WO2015081711A1 - 抗原嵌合体、抗原组合物、疫苗及其制备方法和试剂盒 - Google Patents

抗原嵌合体、抗原组合物、疫苗及其制备方法和试剂盒 Download PDF

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WO2015081711A1
WO2015081711A1 PCT/CN2014/083291 CN2014083291W WO2015081711A1 WO 2015081711 A1 WO2015081711 A1 WO 2015081711A1 CN 2014083291 W CN2014083291 W CN 2014083291W WO 2015081711 A1 WO2015081711 A1 WO 2015081711A1
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antigen
protein
ctb
chimera
ureb
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PCT/CN2014/083291
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English (en)
French (fr)
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李克英
耿雨红
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上海联合赛尔生物工程有限公司
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Priority to BR112016012580-0A priority Critical patent/BR112016012580B1/pt
Priority to AU2014360050A priority patent/AU2014360050B2/en
Priority to EP14868697.5A priority patent/EP3078677B1/en
Priority to US15/102,125 priority patent/US10513543B2/en
Priority to JP2016557173A priority patent/JP6316448B2/ja
Priority to KR1020167018064A priority patent/KR101973079B1/ko
Priority to CA2932211A priority patent/CA2932211C/en
Publication of WO2015081711A1 publication Critical patent/WO2015081711A1/zh

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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
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • 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
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • 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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • 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
    • A61K39/107—Vibrio
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of immunization, particularly antigens, vaccines, and preparation methods and kits thereof for mucosal immunity. Background of the invention
  • the mucosal immune system is a lymphoid tissue widely distributed in the respiratory tract, gastrointestinal tract, genitourinary tract and some exocrine glands, and is the main site for performing local specific immune function.
  • the mucosa is the largest portal for pathogen invasion.
  • infectious diseases that are extremely harmful to animal life and health and difficult to control such as influenza, tuberculosis, epistaxis, and salmonellosis, are diseases caused by mucosal invasion or mucosa, and mucosal damage and mucosa Disorders of immune function often become an important mechanism for opportunistic pathogen infections and even tumorigenesis.
  • the mucosal immune system is the first immune barrier against pathogen invasion.
  • the independent immune system with unique structure and function has positive significance for the prevention and colonization of pathogens.
  • the mucosal immune system is a large number of immune cells and immune molecules dispersed in the mucosal epithelium or submucosal lamina basement (dispersed lymphoid tissue), or a mucosa-associated lymphoid aggregated by single or multiple lymphoid follicles. Tissue, more than 50% of the body's lymphoid tissue and more than 80% of immune cells are concentrated in the mucosal immune system. And antibodies in mucosal secretions are mainly secretory IgA and slgM antibodies.
  • the mucosal immune system can be divided into two parts according to its function: the induction site and the effect site. Between the induction site and the effect site, it is mainly linked by lymphocyte homing.
  • the main function of the mucosal immune system is to recognize and respond to a large variety of antigens inhaled or ingested on the mucosal surface. Can be adjusted for a large number of harmless antigens
  • the epidemic response or tolerance can also produce highly effective humoral and cellular immunity to harmful antigens or pathogens for effective immune rejection or clearance.
  • Mucosal immunity is theoretically the most effective way to prevent the pathogenic pathogens caused by mucosal infections, because when immunized by other routes, the vaccine is difficult to induce a significant mucosal immune response.
  • the vast majority of vaccines that have been approved or are in clinical research so far are still immunized by the injection route, and only a few are mucosal.
  • the main reason for the development of mucosal immune vaccine products is the difficulty of detecting local mucosal immune-specific antibodies. More importantly, because of the host's host defense, mucosal immunity cannot be accurately controlled when immunized by injection. The antigen level of the body.
  • the antigen After the antigen reaches the mucosal surface by oral or topical administration, it will undergo mucosal secretion dilution, mucin gel adsorption, protease/nuclease hydrolysis, and clearance by the endomembrane barrier, so only a very small number of antigens can enter the body through the mucosa. Mucosal immune response. In general, water-soluble and mucosal-free antigens have a low antigenic mucosal uptake rate, and some may cause immune tolerance in the intestine.
  • an antigen chimera comprising: a fusion protein of an antigen and a mucosal immunoadjuvant protein monomer capable of forming a multimer, and the formation of the antigen a mucosal immunoadjuvant protein monomer of a polymer, wherein the mucosal immunoadjuvant protein monomer capable of forming a multimer is selected from the group consisting of cholera toxin B subunit (CTB) and Escherichia coli heat labile enterotoxin B subunit ( In one of LTB), the multimer is a pentamer, and the molar ratio of the fusion protein to the multimeric mucosal immunoadjuvant protein monomer in the chimera is 1: 4 , wherein the antigen chimera is stably present in PH More than 7.0, preferably pH 8.0.
  • CTB cholera toxin B subunit
  • Escherichia coli heat labile enterotoxin B subunit In one of L
  • Both CTB and LTB are very effective mucosal immune adjuvants, and both can form stable pentamers.
  • the antigen chimeras prepared by this property can not only maintain the mucosal immune adjuvant properties, but also increase the antigen volume. It is easy to be taken up by antigen presenting cells (APC), which is beneficial to further promote the stimulation of mucosal immunity and systemic immunity.
  • APC antigen presenting cells
  • the stability of the chimera is closely related to its pH value, and in the present invention, the chimera stably has a pH of more than 7.0, preferably a pH of 8.0. The chimera under these conditions, in particular, the CTB forming a pentameric chimera, can most effectively enhance the mucosa.
  • the CTB and LTB in the antigen chimera are their natural constituent structures. Or a mutant capable of forming a pentamer.
  • CTB and LTB which have glycosylation modifications in eukaryotic expression
  • CTB and LTB and their mutants with natural structure expressed by prokaryotic expression vectors can more effectively stimulate the immune response of antigens and exert their adjuvant functions. .
  • the antigen has a molecular weight in the range of 10 to 100 kD, preferably 16 kD to 65 kD. Antigens in the range of 10-100 kD are better able to ensure that CTB or LTB fold into the correct conformation to facilitate the formation of stable pentamers. If the molecular weight of the antigen is too small, it may be affected by CTB or LTB, which will cause the fusion protein to form a polymer itself, which will not fold properly. If the molecular weight is too large, it may cause steric hindrance and hinder the formation of CTB or LTB pentamer.
  • the antigen is an antigen suitable for mucosal immunity.
  • antigens that infect human or animal pathogens via the mucosal route include, but are not limited to, Helicobacter pylori antigen, typhoid antigen, and influenza HA antigen.
  • the H. pylori antigen is selected from the group consisting of Helicobacter pylori urease B subunit (UreB), Helicobacter pylori cytotoxin associated gene A (CagA) protein, and Helicobacter pylori neutrophils At least one of activating proteins (NAPs). These resistances Originally strong immunogenic, non-toxic, it is a good vaccine candidate antigen.
  • the fusion protein comprises three G4S (Gly-Ser-Ser-Ser-Ser) linkers between the antigen and the mucosal immunoadjuvant protein monomer.
  • G4S Gly-Ser-Ser-Ser-Ser linkers
  • the present invention also provides an antigen composition comprising the antigen chimera as described above, wherein the antigen chimera is stably present at a pH greater than 7.0, preferably at a pH of 8.0.
  • the present invention also provides a vaccine comprising the above antigen composition and an excipient suitable for use in a vaccine.
  • the vaccine is an oral vaccine, a nasal vaccine or a rectal vaccine.
  • the present invention also provides a kit for preparing an antigen composition
  • a kit for preparing an antigen composition comprising a vector expressing the above fusion protein and a vector expressing the above-described multimeric mucosal immunoadjuvant protein monomer.
  • the vectors are all prokaryotic expression vectors.
  • the present invention also provides a method for preparing the above antigen chimera, comprising separately expressing the fusion protein and the multimeric mucosal immunoadjuvant protein monomer by a vector in the kit, and then using a renaturation method
  • the two proteins combine to form the chimera, wherein the renaturation method comprises the step of renaturation of the two proteins together in a reconstituted solution containing urea and DTT (or mercaptoethanol).
  • the concentration of urea in the reconstituted solution is from 1.0 M to 2.5 M.
  • the concentration of the DTT or mercaptoethanol is from 0.2 mM to 1.0 mM.
  • the present invention also provides a method for preparing a chimera by renaturation, the chimera comprising (1) a fusion protein of a protein with a monomeric protein capable of forming a multimer and (2) capable of forming a multimer a monomeric protein, which forms a multimer by forming a multimer of the monomeric protein in the (1) fusion protein and the (2) monomeric protein, the method comprising:
  • the (2) monomeric protein capable of forming a multimer is pre-refolded in a buffer containing 6 M to 9 M, preferably 8 M urea, and having a pH of 3.0 to 4.0, preferably for 0.5 to 3 hours, preferably pre-recovered f born 1 hour; and
  • the (1) fusion protein and the (2) monomeric protein capable of forming a multimer are reconstituted in a reconstituted solution containing 1.0 M to 2.5 M urea and 0.2 mM to 1.0 mM DTT or mercaptoethanol. Forming the chimera,
  • the pH of the reconstituted solution is greater than 7.0, more preferably the pH of the reconstituted liquid is 8.0.
  • the formation of the chimera can be promoted, the efficiency of assembly of the chimera can be improved, and the defect that the assembly efficiency of the chimera is low due to the conventional renaturation treatment can be effectively overcome.
  • This renaturation method can be used to form a plurality of fields in which protein chimeras need to be formed, and is not limited to the vaccine field described herein.
  • Figure 1 is a three-dimensional structure diagram of a computer-simulated cholera toxin (CT);
  • Figure 2 is a three-dimensional structure diagram of CTB pentamer in computer simulation
  • Figure 3 is a three-dimensional structure diagram of a computer-simulated CTB-UreB fusion protein
  • Figure 4 is a perspective view of a computer-simulated CTB-UreB/4CTB chimeric protein
  • Figure 5 is a double-digested electrophoresis pattern of PET-28a-CTB-X plasmid prepared according to an embodiment of the present invention, wherein the samples of each lane are as follows : M: DL 10000 DNA Marker; Lane 1, 2, 3: PET-28a-CTB-UreB double digestion; Lane 4, 5, 6: PET-28a-CTB-CagA double digestion; Lane 7: PET-28a -CTB-NAP double digestion;
  • Figure 6 is a protein electropherogram of the CTB-UreB fusion protein prepared according to an embodiment of the present invention, wherein the samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lanes 1, 2, 3, 4, 5, 6, 7 : PET-28a-CTB-UreB/BL21-DE3 cells after IPTG induction;
  • FIG 7 is a CTB-CagA and CTB-NAP fusion prepared in accordance with an embodiment of the present invention.
