 position is a His tag gene.
- XbaI and XhoI were used to digest pET28a (+) (NOVAGEN, catalog number: 69864) to obtain a large vector fragment; the fusion protein encoding gene CTB-Ata shown in the above sequence 1 was ligated to the vector large fragment to obtain a recombinant expression vector pET28a-CTB-Ata.
- the recombinant expression vector pET28a-CTB-Ata is a vector obtained by replacing the DNA molecule between the XbaI and XhoI digestion sites of the pET28a (+) vector with the fusion protein coding gene CTB-Ata shown in Sequence 1.
- the fusion protein contains the vector. His tag.
- the recombinant bacteria pET28a-CTB-Ata / BL21 was inoculated in 5 ml LB liquid medium containing a final concentration of 50 ⁇ g / mL kanamycin, cultured at 37 ° C overnight, and passaged in LB liquid medium at a volume ratio of 1: 100, 37 When cultured at OD 600 to about 0.6, IPTG was added to a final concentration of 1 mM, and the temperature was lowered to 30 ° C. for 12 hours to obtain a protein-inducing culture solution. Centrifuge (10800 g for 8 min) and collect the precipitate to obtain protein-inducing bacteria.
- A1 solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 10 mM imidazole, adjust pH to 7.0), sonicate (ultrasonic 4s, 5s, cumulative ultrasonic time 2h), use Centrifuge with a centrifugal force of 1,000 g and collect the supernatant.
- the supernatant is a crude extract containing the fusion protein CTB-Ata.
- the above supernatant was purified using a Chelating affinity chromatography column (GE Healthcare, catalog number 17-5203-06) ( ⁇ 1.6 cm * 15 cm).
- a Chelating affinity chromatography column (GE Healthcare, catalog number 17-5203-06) ( ⁇ 1.6 cm * 15 cm).
- the purified fusion protein CTB-Ata was analyzed by 15% SDS-PAGE and the anti-His antibody was used for WB detection. The results are shown in Figure 2. After purification, a relatively pure target fusion protein CTB-Ata (molecular weight is 22.5 KDa). After quantifying the protein by the BCA method, the following animal experiments were performed.
- the empty vector pET28a was transferred into BL21 cells, and expression was induced in the same way. No target protein was obtained.
- the purified fusion protein CTB-Ata prepared in Example 1 was diluted with physiological saline to prepare an immune sample containing the fusion protein CTB-Ata; 2.5 ⁇ g of the fusion protein CTB-Ata per mouse (the weight of the mouse is about 17 g) Immunization was carried out at the same immunization dose, and Ata (39 amino acids in the N-terminal ⁇ -helix) peptide alone was immunized as a control.
- mice Fifty female 5-week-old (no significant differences in body weight) female Balb / c mice (Vitalivar) were randomly divided into 5 groups (10 in each group):
- Intraperitoneal immunization group 2 Each mouse was injected with 100 ⁇ l of immune samples containing the fusion protein CTB-Ata (CTB-ATA (peritoneal cavity)) or Ata peptide (ATA (peritoneal cavity)), the immune dose was 2.5 ⁇ g per mouse;
- Subcutaneous immunization group 2 Each mouse was injected with 100 ⁇ l of immune samples containing the fusion protein CTB-Ata (CTB-ATA (subcutaneous)) or Ata peptide (ATA (subcutaneous)), the immune dose was 2.5 ⁇ g per mouse;
- Blank control group (without injecting any sample, control): female Balb / c mice;
- Each group was immunized on days 1, 14, and 28. Blood was collected from tails 10 days after each immunization injection, and serum from each group of mice was collected.
- the indirect ELISA method was used to measure the titer of anti-Acinetobacter baumannii antibodies in the serum of each group of mice, as follows:
- the expression vector pGEX-4T-Ata was introduced into BL21 cells, and the GST-Ata fusion protein was purified by fermentation and then used to coat the antigen.