  • Protein electrophoresis map of the protein wherein the samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lane 1, 2, 3, 4: PET-28a-CTB-CagA/BL21-DE3 cells after IPTG induction; Lane 5 6,6,8: PET-28a-CTB-NAP/BL21-DE3 cells after IPTG induction;
  • Figure 8 is a purified electropherogram of CTB-UreB fusion protein prepared according to an embodiment of the present invention, wherein samples of each lane As follows: M: Fermentas protein pre-stained Marker; Lane 1: SP HP pre-peak sample; Lane 2, 3: SP HP elution peak sample; Lane 4: SP HP post-peak sample;
  • Figure 9A is prepared in accordance with an embodiment of the present invention The CTB-UreB/4CTB chimeric protein was purified
  • Figure 9B is an electropherogram of the results of the CTB-UreB/4CTB chimeric protein stability study prepared according to an embodiment of the present invention, wherein the samples of each lane are as follows: M: Fermentas protein pre-staining; Lanes 1-7 samples are pH 9. The electrophoresis results of the samples after treatment under conditions of 0, 8.0, 7.0, 6.0, 5.0, 4.0, and 3.0.
  • Figure 10 is a purified electrophoresis pattern of CTB-CagA fusion protein prepared according to an embodiment of the present invention, wherein samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lane 1, 2, 3, 4: QHP elution peak;
  • Figure 11A is a purified electrophoresis pattern of CTB-Ca g A/4 CTB chimeric protein prepared according to an embodiment of the present invention, wherein samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lane 1, 2, 3: SP HP wash De-peaking
  • Figure 11B is an electropherogram of the results of the CTB-Ca g A/4 CTB chimeric protein stability study prepared according to an embodiment of the present invention, wherein the samples of each lane are as follows: M: Fermentas protein pre-staining; lanes 1-7 sample separately 1J is the electrophoresis result of the sample treated under the conditions of pH 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0;
  • Figure 12 is a purified electrophoresis pattern of CTB-NAP fusion protein prepared according to an embodiment of the present invention, wherein samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lane 1: Inclusion body (8M urea); Lane 2, 3, 4: QHP elution peak;
  • Figure 13 is a purified electrophoresis pattern of CTB-NAP/4CTB chimeric protein prepared according to an embodiment of the present invention, wherein samples of each lane are as follows: M: Fermentas protein pre-stained Marker; Lane 1, 2, 3: QHP elution peak;
  • Figure 14 is a graph showing the results of serum specific IgG detection of CTB-UreB/4CTB chimeric protein-immunized mice according to an embodiment of the present invention.
  • Figure 15 is a graph showing the results of detection of intestinal mucosa-specific slgA in mice immunized with CTB-UreB/4CTB chimeric protein according to an embodiment of the present invention
  • Figure 16 is a graph showing the results of detection of intestinal mucosa-specific IgG in mice immunized with CTB-CagA/4CTB chimeric protein according to an embodiment of the present invention
  • Figure 17 is a graph showing the results of detection of intestinal mucosa-specific slgA in mice immunized with CTB-CagA/4CTB chimeric protein according to an embodiment of the present invention
  • Figure 18 is a graph showing the results of serum specific IgG detection of CTB-NAP/4CTB chimeric protein-immunized mice according to an embodiment of the present invention.
  • Figure 19 is a graph showing the results of detection of intestinal mucosa-specific slgA in CTB-NAP/4CTB chimeric protein-immunized mice according to an embodiment of the present invention.
  • Figure 20 is a graph showing the results of detection of specific serum IgG antibodies after challenge with HPSS1 in a CTB-UreB/4CTB chimeric protein-immunized mouse according to an embodiment of the present invention
  • Figure 21 is a graph showing the results of detection of specific intestinal mucosal slgA antibodies after challenge with HPSS1 in a CTB-UreB/4CTB chimeric protein-immunized mouse according to an embodiment of the present invention
  • Figure 22 is a graph showing the results of pathological section examination of gastric tissue after challenge with HPSS1 in a CTB-UreB/4CTB chimeric protein-immunized mouse according to an embodiment of the present invention.
  • B UreB group mice stomach tissue section (x250)
  • C UreB group mice stomach tissue section ( ⁇ 400) - ;
  • Figure 23 ⁇ and 23 ⁇ are the assembly effects of UreB-CTA 2 /5CTB and CTB-UreB/4CTB, respectively.
  • the electropherogram of the rate comparison results Lane 1 is the experimental result of Comparative Example 1
  • Lane 2 is the experimental result according to the embodiment of the present invention. detailed description
  • the present invention mainly provides an antigen which enhances immunogenicity and a preparation method thereof. On the one hand, it enhances the immune response by using mucosal immune adjuvants; on the other hand, the use of CTB or LTB to form pentameric features enhances the mucosal immune adjuvant effect while increasing the volume of the antigen and is easily taken up by APC. More conducive to the occurrence of immune response. Further, the inventors of the present invention have selected to maintain a certain concentration of urea and DTT (or urea and mercaptoethanol) in the reconstituted liquid, and can effectively prevent the monomer forming the multimer from aggregating itself into a pentamer, thereby promoting chimerism. Formation, improving the efficiency of chimer assembly. It effectively overcomes the defects that the conventional renaturation treatment leads to low assembly efficiency of the insert.
  • urea and DTT or urea and mercaptoethanol
  • the present invention selects CTB or LTB to bind to the antigen to form a new antigen composition.
  • Cholera toxin is a potent mucosal immune adjuvant consisting of one A subunit and five B subunits (see Figure 1).
  • the five B subunits are non-covalently combined into a very compact and stable barrel-shaped pentamer (see Figure 2).
  • the B subunit monomer has a three-chain anti-parallel sheet on each side, and in the pentamer each other Adjacent monomers interact through the lamellar layer and a large number of salt bonds, making the B subunit pentamer one of the most stable protein complexes.
  • CTA is the active part of cholera toxin
  • CTB is non-toxic and its primary function is to produce an immune response by binding CTA into cells by binding to monosialoganglioside (GM1) on mammalian intestinal epithelial cells.
  • GM1 monosialoganglioside
  • CTB is highly immunogenic because it increases the uptake rate of the intestinal tract to its oral vaccine by binding to GM1, and also specifically affects the tight junction or small band atresia between small intestinal mucosa cells. Permeability prevents oral vaccines from being digested and decomposed in the small intestinal mucosa, maintaining their antigenicity, thereby increasing the antibody titer produced by the body and allowing the body to produce a good immune response.
  • CTB is currently considered to be one of the most effective and safe mucosal immune adjuvants to date.
  • LTB is also a highly effective mucosal immune adjuvant.
  • Escherichia coli heat-labile enterotoxin (LT) is a heat-labile exotoxin secreted to the periplasm by enterotoxic Escherichia coli. It is composed of A subunit with ADP ribosyltransferase activity and binds to gangliosides.
  • the B subunit consists of the AB 5 hexameric protein, the A subunit is the major unit of its toxicity, and the B subunit has immunogenicity and adjuvant function.
  • LT is not only immunogenic, but also an effective mucosal adjuvant that significantly enhances the body's IgA and IgG responses to candidate antigens, while also reducing the body's immune tolerance to these candidate antigens, inducing long-term effects on them. Memory, so LTB has received extensive attention as a mucosal adjuvant.
  • the present invention provides a recombinant antigen composition
  • a recombinant antigen composition comprising: a fusion protein of an antigen with CTB or LTB; and a corresponding CTB or LTB protein.
  • the combination of five CTB or LTB non-covalent bonds can form a chimeric structure of the complex of the above fusion protein with another four corresponding CTB or LTB monomers, thereby increasing the effective volume of the antigen and facilitating Ingested by APC.
  • CTB and LTB are known to be potent immunological adjuvants, which can reduce the body's immune tolerance to candidate antigens, thereby more effectively exerting its immune-promoting effect.
  • a higher titer antigen is formed by the formation of an antigen and a chimeric protein of five CTB or LTB, thereby enhancing the immunogenicity of the entire antigen composition.
  • Antigens including but not limited to Helicobacter pylori antigen, typhoid antigen, influenza HA antigen.
  • the antigen chimera prepared by the present invention can enhance the immunogenicity of the relevant antigen and facilitate the construction of a more effective vaccine.
  • HP Helicobacter pylori
  • HP is a Gram-negative microaerobic bacterium that is parasitic on the surface of gastric epithelial cells. It was first isolated from human gastric mucosa by Australian researchers Warren and Marshall in 1983. HP can be acidic. Colonization and survival in the environment, the body produces inflammation and immune response, destroying the gastric mucosal barrier, and the balance of apoptosis and proliferation of gastric mucosal epithelial cells. HP is an important pathogen of human upper gastrointestinal diseases and is the main cause of chronic gastritis, gastric ulcer and duodenal ulcer. In 1994, WHO confirmed that it is closely related to the occurrence of gastric cancer and listed it as a class of carcinogenic factors. HP is one of the most infectious bacteria in the world. It is reported that 90% of Asians and 60% of Europeans are infected with HP, so prevention and treatment of HP infection has become the focus of global attention.
  • HP vaccines are mainly whole-bacteria vaccines, subunit vaccines (genetical engineering vaccines), live vector vaccines and DNA vaccines.
  • HP whole or whole lysates or HP toxic proteins such as urease, vacuolating toxin, toxin-related protein, neutrophil activator, etc., alone or in combination with different adjuvants, in animal models. Both can cause a protective response.
  • Prophylactic vaccines work through the body's immune response, so choosing an effective HP antigen to immunize the body can stimulate the body to produce a protective immune response, thus preventing the body from feeling The role of HP.
  • an effective HP antigen to immunize the body can stimulate the body to produce a protective immune response, thus preventing the body from feeling The role of HP.
  • the construction and application of genetic engineering strains provide favorable conditions for screening effective antigens, and it is a key step to prevent and eradicate HP infection.
  • the inventors preferred Helicobacter pylori urease, Helicobacter pylori CagA protein and Helicobacter pylori NAP as effective antigens for Helicobacter pylori.
  • Urease has the function of decomposing urea and neutralizing gastric acid during HP colonization infection, which is important for the pathogenicity of HP.
  • HP urease is both a colonization factor and a virulence factor.
  • the urease gene is co-owned by all HP strains, and the encoded protein has a strong urease activity.
  • the urease is distributed on the surface of HP, accounting for 5-10% of the total protein.
  • the urease gene size is currently about 7.5 kb, and there are 9 reading frames (ORF) UreA, UreB, UreC, UreD, UreE, UreF, UreG, UreH and Urel.
  • ORF 9 reading frames
  • UreA and UreB are two structural subunits of urease, which are highly conserved. They are the target genes for PCR detection of HP infection and the candidate genes for gene vaccines.
  • UreB is HP's main outer membrane antigen component, encoded by 569 amino acid residues. It is the most antigenic protective protein of HP. It is non-toxic, antigenic and relatively conservative, and its pathogenic process in HP. It plays a key role. The amino acid homology of urease B subunit reached 97.9% or more by comparison of different strains, indicating that the antigenic variability of UreB between HP strains was extremely small. In addition, the main active part of UreB is located on the surface of the bacteria, and its large molecular weight and granular structure facilitate mucosal immunization, so urease molecules can be used as vaccine antigens. Reasonable choice.
  • the HP surface has the property of adhering to cytoplasmic proteins, which can adhere the autolyzed released urease to the surface of live bacteria, which provides an important theoretical basis for the research of HP vaccine. It has been confirmed by experiments that UreB protein vaccine can induce and stimulate the protective immune response of the body through mucosal immunization, indicating that UreB is one of the important protective candidate antigens for HP infection, and is also the preferred antigen for genetic engineering vaccines. It has significant advantages. .
  • mice immunized with Helicobacter Felis were orally immunized with recombinant urease subunit B (rUreB), and it was found that the immunized mice not only completely cleared Hf infection, but also Preventing mice from re-infection; Kleanthous et al. used rUreB plus LT as a mucosal adjuvant to immunize mice.
  • Kleanthous et al. used rUreB plus LT as a mucosal adjuvant to immunize mice.
  • the urease activity in the stomach of the immunized group was significantly decreased, and the HP culture of gastric mucosa showed a decrease in HP. More than 100%; Michetti et al.
  • the CagA gene is also called a cytotoxin-related gene, and its expression product is a CagA protein.
  • a toxin secreted by bacteria it is phosphorylated by the type IV secretion system Cag pathogenicity island (PAI) from bacteria to host cells, involved in signal transduction of host cells and causes rearrangement of cytoskeletal structures.
  • PAI Cag pathogenicity island
  • cagA-positive HP is a virulent strain, which is closely related to common digestive tract diseases such as atrophic gastritis, gastric cancer and peptic ulcer. More than 90% of HP clinical isolates are positive for cagA.
  • HP with the CagA gene almost exclusively expresses the CagA protein and produces a measurable antibody response.
  • CagA-positive strains can directly or indirectly (through NF-KB) induce secretion of IL-8 from gastric epithelial cells, and IL-8 plays an important role in neutrophil chemotaxis and activation, leading to gastric mucosal damage.
  • Animal experiments have found that CagA gene and CagA protein have a good correlation with gastritis, peptic ulcer, especially gastric cancer. Marchetti et al. immunized the HP mouse infection model with recombinant CagA, which was shown to induce a protective immune response with a 70% protection rate.
  • CagA has high feasibility as a protective antigen for HP vaccine, but the protective effect of this antigen is only limited to strains producing CagA, and has no protective effect on strains that do not produce CagA. It can be seen that there is currently no satisfactory vaccine against CagA protein.
  • NAP is highly conserved, and clinical studies have found that 60% of HP-infected patients have NAP-specific antibodies in their serum.
  • NAP is a ferritin, and the NAP gene encoding it is detectable in almost all HP, but the activity of NAP expressed in vitro varies greatly. It can pass CDlla/CD18 and CD11b/CD18 interact with endothelial cell adhesion molecule (ICAM-1) to bind leukocytes to endothelial cells; selectively bind to acidic sphingolipids of neutrophils to regulate leukocyte function;
  • IAM-1 endothelial cell adhesion molecule
  • the combination with mucin has laid a foundation for HP colonization in gastric epithelial cells, so NAP plays an important role in its adhesion and pathogenesis. Satm et al. orally immunized 10 mice with purified recombinant NAP, and as a result, 8 mice obtained protective immunity. It is suggested that NAP can be used as an
  • the invention provides a recombinant H. pylori antigen composition
  • a recombinant H. pylori antigen composition comprising a fusion protein of H. pylori antigen and CTB and a CTB protein.
  • CTB itself, the properties of pentamers can be formed, the volume of the antigen is increased, and it is easily taken up by APC.
  • CTB is a good immune adjuvant. Therefore, the recombinant H. pylori antigen composition of the present invention can effectively enhance the immunogenicity of the H. pylori antigen, and is advantageous for constructing a more effective vaccine.
  • the H. pylori antigen is selected from at least one of the Helicobacter pylori UreB protein, the H. pylori CagA protein, and H. pylori NAP. These antigens are currently non-toxic, antigenic and relatively conservative antigens of the currently known H. pylori antigens, and are more suitable for use in vaccines. Selection of the combination of antigen and immune adjuvant
  • Conventional immunological adjuvants are generally used by direct mixing with an antigen and then administered simultaneously with an antigen to stimulate the body's immune response, but its effect of enhancing antigen immunogenicity is not desirable.
  • the invention prepares a chimeric protein by preparing a fusion protein of an antigen and a mucosal immune adjuvant protein monomer, and then forming a chimeric protein by non-covalent binding to the mucosal immunoadjuvant protein monomer capable of forming a multimer, not only can maintain the same
  • the mucosal immune adjuvant properties also increase the antigen volume and are more readily taken up by antigen-presenting cells.
  • the antigen and the protein of CTB (or LTB) in the present invention A flexible linker is added between them so that the respective folds of the antigen and CTB (or LTB) after protein fusion are not affected, thereby forming the correct conformation, respectively.
  • the invention selects three G4S linkers.
  • the present invention is not limited to the use of such a linker, and other linkers such as a linker peptide (A (EAAAK) nA) and a glucocerein A (PA) in a spiral form can also be used in the present invention. .
  • the present invention also provides a kit comprising a vector expressing a fusion protein of the above antigen and CTB (or LTB) and a vector expressing a corresponding mucosal immunoadjuvant monomeric protein (CTB or LTB) protein.
  • CTB mucosal immunoadjuvant monomeric protein
  • the expression vector should also include elements necessary for expression of the protein of interest, such as a promoter, a terminator, an alternative marker gene, and the like.
  • the expression vector is preferably a prokaryotic expression vector, for example, a prokaryotic vector such as the pET series.
  • a prokaryotic vector such as the pET series.
  • Methods and reagents for preparing a protein of interest using a prokaryotic expression vector are well known in the art, and can be found in commonly used reference books in the field, such as the Guide to Molecular Cloning, compiled by Cold Spring Harbor Laboratory ((US) Sambrook et al., Huang Peitang et al. Translation, Beijing, Science Press,), “Cellular Experiment Guide” ((US) DL Spector waiting, Huang Peitang et al., Beijing, Science Press).
  • the inventors of the present invention have selected to maintain a certain concentration of urea and DTT (or urea and mercaptoethanol) in the reconstituted liquid, which can effectively prevent the monomer forming the multimer from aggregating into a pentamer and promoting the formation of the chimera. Improve the efficiency of chimera assembly. It effectively overcomes the defects that the conventional renaturation treatment leads to low assembly efficiency of the chimera.
  • the present invention provides a method for preparing a chimera by renaturation, which comprises (1) a fusion protein of a protein with a monomeric protein capable of forming a multimer and (2) a single molecule capable of forming a multimer a bulk protein, which forms a chimera by forming a multimer of the monomeric protein in the (1) fusion protein and the (2) monomeric protein, the method comprising: forming the multimer by the (2) Monomeric protein is pre-refolded in a buffer containing 6M to 9M, preferably 8M urea, and having a pH of 3.0 to 4.0 for 0.5 to 3 hours, preferably pre-refolded for 1 hour; and the (1) fusion protein and The (2) monomeric protein capable of forming a multimer is renatured to form the chimera in a DTT or mercaptoethanol reconstitution solution containing 1.0 M to 2.5 M urea and 0.2 mM to 1.0 mM.
  • the present invention utilizes a reconstituted solution containing a concentration of urea and a reducing agent to combine the fusion protein and the monomeric protein into a desired chimera during the slow renaturation process.
  • a high yield of chimera is unexpectedly obtained by pre-reversion under certain pH conditions and then slowly renaturation in a slightly lower concentration urea solution in the presence of a reducing agent.
  • the yield directly affects the cost of production and directly affects the possibility of mass production.
  • the UreB DNA sequence is from the HP strain MEL-HP27, and the CTB DNA sequence is from Vibrio cholerae 0395.
  • a glycine/serine-rich (G4S) 3 linker sequence is introduced between the fusion DNA fragments (underlined in italics) ), and synthesize the CTB-UreB gene codon.
  • the gene sequence is SEQ ID No. 1:
  • the target DNA in the agarose gel is recovered and purified, and then the ligation reaction is carried out.
  • the ligation reaction is carried out at 16 ° C for 16 hours.
  • the design of the connection reaction system is as follows:
  • a small strain of DH5a glycerol preserved at -70 °C was picked, and a single colony of E. coli DH5a receptor was picked from a covered tube containing 5 ml of LB medium and shaken at 37 °C (200 rpm). After culturing for 10 to 12 hours, the seed liquid is obtained. 5 ml of the seed liquid is added to 100 ml of fresh LB medium and shaken (200 rpm). When the OD600 of the bacterial solution is 0.4 to 0.9, 1.5 ml of the fresh culture solution is transferred to the Eppendorf tube.
  • the cells were placed on ice for 10 minutes, then centrifuged at 4000 rpm for 10 minutes at 4 ° C, the supernatant was carefully discarded, and the first two steps were repeated to collect the cells.
  • the cells were gently suspended by adding ice-cold 0.1 mol/L CaCl 2 solution to 300 ⁇ l, placed on ice for 30 minutes, centrifuged at 4000 rpm for 10 minutes at 4 ° C, the supernatant was discarded, and the precipitate was added with ⁇ precooled 0.1 mol/L CaCl. 2
  • the solution gently suspends the cells, which is a competent cell.