- Use coating buffer NaHCO 3 29.3g, Na 2 CO 3 19.5g, dissolve with ddH 2 O and make up to 1L and adjust the pH to 9.6 to dilute the coating antigen to 50 ⁇ g / mL, 100 ⁇ l / well, 4 C overnight.
- mice were challenged by intraperitoneal injection with 1.5 times LD 50 of Acinetobacter baumannii, and the injection volume of each mouse was 200 ⁇ L, which was observed and recorded for 7 consecutive days. Number of mice dead in each group.
- the surface protein Ata (Acinetobacter trimeric autotransporter) of Acinetobacter baumannii is cut from 39 amino acids at the N-terminal ⁇ -helix, fused with CTB, and expressed in BL21. Purified by a nickel column, 2.5 ⁇ g / mouse was used to perform intraperitoneal or subcutaneous immunization, and its immunogenicity and immunoprotective properties were verified by animal experiments, which proved that it has a good anti-A. Baumannii infection effect.
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了鲍曼不动杆菌免疫原性蛋白及其组合物和应用。本发明提供了一种蛋白质,包括鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸和佐剂蛋白;所述鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸的氨基酸序列分别为序列2第127-165位。本发明将鲍曼不动杆菌的表面蛋白Ata(Acinetobacter trimeric autotransporter),截取N-端α-螺旋部分39个氨基酸,与CTB进行融合,在BL21中表达。经镍柱纯化,以2.5μg/只小鼠进行腹腔免疫,通过动物实验验证其免疫原性和免疫保护性,证明其具有良好的抗鲍曼不动杆菌的侵染。
Description
本发明属于生物技术领域,涉及鲍曼不动杆菌免疫原性蛋白及其组合物和应用,尤其涉及一种包含鲍曼不动杆菌Ata蛋白组成部分的免疫原性融合蛋白、包含该蛋白的疫苗组合物及其在使动物或人免疫以抵抗鲍曼不动杆菌感染的方法中的应用。
鲍曼不动杆菌(Acinetobacter baumannii,Ab)是自然界中广泛存在的一种革兰氏阴性菌,属于一种机会致病菌,极易感染免疫功能低下的人,同时也是医院感染中最典型的临床病原菌之一。因其增殖速度快,粘附力极强,因此在医院环境中广泛分布,并且随着抗生素的大量使用,出现了越来越多的耐药菌株,鲍曼不动杆菌已经对全球医疗卫生系统构成了严重威胁。目前,临床上主要利用抗生素对抗鲍曼不动杆菌的感染,然而多重耐药菌株的出现,使得医生在治疗时不得不联合用药。可是联合用药存在许多弊端,因此相关疫苗的研制,对于预防鲍曼不动杆菌的疫情刻不容缓。可惜的是,目前还没有一种针对鲍曼不动杆菌的疫苗上市。
因此,对可用于制备针对鲍曼不动杆菌的疫苗的免疫原性蛋白和包含该蛋白的疫苗存在未满足的需求。
发明概述
本发明旨在提供一种包含鲍曼不动杆菌Ata蛋白片段的免疫原性融合蛋白、包含该蛋白的疫苗组合物及其在使哺乳动物免疫以抵抗鲍曼不动杆菌感染的方法中的应用。
本发明人通过深入实验研究发现,鲍曼不动杆菌的表面蛋白Ata(Acinetobacter trimeric autotransporter)的组成部分与佐剂蛋白融合形成融合蛋白后对鲍曼不动杆菌具有符合疫苗需求的免疫原性,可用于制备预防鲍曼不动杆菌感染的疫苗及使用该疫苗。基于上述发现,本发明人完成了本发明。
可以从不同方面描述本发明,这些方面及其任何形式中所描述的发明相互独立又彼此关联,相互结合构成本发明的内容。
本发明一个方面提供一种免疫原性融合蛋白,它包括鲍曼不动杆菌Ata蛋白的组成部分,该组成部分包含鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分的氨基酸序列片段;以及佐剂蛋白或具有佐剂功能的蛋白片段。
本发明另一方面提供编码上述融合蛋白的核酸分子。
本发明另一方面提供一种生物材料,该生物材料选自:
1)含有上述核酸分子的表达盒;
2)含有上述核酸分子的重组载体;以及
3)含有上述核酸分子的重组菌或转基因细胞系。
本发明另一方面提供了一种使动物或人抗鲍曼不动杆菌侵染的方法,包括如下步骤:向有需要的动物或人免疫上述蛋白组合物或上述融合蛋白或上述核酸分子或上述的生物材料,实现抗鲍曼不动杆菌侵染。
本发明另一方面提供了一种产生鲍曼不动杆菌抗体的方法,包括如下步骤:向动物或人有效免疫上述蛋白组合物或上述融合蛋白或上述核酸分子或上述的生物材料,得到鲍曼不动杆菌抗体。
上述免疫可以为腹腔免疫或皮下免疫。
本发明另一方面提供一种产品,其包括上述蛋白质或上述蛋白组合物或上述融合蛋白或上述核酸分子或上述的生物材料或上述抗体。
本发明还一方面提供本发明的融合蛋白或其编码核酸分子在制备用于对抗鲍曼不动杆菌感染的疫苗组合物中的用途。
图1为蛋白Ata转运功能区N-端α-螺旋部分39个氨基酸的示意图。
图2为纯化的融合蛋白CTB-Ata的SDS-PAGE分析结果。
图3为间接ELISA法测各组小鼠血清中抗鲍曼不动杆菌的抗体滴度结果。
图4为融合蛋白CTB-Ata免疫后小鼠攻毒鲍曼不动杆菌的实验结果。
发明的详细说明
上文已经概述了本发明,下面将举例说明以进一步详细描述本发明。
为准确理解本发明中所使用的术语,下面特别定义部分术语的含义。 对于在此没有特别定义的术语,它们具有本领域技术人员普遍理解和接受的含义。如果在此所定义的某个术语的含义与本领域技术人员普遍理解和接受的含义不一致,则该术语的含义以在此所定义的含义为准。
本发明中所使用的术语“蛋白质”是指氨基酸的分子链,包括肽、寡肽和多肽。根据需要,可以通过例如糖基化,酰胺化,羧化或磷酸化在体内或体外对所述的蛋白质进行修饰。蛋白质或肽可以是天然的或合成的。
本发明中所使用的术语“融合蛋白”意指由两种或更多种多肽通过一定的方式直接或间接地彼此共价连接在一起形成的蛋白质。
本发明中所使用的术语“免疫保护性”意指在免疫期间诱导的血清抗体和/或细胞毒性T细胞应答的能力(部分或全部)保护免受由诸如鲍曼不动杆菌引起的疾病。
本发明中所使用的术语“同源性”意指DNA核苷酸和蛋白质氨基酸序列的同源性,前者可通过DNA测序方法测定,后者可通过质谱或Edman降解法等方法测定。
根据本发明的一个方面,提供一种免疫原性蛋白组合物,其包括鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸以及佐剂蛋白或具有佐剂功能的蛋白片段。