  • competent cells need to be stored for later use, resuspend the cells with pre-cooled 30 ⁇ 150% glycerol and 70 ⁇ l 0.1mol/L CaCl 2 solution (final glycerol concentration 15%), and store at -70 °C. Save for half a year), spare.
  • the competent cell suspension was taken out from the -70 °C refrigerator, thawed at room temperature, immediately placed on ice after thawing, and the reaction solution was added (content not exceeding 50 ng, volume not exceeding 10 ⁇ 1), gently bombed, iced After standing for 30 minutes, heat shock 90 in a 42 °C water bath, quickly set on ice for 5 minutes, add 800 ⁇ l of ⁇ liquid medium to the tube, mix and shake at 37 ° C for 150 hours at 150 rpm for 1.5 hours. Screening plates containing kanamycin (Kan) were incubated overnight at 37 °C.
  • the transformed DH5a single colony was picked and the plasmid was extracted. After double digestion, the fragment size was
  • the seed was activated with LB (tryptone 10 g / L; yeast extract 5 g / L; NaCl 10 g / L) medium, prepared in a volume of 50 mL, sterilized at 121 ° C for 20 minutes, cooled and added to Kan (50 mg / L) And the working seed batch liquid solution is 50 ⁇ 1, 37 °C ⁇ 1 °C, and cultured for 15-16 hours.
  • LB tryptone 10 g / L
  • NaCl 10 g / L NaCl 10 g / L
  • the seed was treated with 2 YT medium (tryptone 16 g/L; yeast extract 10 g / L; NaCl 5 g/L), and the preparation volume was 3 L. After sterilization at 121 °C for 20 minutes, the activated seed solution was added 3 ml, 37 °C ⁇ 1 °C, culture for 4 to 6 hours.
  • 2 YT medium tryptone 16 g/L
  • yeast extract 10 g / L yeast extract 10 g / L
  • NaCl 5 g/L NaCl 5 g/L
  • the sterilized purified water was used to dissolve 5.0 g of IPTG, filtered through a 0.22 ⁇ m filter, and placed in an ice bath at 2-8 ° C for a storage period of 24 h.
  • the fermentation broth was transferred to a pre-cooled storage tank, and after the fermentation broth was cooled to below 15 ° C, the cells were collected by centrifugation using a tube centrifuge.
  • the fermentation broth is transferred to a pre-cooled storage tank, and after the fermentation liquid is cooled to below 15 ° C, the cells are collected by centrifugation using a tube centrifuge. .
  • the cells were suspended in a ratio of 1:10 (W/V) with 50 mM Tris-HCl, 0.5% Triton X-100, 150 mM NaCl, ImM EDTA pH 8.0 buffer, and pre-cooled at 4 ° C, and the high-pressure homogenate was broken for 900 bar. Three times, centrifuged at 14,000 g for 20 min, and the precipitate was collected.
  • the washing conditions were as follows: stirring at room temperature for 30 min, and centrifugation at 6000 g for 20 min.
  • Washing solution 20 mM PB, 8 M urea, 30 mM NaCl, 5 mM DTT, 10% glycerol pH 7.2, conductance less than 3.7 ms/m
  • the CTB stock solution was diluted with 50 mM Tris-HCl, 8 M urea, pH 8.0 buffer to adjust the pH to 3.0-4.0, allowed to stand at room temperature for 1 hour, then adjusted to pH 8.0, and placed at 4 ° C until use.
  • the two proteins CTB-UreB and CTB monomer were mixed at a molar ratio of 1:4, and then diluted 5 times into the final reconstituted solution (50 mM Tris-HCl pH 8.0 + 10% glycerol + 150 mM NaCl).
  • the concentration of DTT ranges from 0.2 to 1.0 mM and the urea concentration ranges from 1.0 M to 2.5 M. Leave at room temperature overnight.
  • Buffer A 20 mM Tris-HCl + 5% glycerol pH 7.5
  • Buffer B 20 mM Tris-HCl + 5% glycerol + 0.3 M NaCl pH 7.5
  • B 10CV elutes the protein of interest.
  • the CTB-UreB/4CTB purified electrophoresis is shown in Figure 9A.
  • the purified CTB-UreB/4CTB antigen chimera was adjusted to different pH values with dilute hydrochloric acid. After standing at room temperature for 1 hour, the sample was taken for SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 9B.
  • the samples of each lane are as follows: M: Pre-staining of Fermentas protein; samples of lanes 1-7 were electrophoresis results of samples treated under conditions of pH 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, and 3.0, respectively.
  • the chimeric protein is stable above pH 7.0, especially pH 8.0, and no protein bands having a molecular weight smaller than the chimera are present.
  • the CTB-UreB/4CTB antigen chimera is stable in a neutral alkaline environment and is easily dissociated in an acidic environment. Specifically, it is relatively stable in the case of pH > 7.0, and preferably pH is 8.0.
  • the gene sequence is SEQ ID No.: 2, where linker is underlined in italics:
  • the inclusion bodies were mixed with the inclusion body solution at a ratio of 1:20 (W/V), stirred at 4 °C for 3 hours, and overnight, and centrifuged at 12,000 rpm for 30 minutes to collect the supernatant.
  • Buffer A 20 mM Tirs-HCl + 8 M urea + 5 mM DTT 5% glycerol H7.5
  • Buffer B 20 mM Tirs-HCl, 8 M urea, l. OM NaCl, 5 mMDTT 5% glycerol, pH 7.5
  • Buffer A 20 mM Tris-HCl, 8 M urea, 5 mM DTT, 5% glycerol, pH 7.5
  • Buffer B 20 mM Tirs-HCl, 8 M urea, 150 mM NaCl, 5 mM DTT 5% glycerin, pH 7.5
  • the CTB-CagA purification electrophoresis is shown in Figure 10.
  • the CTB stock solution was diluted with 50 mM Tris-HCl, 8 M urea, pH 8.0 buffer to adjust the pH to 3.0-4.0, allowed to stand at room temperature for 1 hour, then adjusted to pH 8.0, and placed at 4 ° C until use.
  • the two proteins CTB-CagA and CTB monomer were mixed at a molar ratio of 1:4, and then diluted 5 times into the final reconstituted solution (50 mM Tris-HCl pH 8.0 + 10% glycerol + 150 mM NaCl).
  • the concentration of DTT ranges from 0.2 to 1.0 mM and the urea concentration ranges from 1.0 M to 2.5 M. Leave at room temperature overnight.
  • Buffer B 20 mM Tris-HCl + 5% glycerol + 200 mM imidazole eluted protein of interest 100% B wash
  • Buffer A 20 mM Tris-HCl + 5% glycerol pH 7.5
  • Buffer B 20 mM Tris-HCl + 5% glycerol + 0.3 M NaCl pH 7.5
  • the purified CTB-CagA/4CTB antigen chimera was adjusted to different pH values with dilute hydrochloric acid. After standing at room temperature for 1 hour, the sample was taken for SDS-PAGE electrophoresis. The electrophoresis results are shown in Fig. 11B.
  • the samples of each lane are as follows: M: Pre-staining of Fermentas protein; samples of lanes 1-7 were electrophoresis results of samples treated under conditions of pH 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, and 3.0, respectively.
  • the chimeric protein is stable above pH 7.0, especially pH 8.0, and no protein bands having a molecular weight smaller than the chimera are present.
  • the CTB-CagA/4CTB antigen chimera is stable in a neutral alkaline environment and is easily dissociated in an acidic environment. Specifically, it is relatively stable in the case of pH > 7.0, and preferably pH is 8.0.
  • the CTB-NAP was prepared in the same manner as in Example 1, except that the NAP gene was used instead of the UreB gene.
  • the constructed engineered plasmid was double-digested and the fragment size was 789 bp (see Figure 5).
  • the expression of the fusion protein is shown in Figure 7.
  • the gene sequence is SEQ ID No.: 3, wherein the linker is drawn in italics
  • Buffer A 20 mM Tris-HCl + 5% glycerol pH 7.5
  • Buffer B 20 mM Tris-HCl + 5% glycerol + 0.3 M NaCl pH 7.5
  • CCTTBB--NNAAPP treatment : Using 2200mmMM TTrriiss--HHCCll, 88MM urinary urea, ppHH88..00 buffer buffer will be above CCTTBB--
  • the NNAAPP-like sample was diluted and diluted, and the ppHH was adjusted to 88..00, and the addition of thioglycolethanol was added to make it a final concentration of 33 mmMM. .
  • CCTTBB treatment :: Using 2200mmMM TTrriiss--HHCCll, 88MM urinary urea, ppHH88..00 buffer will be
  • the CCTTBB original stock solution is diluted and diluted, and the ppHH is adjusted to 33..55 ⁇ 33..77, and placed for 11 hours, then ppHH is adjusted to 88..00, and The addition of hydrazinyl glycol alcohol was made to have a final concentration of 33 mmMM. .
  • ((33)) Refractory Take the above two kinds of egg white protein CCTTBB--NNAAPP and the CCTTBB single monomer to the molar ratio of 11 :: 44 mixed mix, use 5500mmMM TTrriiss--HHCCll,, 55%% glycerol oil, 00..0033mmMM GGSSSSGG,, ppHH88..00 iioo buffer solution to dilute at 11::66 Release, and control and control of the concentration range of the thiol glycol ethanol range between 00..22--11.. OOmmMM, the concentration range of urinary urea concentration range Between 11..00MM--22..55MM. . Place the 2211 ° ° CC placed overnight night. .
  • Slow buffering liquid AA :: 5500mmMM TTrriiss--HHCCll,, 55%% glycerol oil, 2200mmMM imidazole, PPHH88..00
  • buffer buffer BB : 5500mmMM TTrriiss--HHCCll ,, 55% % glycerol oil, 330000mmMM imidazole, PPHH88..00
  • Replicative sample sample supplementation plus end concentration concentration is 2200mmMM imidazole, upper NNII FFFF, flow velocity 88mmll//mmiinn, use
  • Buffer buffer BB 5500mmMM TTrriiss--HHCCll ,, 55%% glycerol oil, 11 ⁇ NNaaCCll PPHH88..00
  • CCTTBB--NNAAPP//44CCTTBB embedded chimeric egg protein white pure purified electrophoresis swimming chart is shown in Figure 1133. .
  • Test animals 8-10 weeks, body weight 18-20g, SPF female B ALB/c mice,
  • Test drug UreB, CTB-UreB fusion protein, CTB-UreB /4CTB chimeric protein were diluted with Tris-HCl buffer (pH 8.0), UreB protein immunization dose was 20 ( g / only, CTB-UreB Both the fusion protein and the CTB-UreB/4CTB chimeric protein were administered in equimolar amounts with the UreB protein, and the antacid was diluted to 50 mg/ml with PBS.
  • Tests 4 groups UreB IS, CTB-UreB group, CTB-UreB / 4CTB group and saline group (or, blank control group (CON, Control)).
  • mice were immunized 4 times and immunized once every 7 days.
  • 0.2 ml of the test drug was administered orally to each animal, and 0.2 ml of the antacid was administered 10 mm before administration. All animals were fasted for 12 hours before administration, and water could not be forbidden. After 1 hour of administration, they were fed with water.
  • 96-well microtiter plate was added with ⁇ Ure B antigen (1.25 g / ml) per well, and coated at 4 °C overnight. The solution was removed from the wells the next day and washed 3 times with PBST; each well was filled with blocking solution 200 ⁇ (including 2 %BSA), 37.
  • the BALB/C mice were orally immunized with the HP vaccine, and their serum IgG and intestinal mucosal slgA antibody responses are shown in Figures 14 and 15.
  • the antibodies in the blank control group and the UreB protein group did not increase after administration.
  • the serum IgG antibodies of the CTB-UreB fusion protein and the CTB-UreB/4CTB chimeric protein group were significantly 4 weeks after the first administration.
  • the increase reached a peak at 8 weeks, and there was a significant difference (PO.001) compared with the blank control group.
  • the intestinal mucosal slgA antibody was also significantly increased, which was significantly different from the blank control group (P ⁇ 0.001).
  • the anti-UreB antibody of the CTB-UreB fusion protein group had a positive conversion rate of 60%
  • the anti-UreB antibody of the CTB-UreB/4CTB chimeric protein group had a positive conversion rate of 95%, indicating that CTB-UreB
  • the /4CTB chimeric protein vaccine induces a significant immune response in the body.
  • Oral administration of recombinant CTB-UreB fusion protein and CTB-UreB/4CTB chimeric protein can stimulate the body to produce specific antibodies against UreB antigen.
  • the CTB-UreB/4CTB chimeric protein has a significantly better immune response than the CTB-UreB fusion protein, indicating that the CTB-UreB/4CTB chimeric protein has better immunoreactivity and antigenicity and can be used as a candidate for the prevention of Helicobacter pylori. vaccine.
  • the immunization method is basically the same as 2.1.1.
  • the test drug CagA protein, CTB-CagA fusion protein, CTB-Ca g A/4CTB chimeric protein were diluted with Tris-HCl buffer (pH 8.0), CagA protein immunization dose was 200 ⁇ , CTB-CagA fusion protein and CTB-CagA/4CTB chimeric proteins are administered in equimolar amounts with CagA protein, and the drug delivery capacity It is 0.2ml/only. Plates were coated with CagA antigen during ELISA. The serum IgG and intestinal mucosal slgA antibody responses are shown in Figures 16 and 17.
  • CTB-CagA/4CTB chimeric protein can stimulate the body to produce specific antibodies against CagA antigen. It indicates that the CTB-CagA/4CTB chimeric protein has better immunoreactivity and antigenicity and can be used as a vaccine candidate for the prevention of Helicobacter pylori.
  • the immunization method is basically the same as 2.1.1.
  • the test drug NAP protein, CTB-NAP fusion protein, CTB-NAP/4CTB chimeric protein were diluted with Tris-HCl buffer (pH 8.0), NAP fusion protein immunization dose was 200 ⁇ , CTB-NAP fusion protein and CTB.
  • the -NAP/4CTB chimeric protein was administered in an equimolar amount with the UreB protein, and the administration capacity was 0.2 ml/mouse. Plates were coated with CagA antigen during ELISA.
  • mice were orally immunized with the HP vaccine, and their serum IgG and intestinal mucosal slgA antibody responses are shown in Figures 18 and 19.
  • CTB-NAP/4CTB chimeric protein can stimulate the body to produce specific antibodies against NAP antigen. It indicates that the CTB-NAP/4CTB chimeric protein has better immunoreactivity and antigenicity and can be used as a vaccine candidate for the prevention of Helicobacter pylori.
  • the experimental group and the negative control group were intragastrically administered with 0.2 ml ( ⁇ ⁇ 9 CFU/ml) of HP SSI for 2 consecutive days after the last immunization, once a day, and given 0.2 ml before ligation of HP SSI for 10 min. Antacid.
  • Serum and intestinal mucosa collection and detection methods are the same as 2 ⁇ 1.5.
  • the stomach was taken by laparotomy.
  • the stomach tissue was divided into three parts along the vertical axis. Each part included the fundus, corpus and antrum.
  • One for quantitative bacterial culture The antrum tissue was taken with a sterile sputum, and the mucosa was facing down. It was applied to a HP selective culture plate at 37 ° C, and cultured under microaerobic conditions for about 1 day. See transparent and smooth. , needle-shaped small colonies, that is, positive.
  • a 96-well plate rapid urease test the urease reagent was added to each well of a 96-well plate containing rat stomach tissue, and each well was quantified to ⁇ , and allowed to stand at room temperature for 0.5 h, and the reagent was changed from yellow to red. Positive, that is, the sample has Hp infection.
  • the other was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, HE stained, and stained with Gmisa.
  • the colonization of HP SSI and the inflammatory response of the tissue were observed under a microscope.
  • mice in each group had good mental state and no adverse reactions such as wilting and diarrhea were found.
  • mice in each group showed that 8 animals in the model group were infected with HPSS1, urease and tissue smear were positive; blank control group (not given HPSS) Attack) All animals had negative results for urease and tissue smears; 7 animals in the UreB group showed positive results for urease and tissue smear; CTB-UreB group showed positive results for 5 animals with urease, 4 Animal smear results were positive; only one animal in the CTB-UreB/4CTB group showed positive urease test, while all animal smear results were negative. The results are shown in Table 1.
  • Table 1 Example of the present invention rHP vaccine was orally administered to mice, and HPSS1 was used after HP test results.
  • the CTB-UreB/4CTB chimeric protein can effectively induce the immune response by oral immunization, and has a good protective effect on mice challenged by HP SSI. It has a good application prospect as an oral vaccine against HP infection.
  • the results of the above test examples indicate that oral administration of recombinant CTB-UreB fusion protein and CTB-UreB/4CTB chimeric protein can stimulate the body to produce specific antibodies against UreB antigen; CTB-UreB/4CTB chimeric protein is used to immunize mice.
  • Serum anti-UreB antigen-specific IgG and gastric mucus s-IgA antibody titers and positive conversion rates were significantly higher than CTB-UreB fusion protein, which was significantly different from the blank control group, indicating that the above antigen has good immunoreactivity. And antigenicity, can be used as a candidate vaccine for HP.
  • results of the secretory IgA assays of Table 2 and Figure 14 also indicate that the chimeric protein composed of the recombinant antigen of the present invention facilitates the penetration of the antigen into the intestinal mucosa and stimulates the mucosa to produce secretory IgA immunity.
  • Comparative example 1
  • the inventors constructed vectors and expressed fusion proteins of Helicobacter pylori antigens such as UreB and cholera toxin into 2 subunits (CTA 2 ), and combined them with the expressed CTB protein in vitro to form UreB-CTA 2 /5 CTB. Protein.
  • the inventors attempted to simultaneously express CTB-antigen fusion protein and CTB using a eukaryotic expression system represented by yeast, and it is desired to obtain a naturally occurring chimeric structure having a desired effect.
  • the inventors found that when expressed in a yeast system, CTB correctly formed a pentameric structure composed of five identical CTB monomers, but the expressed CTB molecular weight (about 65 KD) was significantly greater than in the native state (55 KD). It is suggested that when expressed in eukaryotic system, the glycosylation post-translational modification of CTB may occur, resulting in a large difference between the expressed CTB and the natural CTB molecule, which in turn affects the binding ability to the GM1 receptor.
  • the size of the antigen fused to CTB is strictly required, and when this expression is used, the fusion protein and the CTB monomer are naturally assembled in a low efficiency, resulting in a low yield of the entire expression system. , does not apply to the preparation of large-scale vaccines.
  • the inventors attempted to simultaneously express the CTB-antigen fusion protein and CTB in a prokaryotic expression system represented by Escherichia coli, and it is expected that a naturally occurring chimera can be obtained.
  • Cholera toxin B The assembly mechanism of the pentamer of the subunit is unknown, and the study agrees that the assembly of the chimera occurs during the transmembrane process.
  • the CTB-antigen fusion protein with a signal peptide fails to penetrate the membrane correctly, forming the desired chimeric structure.
  • the CTB-X-4CTB system of the present invention can also be applied to a variety of other mucosal immune antigens, including but not limited to Helicobacter pylori antigen, typhoid antigen, influenza HA antigen, The immunogenicity of these antigens, and the preparation of high titer vaccines.
  • the corresponding vector selection and preparation, protein expression and purification are all well-known in the art, and those skilled in the art can use the CTB-X-4CTB system of the present invention to prepare corresponding ones after understanding the invention of the present invention.
  • the protein and antigen compositions are fused and tested for their corresponding increase in immunogenicity.