根据本发明的另一个方面,提供一种免疫原性融合蛋白,它包括鲍曼不动杆菌Ata蛋白的组成部分,该组成部分包含鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分的39个氨基酸,即序列2第127-165位氨基酸残基;以及佐剂蛋白或具有佐剂功能的蛋白片段。
在一个方案中,佐剂蛋白具体为霍乱毒素B亚单位,即CTB蛋白。
在另一个方案中,上述融合蛋白为如下a)-e)中任一种蛋白质:
a)氨基酸序列包括序列表中序列2所示的氨基酸序列的蛋白质;
b)氨基酸序列由序列表中序列2所示的氨基酸残基组成;
c)将a)或b)所限定的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有提高动物免疫能力功能的蛋白质;
d)与a)或b)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有提高动物免疫能力功能的蛋白质;
e)a)-d)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
根据本发明的另一方面,提供编码上述融合蛋白的核酸分子,该核酸分子为如下1)-4)中任一种所示的核酸分子:
1)其编码序列包括序列表中序列1;
2)其编码序列为序列表中序列1;
3)在严格条件下与1)或2)限定的DNA分子杂交且编码上述蛋白的DNA分子;
4)与1)或2)限定的DNA分子具有80%以上或90%以上的同源性且编码上述蛋白的DNA分子。
根据本发明的另一方面,提供一种生物材料,该生物材料选自:
1)含有上述核酸分子的表达盒;
2)含有上述核酸分子的重组载体;
3)含有上述核酸分子的重组菌或转基因细胞系。
根据本发明的另一方面,提供所述的蛋白质、上述蛋白组合物、上述融合蛋白、上述核酸分子或生物材料在如下1)-6)中至少一种中的应用:
1)制备促进动物或人产生拮抗鲍曼不动杆菌的抗体产品;
2)制备预防和/或治疗鲍曼不动杆菌所致疾病产品;
3)制备抗鲍曼不动杆菌侵染或感染的产品;
4)促进动物或人产生拮抗鲍曼不动杆菌的抗体;
5)预防和/或治疗鲍曼不动杆菌所致疾病;
6)抗鲍曼不动杆菌侵染或感染。
根据本发明的另一方面,提供一种使动物或人抗鲍曼不动杆菌侵染的方法,它包括如下步骤:向动物或人免疫上述任一方案的蛋白质或蛋白组合物或融合蛋白或核酸分子或生物材料,以实现抗鲍曼不动杆菌侵染。
根据本发明的另一方面,提供一种产生鲍曼不动杆菌抗体的方法,它包括如下步骤:向动物或人免疫上述任一方案的蛋白质或蛋白组合物或融合蛋白或核酸分子或生物材料,以得到鲍曼不动杆菌抗体。
由上述方法制备的抗体也是本发明保护的范围。
上述抗体在如下1)-6)中至少一种中的应用也是本发明保护的范围:
1)制备促进动物或人产生拮抗鲍曼不动杆菌的抗体产品;
2)制备预防和/或治疗鲍曼不动杆菌所致疾病产品;
3)制备抗鲍曼不动杆菌侵染或感染的产品;
4)促进动物或人产生拮抗鲍曼不动杆菌的抗体;
5)预防和/或治疗鲍曼不动杆菌所致疾病;
6)抗鲍曼不动杆菌侵染或感染。
根据本发明的另一方面,提供一种产品,该产品包括上述任一方案的蛋白质或蛋白组合物或融合蛋白或核酸分子或生物材料或抗体。
在一个方案中,本发明的产品具有如下a-c中至少一种功能:a)、促进动物或人产生拮抗鲍曼不动杆菌的抗体;b)预防和/或治疗鲍曼不动杆菌所致疾病;c)抗鲍曼不动杆菌侵染或感染。
在一个方案中,所述产品为药品或保健品或检测试剂盒或疫苗。
在一个方案中,所述的动物为小鼠。
为进一步说明本发明,提供下面的实施例。这些实施例仅用于举例说明本发明,不应对本发明构成任何限制。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、融合蛋白CTB-Ata的获得
融合蛋白CTB-Ata包括CTB氨基酸序列和鲍曼不动杆菌的表面蛋白Ata(Acinetobacter trimeric autotransporter)转运功能区N-端α-螺旋部分39个氨基酸。