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Abstract

本发明提供了一种抗原嵌合体，包括：抗原与能形成多聚体的黏膜免疫佐剂蛋白质单体的融合蛋白，和所述能形成多聚体的黏膜免疫佐剂蛋白质单体，其中，所述能形成多聚体的黏膜免疫佐剂蛋白质单体选自霍乱毒素B亚单位（CTB）和大肠杆菌不耐热肠毒素B亚单位（LTB）中的一种，所述多聚体为五聚体，而且在所述嵌合体中所述融合蛋白与所述能形成多聚体的黏膜免疫佐剂蛋白质单体的摩尔比为1:4。本发明利用黏膜免疫佐剂蛋白质能形成五聚体的特性，形成嵌合结构，从而形成效价更高的抗原。同时，利用黏膜免疫佐剂蛋白质增强免疫的作用，起到增强抗原免疫原性的效果。另外，本发明的重组抗原构成的嵌合蛋白抗原刺激黏膜产生分泌型IgA，诱导黏膜免疫产生。

Description

抗原嵌合体、 抗原组合物、 疫苗及其制备方法和试剂盒 技术领域
本发明涉及免疫领域， 特别是用于黏膜免疫的抗原、 疫苗及其制备 方法和试剂盒。 发明背景
黏膜免疫系统是广泛分布于呼吸道、 胃肠道、 泌尿生殖道黏膜下及 一些外分泌腺处的淋巴组织， 是执行局部特异性免疫功能的主要场所。
由于 95%以上的机体相关感染发生在黏膜部位或经由黏膜入侵机 体， 因此黏膜是病原体侵入体内的最大门户。 当前对动物生命与健康危 害极大的以及难以防治的重要传染病， 如流感、 结核、 鼻疽、 沙门氏菌 病等都属于由黏膜入侵或与黏膜相关的疾病， 且由此引起的黏膜损伤及 黏膜免疫功能的紊乱， 常常成为机会性病原菌感染， 甚至肿瘤发生的重 要机制。
黏膜免疫系统是机体抵抗病原体入侵的第一道免疫屏障， 具有独特 结构和功能的独立免疫体系， 对于防治病原体的定植和侵入有着积极的 意义。 有别于传统的系统免疫系统， 黏膜免疫系统是大量免疫细胞和免 疫分子弥散在黏膜上皮内或黏膜下固有层 （弥散淋巴组织）， 或由单个 或多个淋巴滤泡聚集成的黏膜相关淋巴组织，机体 50%以上的淋巴组织 和 80%以上的免疫细胞集中于黏膜免疫系统。并且黏膜分泌物中的抗体 以分泌型 IgA和 slgM抗体为主。 黏膜免疫系统按功能不同可分为两个 部位： 诱导部位和效应部位。 在诱导部位和效应部位间， 主要通过淋巴 细胞归巢发生联系。 黏膜免疫系统主要功能是对黏膜表面吸入或摄入的 大量种类繁多的抗原进行识别并做出反应。 既可对大量无害抗原下调免 疫反应或产生耐受， 也可对有害抗原或病原体产生高效体液和细胞免 疫， 进行有效免疫排斥或清除。
黏膜免疫理论上是预防经黏膜感染产生致病作用病原体最为有效的 免疫途径， 因为通过其它途径免疫时， 疫苗难以诱导产生明显的黏膜免 疫反应。 然而， 迄今为止已获批准或正在临床研究中的疫苗， 绝大多数 却仍旧釆用的是注射途径免疫， 仅有少数为黏膜途径免疫。 究其原因， 困扰黏膜免疫疫苗产品开发的主要困难除了局部黏膜免疫特异性抗体 检测的困难外， 更为重要的是因为机体的宿主防御作用， 黏膜免疫无法 如注射途径免疫时能准确的控制进入机体的抗原水平。抗原通过口服或 者局部给药达到黏膜表面后， 将经历黏膜分泌液稀释、 粘液凝胶吸附、 蛋白酶 /核酸酶水解以及被内膜屏障清除，因此只有极少数的抗原能通过 黏膜进入体内， 发挥有效的黏膜免疫反应。 一般， 水溶性以及无黏膜作 用的抗原黏膜摄取率低， 并且部分可能会在肠内产生免疫耐受现象。 解 决黏膜免疫摄取率低最为有效的方法是通过对天然黏膜病原体的特征 模拟， 以及应用有效的黏膜佐剂， 增强与机体黏膜表面结合水平 （最好 能选择性的粘附于 M细胞）。 目前仍然需要更为有效的黏膜免疫疫苗。 发明内容
为了增强用于黏膜免疫抗原的免疫原性， 本发明提供了一种抗原嵌 合体， 其包括： 抗原与能形成多聚体的黏膜免疫佐剂蛋白质单体的融合 蛋白， 和所述能形成多聚体的黏膜免疫佐剂蛋白质单体， 其中， 所述能 形成多聚体的黏膜免疫佐剂蛋白质单体选自霍乱毒素 B亚单位 （CTB) 和大肠杆菌不耐热肠毒素 B亚单位 （LTB) 中的一种， 所述多聚体为五 聚体， 并且在所述嵌合体中所述融合蛋白与所述能形成多聚体的黏膜免 疫佐剂蛋白质单体的摩尔比为 1:4 ,其中所述抗原嵌合体稳定存在的 PH 大于 7.0， 优选 pH为 8.0。 CTB和 LTB都是非常有效的黏膜免疫佐剂， 而且都能够形成稳定的五聚体， 利用这一性质制备的抗原嵌合体， 不但 可保持其黏膜免疫佐剂性质， 还增大了抗原体积， 易于被抗原递呈细胞 (antigen presenting cell , APC) 摄取， 有利于进一步促进黏膜免疫和系 统免疫的激发。 并且， 嵌合体的稳定性与其 pH值密切相关， 在本发明 中， 所述嵌合体稳定存在的 PH大于 7.0， 优选 pH为 8.0。 而此条件下 的嵌合体，特别是 CTB形成五聚体的嵌合体，能够最有效地增强黏膜免 在本发明一个实施方式中， 所述的抗原嵌合体中的 CTB和 LTB为 其天然组成结构或其能够形成五聚体的突变体。 与真核表达时发生糖基 化修饰的 CTB和 LTB相比， 通过原核表达载体表达的具有天然结构的 CTB和 LTB及其突变体能够更加有效地激发抗原的免疫反应， 发挥其 佐剂的功能。
在本发明一个实施方式中， 所述抗原嵌合体中所述抗原的分子量在 10至 lOOkD范围内，优选 16kD至 65kD。在 10-lOOkD范围内的抗原能 够更好地保证 CTB或 LTB折叠成正确的构象， 以易于形成稳定的五聚 体。 抗原分子量过小可能会受 CTB或 LTB影响， 促使融合蛋白其自身 形成聚体， 从而不能正确折叠， 分子量过大则可能产生位阻效应， 阻碍 CTB或 LTB五聚体的形成。
在本发明的实施方式中， 所述抗原为适用于黏膜免疫的抗原。 特别 是经由黏膜途径感染人体或动物体的病原菌的抗原， 包括但不限于幽门 螺杆菌抗原、 伤寒抗原、 流感 HA抗原。
在本发明的一个优选实施方式中， 所述幽门螺杆菌抗原选自幽门螺 杆菌尿素酶 B亚单位（UreB)、幽门螺杆菌细胞毒素相关基因 A (CagA) 蛋白和幽门螺杆菌中性粒细胞激活蛋白 （NAP) 中的至少一种。 这些抗 原具较强的免疫原性， 无毒性， 是良好的疫苗候选抗原。
在本发明的实施方式中， 所述融合蛋白包含位于所述抗原与所述黏 膜免疫佐剂蛋白质单体之间的 3个 G4S (Gly-Ser-Ser-Ser-Ser) 连接体。 此柔性连接体的加入可以避免抗原蛋白质与 CTB或 LTB在复性折叠时 的相互影响， 有助于形成正确的构象。
本发明还提供了一种抗原组合物， 包含如上所述的抗原嵌合体， 其 中所述抗原嵌合体稳定存在的 PH大于 7.0 , 优选 pH为 8.0。
本发明还提供了一种疫苗， 包含上述抗原组合物和适用于疫苗的赋 形剂。 优选地， 所述疫苗为口服疫苗、 经鼻给药的疫苗或经直肠给药的 疫苗。
本发明还提供了一种用于制备抗原组合物的试剂盒， 包括表达上述 融合蛋白的载体和表达上述能形成多聚体的黏膜免疫佐剂蛋白质单体 的载体。 优选地， 其中所述载体均为原核表达载体。
本发明还提供了制备上述抗原嵌合体的方法， 包括用上述试剂盒中 的载体分别表达所述融合蛋白和所述能形成多聚体的黏膜免疫佐剂蛋 白质单体， 然后通过复性方法使这两种蛋白结合形成所述嵌合体， 其中 所述复性方法包括将所述两种蛋白共同放置在含有尿素和 DTT (或巯基 乙醇） 的复性液中进行复性的步骤。 优选所述复性液中尿素的浓度为 1.0M至 2.5M。 所述 DTT或巯基乙醇的浓度为 0.2mM至 1.0mM。
本发明还提供了一种通过复性制备嵌合体的方法， 所述嵌合体包含 ( 1) 一种蛋白质与能形成多聚体的单体蛋白质的融合蛋白和 （2) 能形 成多聚体的单体蛋白质， 通过所述 （1 ) 融合蛋白中的单体蛋白质和所 述 （2) 单体蛋白质形成多聚体来形成嵌合体， 所述方法包括：
将所述 （2) 能形成多聚体的单体蛋白质在含 6M至 9M, 优选 8M 的尿素， 且 pH为 3.0至 4.0的缓冲液中预复性 0.5至 3小时， 优选预复 f生 1小时； 和
将所述 （1 ) 融合蛋白和所述 （2) 能形成多聚体的单体蛋白质在含 有 1.0M至 2.5M的尿素和 0.2mM至 l.OmM的 DTT或巯基乙醇的复性 液中复性形成所述嵌合体，
优选地，所述复性液的 pH大于 7.0，更优选所述复性液的 pH为 8.0。 在本发明中， 我们选择在复性液中保持一定浓度的尿素和 DTT (或 巯基乙醇）， 能够有效避免形成多聚体的单体其自身聚集成为五聚体， 并且在一定 pH条件下， 能够促进嵌合体的形成， 提高嵌合体组装的效 率， 有效克服了釆用常规复性处理导致嵌合体组装效率较低的缺陷。 该 复性方法可用于形成需要形成蛋白质嵌合体的多个领域， 并不限于此处 所述的疫苗领域。 附图简要说明
图 1为电脑模拟的霍乱毒素 （CT) 立体结构图；
图 2为电脑模拟的 CTB五聚体立体结构图；
图 3为电脑模拟的 CTB-UreB融合蛋白立体结构图；
图 4为电脑模拟的 CTB-UreB/4CTB嵌合体蛋白的立体结构图； 图 5 为根据本发明的实施方式制备的 PET-28a-CTB-X质粒双酶切 电泳图， 其中各个泳道的样品如下： M: DL 10000 DNA Marker; 泳道 1,2,3 : PET-28a-CTB-UreB双酶切；泳道 4,5,6 : PET-28a-CTB-CagA双酶 切； 泳道 7 : PET-28a-CTB-NAP双酶切；
图 6为根据本发明的实施方式制备的 CTB-UreB融合蛋白的蛋白质 电泳图， 其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳 道 1,2,3,4,5,6,7： IPTG诱导后 PET-28a-CTB-UreB/BL21-DE3菌体；
图 7为根据本发明的实施方式制备的 CTB-CagA和 CTB-NAP融合 蛋白的蛋白电泳图，其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳道 1,2,3,4 : IPTG诱导后含 PET-28a-CTB-CagA/BL21-DE3菌 体； 泳道 5,6,7,8： IPTG诱导后含 PET-28a-CTB-NAP/BL21-DE3菌体； 图 8为根据本发明的实施方式制备的 CTB-UreB融合蛋白纯化电泳 图，其中各个泳道的样品如下： M:Fermentas蛋白预染 Marker;泳道 1:SP HP峰前样品； 泳道 2,3:SP HP洗脱峰样品； 泳道 4 : SP HP峰后样品； 图 9A为根据本发明的实施方式制备的 CTB-UreB/4CTB嵌合体蛋白 纯化电泳图，其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳道 1,2,3,4 : QHP洗脱峰；
图 9B为根据本发明的实施方式制备的 CTB-UreB/4CTB嵌合体蛋白 稳定性研究结果的电泳图， 其中各个泳道的样品如下： M: Fermentas蛋 白预染； 泳道 1-7样品分别为 pH9.0、 8.0、 7.0、 6.0、 5.0、 4.0、 3.0条件 下处理后的样品电泳结果。
图 10为根据本发明的实施方式制备的 CTB-CagA融合蛋白纯化电 泳图， 其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳道 1,2,3,4 : QHP洗脱峰；
图 11A为根据本发明的实施方式制备的 CTB-CagA/4CTB嵌合体蛋 白纯化电泳图， 其中各个泳道的样品如下： M: Fermentas 蛋白预染 Marker; 泳道 1,2,3 : SP HP洗脱峰；
图 11B为根据本发明的实施方式制备的 CTB-CagA/4CTB嵌合体蛋 白稳定性研究结果的电泳图， 其中各个泳道的样品如下： M: Fermentas 蛋白预染； 泳道 1-7样品分另 1J为 pH9.0、 8.0、 7.0、 6.0、 5.0、 4.0、 3.0条 件下处理后的样品电泳结果；
图 12为根据本发明的实施方式制备的 CTB-NAP融合蛋白纯化电泳 图，其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳道 1： 包涵体 （8M尿素）； 泳道 2,3,4 : QHP洗脱峰；
图 13为根据本发明的实施方式制备的 CTB-NAP/4CTB嵌合体蛋白 纯化电泳图，其中各个泳道的样品如下： M: Fermentas蛋白预染 Marker; 泳道 1,2,3 : QHP洗脱峰；
图 14为根据本发明的实施方式 CTB-UreB/4CTB嵌合体蛋白免疫小 鼠血清特异性 IgG检测结果；
图 15为根据本发明的实施方式 CTB-UreB/4CTB嵌合体蛋白免疫小 鼠肠黏膜特异性 slgA检测结果；
图 16为根据本发明的实施方式 CTB-CagA/4CTB嵌合体蛋白免疫小 鼠肠黏膜特异性 IgG检测结果；
图 17为根据本发明的实施方式 CTB-CagA/4CTB嵌合体蛋白免疫小 鼠肠黏膜特异性 slgA检测结果；
图 18为根据本发明的实施方式 CTB-NAP/4CTB嵌合体蛋白免疫小 鼠血清特异性 IgG检测结果；
图 19为根据本发明的实施方式 CTB-NAP/4CTB嵌合体蛋白免疫小 鼠肠黏膜特异性 slgA检测结果；
图 20为根据本发明的实施方式 CTB-UreB/4CTB嵌合体蛋白免疫小 鼠， 用 HPSS1攻击后其特异性血清 IgG抗体检测结果图；
图 21为根据本发明的实施方式 CTB-UreB/4CTB嵌合体蛋白免疫小 鼠， 用 HPSS1攻击后其特异性肠黏膜 slgA抗体检测结果图；
图 22为根据本发明的实施方式 CTB-UreB/4CTB嵌合体蛋白免疫小 鼠,用 HPSS1攻击后胃组织病理切片检测结果。其中 A: CTB-UreB/4CTB 嵌合体蛋白组小鼠胃组织切片 （x250 ) ; B: UreB 组小鼠胃组织切片 ( x250) ; C:UreB组小鼠胃组织切片 （χ400) - ;
图 23Α和 23Β分别为 UreB-CTA2/5CTB与 CTB-UreB/4CTB组装效 率比较结果的电泳图， 泳道 1为对比例 1的实验结果， 泳道 2为根据本 发明的实施例的实验结果。 具体实施方式
为了更加清楚地阐述本发明， 下文将详细地说明本发明的各个优选 的实施方式， 以及各个实施方式的技术效果。
本发明主要提供了增强免疫原性的抗原及其制备方法。 一方面， 其 通过利用黏膜免疫佐剂加强免疫反应； 另一方面， 利用 CTB或 LTB形 成五聚体的特点， 提高黏膜免疫佐剂效果的同时， 增大了抗原的体积， 易于被 APC摄取，更加有利于免疫应答的发生。另外，本发明的发明人 选择在复性液中保持一定浓度的尿素和 DTT (或尿素和巯基乙醇）， 能 够有效避免形成多聚体的单体其自身聚集成为五聚体， 从而促进嵌合体 的形成， 提高嵌合体组装的效率。 有效克服了釆用常规复性处理导致嵌 合体组装效率较低的缺陷。
下面从免疫佐剂的选择、 抗原的选择和复性方法等几个方面更加详 细地说明本发明的技术方案。 关于免疫佐剂
为了增强抗原的免疫原性， 本发明选用了 CTB或 LTB分别与抗原 结合形成新的抗原组合物。
霍乱毒素是一种强有力的黏膜免疫佐剂， 由 1个 A亚单位和 5个 B 亚单位 （见图 1) 组成。 5个 B亚单位以非共价结合成非常紧凑稳定的 圆桶状五聚体 （见图 2) , B亚单位单体两侧各有一个三链反平行片层， 在五聚体中彼此相邻的单体通过片层和大量的盐键相互作用，使 B亚单 位五聚体成为最稳定的蛋白复合物之一。 CTA是霍乱毒素的活性部分， CTB无毒性，其主要功能是通过与哺乳动物小肠上皮细胞上的单唾液酸 神经节苷脂 （GM1) 结合而使得 CTA进入细胞而产生免疫反应。 CTB 具有很强的免疫原性， 因为它除了通过与 GM1结合而提高肠道对经它 协助的口服疫苗的摄取率， 还能特异地影响小肠黏膜细胞间的紧密联结 或小带闭锁结构，增加通透性，防止口服疫苗在小肠黏膜处被消化分解 , 保持其抗原性， 从而提高机体产生的抗体效价， 使机体产生良好的免疫 反应。 目前认为 CTB是至今为止最有效、 最安全的黏膜免疫佐剂之一。
LTB也是高效的黏膜免疫佐剂。 大肠杆菌不耐热肠毒素 （LT) 为产 肠毒性大肠杆菌分泌到胞周质的一种热不稳定外毒素， 是由具有 ADP 核糖基转移酶活性的 A亚单位以及与神经节苷脂结合的 B亚单位组成的 AB5型六聚体蛋白， A亚单位是其毒性的主要单位， B亚单位具有免疫 原性和佐剂功能。 LT不仅具有免疫原性， 而且是一种有效的黏膜佐剂， 能明显增强机体针对候选抗原的 IgA和 IgG反应， 同时还能降低机体对 这些候选抗原的免疫耐受性，诱发针对它们的长期记忆， 因此 LTB作为 黏膜佐剂得到了广泛关注。
本发明提供了一种重组抗原组合物， 包括： 抗原与 CTB或 LTB的 融合蛋白； 和相应的 CTB或 LTB蛋白。 禾 IJ用 5个 CTB或 LTB非共价 键结合的特性， 能够形成上述融合蛋白与另外 4个相应的 CTB或 LTB 单体的复合物的嵌合结构， 从而增大了抗原的有效体积， 易于被 APC 摄取。 同时， 已知 CTB和 LTB是强有力的免疫佐剂， 能够降低机体对 候选抗原的免疫耐受， 从而更有效地发挥其免疫促进作用。 并由于形成 抗原与 5个 CTB或 LTB的嵌合蛋白而形成了效价更高的抗原， 从而增 强了整个抗原组合物的免疫原性。
关于抗原
为了更好地发挥黏膜免疫的作用， 优先选用经黏膜感染的病原菌的 抗原， 包括但不限于幽门螺杆菌抗原、 伤寒抗原、 流感 HA抗原。 利用 本发明制备的抗原嵌合体， 能够增强相关抗原的免疫原性， 利于构建更 加有效的疫苗。
下文中以幽门螺杆菌为例， 详细说明和验证了本发明的技术方案和 技术效果。
幽门螺杆菌（Helicobacter pylori, HP)是一种是寄生于胃上皮细胞表 面的革兰氏阴性微需氧菌， 1983年由澳大利亚学者 Warren和 Marshall 首先从人胃黏膜中分离出， HP能在酸性环境下定植和生存， 使机体产 生炎症和免疫反应， 破坏胃黏膜屏障， 使胃黏膜上皮细胞的凋亡增殖平 衡失调。 HP是人类上消化道疾病的重要致病菌， 是慢性胃炎、 胃溃疡 及十二指肠溃疡的主要病因。 1994年 WHO已确认其与胃癌的发生密切 相关， 并将其列为一类致癌因子。 HP是全球感染率最高的细菌之一， 据报道， 90%的亚洲人和 60%的欧洲人感染 HP, 因此预防和治疗 HP 感染成为了全球关注的焦点。
目前临床上主要使用二联或三联抗生素治疗 HP感染。 但存在以下 明显不足之处： 1)耐药菌株产生； 2)易复发与再感染； 3)毒副作用与费 用较高； 4)无法达到群体防治效果， 无法有效控制 HP传播与感染。 因 此，研制疗效明确的疫苗对于预防和控制 HP感染具有十分重要的意义。 目前所研究的 HP疫苗主要为全菌疫苗、亚单位疫苗 (基因工程疫苗)、活 载体疫苗和 DNA疫苗。 大多数研究釆用 HP全菌或全菌裂解物， 或 HP 毒性蛋白如尿素酶、 空泡毒素、 毒素相关蛋白、 中性粒细胞激活蛋白等 单独或联合不同佐剂进行接种， 在动物模型中均能引起保护性应答反 应。