1、融合蛋白CTB-Ata
对于鲍曼不动杆菌的表面蛋白Ata(Acinetobacter trimeric autotransporter),截取其转运功能区N-端α-螺旋部分39个氨基酸(图1)使用。
再通过全基因合成Ata蛋白的α-螺旋部分39个氨基酸、tac启动子以及融合表达的CTB蛋白,构成融合蛋白CTB-Ata,该融合蛋白的氨基酸序列为序列表中序列2。
序列2中第20-123位为融合表达的CTB蛋白,第127-165位为Ata蛋白的α-螺旋部分,第201-206位为His标签。
融合蛋白编码基因CTB-Ata的核苷酸序列为序列表中序列1,其中,第103-131位为tac启动子,第235-543位为CTB蛋白的编码基因,第556-672位为Ata蛋白的α-螺旋部分编码基因,第778-795位为His标签基因。
2、表达融合蛋白CTB-Ata的载体
用XbaI和XhoI酶切pET28a(+)(NOVAGEN公司,产品目录号:69864),得到载体大片段;将上述序列1所示的融合蛋白编码基因CTB-Ata与载体大片段连接,得到重组表达载体pET28a-CTB-Ata。
重组表达载体pET28a-CTB-Ata为将序列1所示的融合蛋白编码基因CTB-Ata替换pET28a(+)载体的XbaI和XhoI酶切位点间的DNA分子,得到的载体,此融合蛋白含有载体上的His标签。
3、融合蛋白CTB-Ata的表达与纯化
将上述2得到的重组表达载体pET28a-CTB-Ata导入到BL21细胞中,得到重组菌pET28a-CTB-Ata/BL21;
将重组菌pET28a-CTB-Ata/BL21接种于含有终浓度为50μg/mL卡那霉素的5ml LB液体培养基中,37℃培养过夜,以体积比1:100传代于LB液体培养基,37℃培养至OD
600约为0.6时,加入终浓度为1mM的IPTG,并降温至30℃诱导12h,得到蛋白诱导培养液,离心(10800g离心8min),收集沉淀得到蛋白诱导菌体。
取蛋白诱导菌体30g,加入100ml A1液(20mM pH7.5 Tris-HCl、0.5M NaCl、10mM咪唑,调pH至7.0),超声破菌(超声4s暂停5s,累计超声时间2h),用12 000g的离心力离心,收集上清液,此上清为含融合蛋白CTB-Ata的粗提液。
利用Chelating亲和层析柱(GE Healthcare,产品目录号为17-5203-06)(Φ1.6cm*15cm)纯化上述上清液。首先用0.5M的NaOH水溶液冲洗柱床至少3个柱床体积,然后用去离子水平衡至pH中性,然后用0.5M的NiSO
4水溶液平衡至少3个柱床体积,再用B1液(20mM pH7.5 Tris-HCl、0.5M NaCl、500mM咪唑,调pH至7.0)平衡至少一个柱床体积,最后用上述A1液平衡至少3个柱床体积,以上流速均为4mL/min。从A管道将粗提液上样,再用A1液洗去未结合蛋白,冲洗至紫外吸收(280nM)接 近于0mAU,最后用含0%-100%(体积比)的B1液的溶液进行线性洗脱(A管道进A1液,B管道进B1液,纯化仪自动混合),收集洗脱液80mL,得到纯化的融合蛋白CTB-Ata。
将纯化的融合蛋白CTB-Ata用15%SDS-PAGE分析,并利用anti-His抗体进行WB检测,结果如图2所示,纯化后成功得到了比较纯的目的融合蛋白CTB-Ata(分子量为22.5KDa)。经BCA法蛋白定量后,进行了以下动物实验。
将空载体pET28a转入BL21细胞中,同样的方法诱导表达,没有得到目的蛋白。
实施例2、融合蛋白CTB-Ata的功能验证
1、动物免疫
将实施例1制备的纯化的融合蛋白CTB-Ata用生理盐水稀释,制成含有融合蛋白CTB-Ata的免疫样品;按照每只小鼠(小鼠体重为17g左右)2.5μg融合蛋白CTB-Ata的免疫剂量进行免疫,同时单独Ata(N-端α-螺旋部分39个氨基酸)肽段进行免疫作为对照。
取50只5周龄大(体重无显著差异)的雌性Balb/c小鼠(维通利华),随机分为5组(每组10只):
腹腔免疫2组:每只小鼠分别注射100μl含有融合蛋白CTB-Ata(CTB-ATA(腹腔))或Ata肽段(ATA(腹腔))的免疫样品,免疫剂量为每只小鼠2.5μg;
皮下免疫2组:每只小鼠分别注射100μl含有融合蛋白CTB-Ata(CTB-ATA(皮下))或Ata肽段(ATA(皮下))的免疫样品,免疫剂量为每只小鼠2.5μg;
空白对照组(不注射任何样品,control):雌性Balb/c小鼠;
各组分别在第1、14、28天进行免疫,每次免疫注射后10天断尾取血,收集各组小鼠血清。
2、抗鲍曼不动杆菌的抗体滴度检测
用间接ELISA法测各组小鼠血清中抗鲍曼不动杆菌的抗体滴度,具体如下:
将表达载体pGEX-4T-Ata导入BL21细胞中,发酵纯化GST-Ata融合蛋白后,用于包被用抗原。用包被缓冲液(NaHCO
3 29.3g、Na
2CO
3 19.5g,用ddH
2O溶解并定容到1L并调pH值为9.6)将包被抗原稀释为50μg/mL,100μl/孔,4℃过夜。用PBST(1L PBS加500μl吐温-20)洗3次,拍干,每孔加入含5%(质量百分比)脱脂奶粉的PBST 200μl,37℃孵育2h,再用PBST洗3次,拍干,加入倍比稀释的上述1得到的免疫组的小鼠血清(用含5%(质量百分比)脱脂奶粉的PBST稀释),100μl/孔,37℃孵育60min。PBST洗3次,拍干,加入驴抗鼠抗体(abcam,产品目录号ab6820)(用含5%(质量百分比)脱脂奶粉的PBST按1:10 000稀释),100μl/孔,37℃孵育1h。PBST洗3次,拍干,加入100μl/孔的OPD-H
2O
2显色液,避光显色15min,加入50μl终止液,测OD
490值。
结果如图3所示,可以看出,CTB-Ata融合蛋白免疫后抗体滴度高于Ata肽段组和空白对照组,且有显著性差异。CTB-Ata融合蛋白经腹腔免疫后抗体滴度最高,皮下免疫抗体滴度次之。说明融合蛋白CTB-Ata免疫能够成功使小鼠产生拮抗鲍曼不动杆菌的抗体。
3、小鼠攻毒实验
在末次免疫后14天,给各组(10只)每只小鼠以1.5倍LD
50的鲍曼不动杆菌进行腹腔注射攻毒,每只小鼠注射体积为200μL,连续7天观察和记录每组小鼠死亡数。
结果如图4所示,可以看出,空白对照组(对照组)只存活了3只,而CTB-Ata腹腔免疫组全部存活,CTB-Ata皮下免疫组存活7只;无CTB辅助的Ata腹腔免疫组有一半小鼠存活(5只),皮下组存活4只。因此可见注射融合蛋白CTB-Ata对小鼠具有良好的保护性,表明融合蛋白CTB-Ata可以用来预防鲍曼不动杆菌所致疾病。
工业应用
本发明将鲍曼不动杆菌的表面蛋白Ata(Acinetobacter trimeric autotransporter),截取N-端α-螺旋部分39个氨基酸,与CTB进行融合,在BL21中表达。经镍柱纯化,以2.5μg/只小鼠进行腹腔或皮下免疫,通过动物实验验证其免疫原性和免疫保护性,证明其具有良好的抗鲍曼不动 杆菌的侵染的作用。
Claims (18)
- 一种蛋白质,包括鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸。
- 一种蛋白组合物,其特征在于:所述蛋白组合物包括鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸以及佐剂蛋白或具有佐剂功能的蛋白片段。
- 一种融合蛋白,其特征在于:所述融合蛋白包括鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸以及佐剂蛋白或具有佐剂功能的蛋白片段。
- 根据权利要求1所述的蛋白质或权利要求2所述蛋白组合物或权利要求3所述融合蛋白,其特征在于:所述鲍曼不动杆菌Ata蛋白转运功能区N-端α-螺旋部分39个氨基酸的氨基酸序列为序列2第127-165位。
- 根据权利要求2所述蛋白组合物或权利要求3所述融合蛋白,其特征在于:所述佐剂蛋白为CTB蛋白。
- 根据权利要求3所述的融合蛋白,其特征在于:所述融合蛋白为如下a)-e)中任一种蛋白质:a)氨基酸序列包括序列表中序列2所示的氨基酸序列的蛋白质;b)氨基酸序列由序列表中序列2所示的氨基酸残基组成;c)将a)或b)所限定的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有提高动物免疫能力功能的蛋白质;d)与a)或b)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有提高动物免疫能力功能的蛋白质;e)a)-d)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
- 编码权利要求3-6中任一所述融合蛋白的核酸分子。
- 根据权利要求7所述的核酸分子,其特征在于:所述核酸分子为如下1)-4)中任一种所示的核酸分子:1)其编码序列包括序列表中序列1;2)其编码序列为序列表中序列1;3)在严格条件下与1)或2)限定的DNA分子杂交且编码权利要求1所述蛋白的DNA分子;4)与1)或2)限定的DNA分子具有80%以上或90%以上的同源性且编码权利要求1所述蛋白的DNA分子。
- 下述1)-3)中的任一种生物材料:1)含有权利要求7或8所述核酸分子的表达盒;2)含有权利要求7或8所述核酸分子的重组载体;3)含有权利要求7或8所述核酸分子的重组菌或转基因细胞系。
- 权利要求1所述的蛋白质或权利要求2所述蛋白组合物或权利要求3-6所述融合蛋白或权利要求7或8所述核酸分子或权利要求9所述的生物材料在如下1)-6)中至少一种中的应用:1)制备促进动物或人产生拮抗鲍曼不动杆菌的抗体产品;2)制备预防和/或治疗鲍曼不动杆菌所致疾病产品;3)制备抗鲍曼不动杆菌侵染或感染的产品;4)促进动物或人产生拮抗鲍曼不动杆菌的抗体;5)预防和/或治疗鲍曼不动杆菌所致疾病;6)抗鲍曼不动杆菌侵染或感染。
- 一种使动物或人抗鲍曼不动杆菌侵染的方法,包括如下步骤:向动物或人免疫权利要求1所述的蛋白质或权利要求2所述蛋白组合物或权利要求3-6所述融合蛋白或权利要求7或8所述核酸分子或权利要求9所述的生物材料,实现抗鲍曼不动杆菌侵染。
- 一种制备鲍曼不动杆菌抗体的方法,包括如下步骤:向动物或人免疫权利要求1所述的蛋白质或权利要求2所述蛋白组合物或权利要求3-6所述融合蛋白或权利要求7或8所述核酸分子或权利要求9所述的生物材料,得到鲍曼不动杆菌抗体。
- 由权利要求12所述方法制备的抗体。
- 权利要求13所述抗体在如下1)-6)中至少一种中的应用:1)制备促进动物或人产生拮抗鲍曼不动杆菌的抗体产品;2)制备预防和/或治疗鲍曼不动杆菌所致疾病产品;3)制备抗鲍曼不动杆菌侵染或感染的产品;4)促进动物或人产生拮抗鲍曼不动杆菌的抗体;5)预防和/或治疗鲍曼不动杆菌所致疾病;6)抗鲍曼不动杆菌侵染或感染。
- 一种产品,其包括权利要求1所述的蛋白质或权利要求2所述蛋白组合物或权利要求3-6所述融合蛋白或权利要求7或8所述核酸分子或权利要求9所述的生物材料或权利要求13所述的抗体。
- 根据权利要求15所述的产品,其特征在于:所述产品具有如下a-c中至少一种功能:a)、促进动物或人产生拮抗鲍曼不动杆菌的抗体;b)预防和/或治疗鲍曼不动杆菌所致疾病;c)抗鲍曼不动杆菌侵染或感染。
- 根据权利要求10或14所述应用或权利要求15或16所述的产品,其特征在于:所述动物为小鼠。
- 根据权利要求10或14所述应用或权利要求15或16所述的产品,其特征在于:所述产品为药品或保健品或检测试剂盒或疫苗。
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