预防性疫苗是通过机体的免疫应答发生作用， 因此选择有效的 HP 抗原免疫机体， 可刺激机体产生保护性免疫应答， 从而起到预防机体感 染 HP的作用。 随着 HP致病机制研究的深入以及分子生物学技术的发 展-基因工程菌株的构建和运用为有效抗原的筛选提供了有利条件，同时 为预防和根除 HP感染迈出了关键的一步。
以幽门螺杆菌相关疫苗的制备为例， 发明人优先选择了幽门螺杆菌 尿素酶、幽门螺杆菌 CagA蛋白和幽门螺杆菌 NAP作为用于幽门螺杆菌 的有效抗原。本发明应用基因生物工程技术,制备了三种 HP抗原和 CTB 的融合蛋白 （CTB-X(X=UreB , CagA, NAP)) (见图 3)， 并利用 5个 CTB非共价键结合的特性， 形成 CTB-X(X=UreB , CagA, NAP) -4CTB 的嵌合结构 （见图 4) , 从而通过 HP抗原与 5个 CTB的嵌合蛋白形成 效价更高的抗原， 并进行了相关的动物免疫原性试验验证。
尿素酶
尿素酶 (Ure)在 HP定植感染过程中有分解尿素、 中和胃酸的作用， 对 HP的致病性具有重要意义， HP尿素酶既是定植因子又是毒力因子。 尿素酶基因是所有 HP菌株共同拥有的， 其编码的蛋白具有很强的尿素 酶活性， 尿素酶分布于 HP表面， 占全菌总蛋白量的 5~10%。 目前发现 尿素酶基因大小约为 7.5 kb,共有 9个读码框 (ORF)分别为 UreA、UreB、 UreC、 UreD、 UreE、 UreF、 UreG、 UreH和 Urel。其中 UreA和 UreB 为 尿素酶的两个结构亚单位，具有高度的保守性，是 PCR检测 HP感染常 选择的目的基因， 以及基因疫苗的侯选基因。
UreB是 HP主要的外膜抗原成分， 由 569个氨基酸残基编码产生， 是 HP抗原性最强的保护性蛋白， 具有无毒性、 抗原性强且相对保守等 特点， 其在 HP的致病过程中起关键作用。 通过不同菌株的比较， 其尿 素酶 B亚单位氨基酸同源性达到 97.9%以上，表明 HP菌株间 UreB的抗 原变异性极小。此外， UreB的主要活性部分位于菌体表面，其大分子量 和颗粒状结构有利于黏膜免疫接种， 因此尿素酶分子可以作为疫苗抗原 的合理选择。 同时在体内 HP表面具有黏附胞质蛋白的特性， 能够将自 溶释放的尿素酶黏附到活菌表面， 这给 HP疫苗的研究又提供了一个重 要的理论依据。 经实验证实， 通过黏膜免疫途径给予 UreB蛋白疫苗,能 够诱导并激发机体的保护性免疫应答,表明 UreB是 HP感染重要的保护 性候选抗原之一， 也是基因工程疫苗的首选抗原， 具有显著的优势。
大量的文献报道， 用重组的尿素酶亚单位 B(rUreB)口服免疫预先感 染了猫胃螺杆菌（Helicobacter Felis， Hf) 的小鼠， 结果发现免疫的小鼠 不仅彻底清除了 Hf感染，而且能够预防小鼠再次感染； Kleanthous等用 rUreB加上 LT作为黏膜佐剂免疫小鼠，与对照组相比结果发现：免疫组 胃内尿素酶活性显著降低,且胃黏膜组织 HP定量培养显示 HP减少 97% 以上； Michetti等对口服不同剂量 (180mg、 60mg、 20mg, 4次 /天)的尿 素酶与 υΤ(5μ^的安全性和免疫原性进行了临床实验研究， 结果发现： 所有的志愿者都能够耐受， 且能够减少胃内 HP的定植， 从而减轻了胃 炎的程度； Saldinger等釆用 rUreB结合霍乱毒素 (CT)免疫感染了 Hf 的 BALB/c^j、鼠，结果发现：免疫组小鼠全部清除了 Hf;进一步研究发现, 脾脏 CD4+T细胞增生， 血清中 lgGl增加， 同时伴随 IL-4的增高， γ-干 扰素的减少，从而推测 rUreB可能诱导了 Th2 CD4+ T细胞的增生，有助 于清除 ΗΡ。 有学者对 HP感染志愿者进行了 I期临床实验， 用 UreB结 合 LT免疫后，同样发现 HP感染的密度显著减少; Lee等对非人灵长类动 物进行的类似研究也表明， 通过减少胃内 HP的密度有助于减轻胃炎的 程度。 然而 Solmck等报道用尿素酶口服和肌肉注射免疫恒河猴， 而后 进行 HP攻击试验， 结果发现： UreB虽然能够诱导体液免疫， 但是所有 给予 HP攻击的恒河猴都感染了 HP,而且细菌密度在实验组和对照组中 没有统计学差异。上述结果显示目前的 UreB相关的 HP疫苗还不够完善， 有待于进一步研究。 CagA蛋白
CagA基因也称为细胞毒素相关基因，其表达产物为 CagA蛋白。作 为细菌分泌的一种毒素， 它由 IV型分泌系统 Cag 致病岛 (pathogenecityisland, PAI) 从细菌转运到宿主细胞中被磷酸化， 参与 宿主细胞的信号转导及引起细胞骨架结构的重排。研究表明， cagA阳性 HP为毒力菌株， 与萎缩性胃炎、 胃癌与消化道溃疡等常见的消化道疾 病密切相关， 90%以上的 HP临床分离菌株 cagA呈阳性表达。具有 CagA 基因的 HP几乎都表达 CagA蛋白， 并产生可测的抗体反应。 据文献报 道 CagA阳性菌株能够直接或间接 （通过 NF-KB) 诱导胃上皮细胞分泌 IL-8, 而 IL-8在中性粒细胞趋化和激活过程中起重要作用， 可导致胃黏 膜的损伤。动物实验发现 CagA基因和 CagA蛋白与胃炎、消化性溃疡， 尤其是胃癌的发生有良好的相关性。 Marchetti等用重组 CagA免疫 HP 小鼠感染模型， 证实能产生诱导保护性免疫反应， 保护率达到了 70%。 Crabtree等应用重组 CagA作为抗原，经胃免疫长期感染 HP的小鼠，结 果成功清除了 HP, 且有效保护了小鼠免遭 HP的再次感染。 提示 CagA 作为 HP疫苗保护性抗原具有较高的可行性， 但这种抗原产生的保护作 用仅局限于产生 CagA的菌株有效，对不产生 CagA的菌株无保护作用。 可见， 目前针对 CagA蛋白也尚无满意的疫苗。
NAP
Evans等在 HP的水溶性抽提物中发现一种 NAP, 它能够募集并活 化嗜中性粒细胞， 促进中性粒细胞对胃上皮细胞的黏附， 激活中性粒细 胞释放反应性氧代谢物， 而引起胃黏膜炎症病变。
NAP高度保守， 临床研究发现 60%的 HP感染者血清中存在 NAP 特异抗体。 NAP是一种铁蛋白， 编码它的 NAP基因几乎在所有 HP中 可检测到， 但是在体外表达的 NAP其活性存在很大差异。 它能够通过 CDlla/CD18和 CDllb/CD18与内皮细胞间粘附分子（ICAM-1)相互作 用， 而使白细胞黏附内皮细胞； 选择性地与中性粒细胞的酸性糖鞘脂相 结合而调节白细胞的功能； 与粘蛋白相结合为 HP定植于胃黏膜上皮细 胞埋下了伏笔， 因此 NAP在其黏附和致病过程中起重要作用。 Satm等 釆用纯化重组的 NAP口服免疫了 10只小鼠， 结果 8只小鼠获得了保护 性免疫。 提示 NAP可作为有效的保护性抗原用于 HP感染的疫苗防治。
在本发明优选的实施方式中， 本发明提供了一种重组幽门螺杆菌抗 原组合物， 包括幽门螺杆菌抗原与 CTB的融合蛋白和 CTB蛋白。 借助 CTB本身能够形成五聚体的性质， 增大抗原的体积，易于被 APC摄取。 另外， CTB是良好的免疫佐剂。 因此， 本发明的重组幽门螺杆菌抗原组 合物能够有效增强幽门螺杆菌抗原的免疫原性， 利于构建更加有效的疫 苗。
在进一步的实施方式中，所述幽门螺杆菌抗原选自幽门螺杆菌 UreB 蛋白、幽门螺杆菌 CagA蛋白和幽门螺杆菌 NAP中的至少一种。这几种 抗原均是目前已知的幽门螺杆菌抗原中无毒性、 抗原性强而且相对保守 的抗原， 更加适合用于疫苗。 抗原与免疫佐剂的结合方式的选择
常规免疫佐剂的使用一般是通过与抗原的直接混合， 然后与抗原同 时给药以激发机体的免疫反应， 但是其增强抗原免疫原性的效果不理 想。 本发明通过制备抗原与黏膜免疫佐剂蛋白质单体的融合蛋白， 再将 其与这种能形成多聚体的黏膜免疫佐剂蛋白质单体通过非共价结合形 成嵌合蛋白， 不但可保持其黏膜免疫佐剂性质， 还可增大了抗原体积， 更易于被抗原递呈细胞摄取。
对于融合蛋白的制备， 本发明中在抗原和 CTB (或 LTB) 的蛋白之 间加入柔性连接体， 以使得蛋白融合后抗原和 CTB (或 LTB)各自的折 叠不会受到影响， 从而分别形成正确的构象。 在一个实施方式中， 本发 明选择了 3个 G4S的连接体。 但是， 本发明中并不限于用此种连接体， 其余连接体， 比如螺旋形式的连接体 （linker) 肽 （A (EAAAK) nA) 以及葡萄糖球菌蛋白 A (PA) 等也可用于本发明中。
进一步地， 在保证了融合蛋白两个部分， 尤其是 CTB (或 LTB) 构 象的基础上， 有利于五聚体的正确形成和形成五聚体的效率。 嵌合体的制备方法
本发明还提供了一种试剂盒， 包括表达上述抗原与 CTB (或 LTB) 的融合蛋白的载体和表达相应黏膜免疫佐剂单体蛋白 （CTB或 LTB)蛋 白的载体。 如本领域普通技术人员所公知， 除了目的蛋白之外， 所述表 达载体中还应包括表达目的蛋白质所必需的元件，例如启动子、终止子、 可选的标记基因等。
所述表达载体优选是原核表达载体， 例如， pET系列等原核载体。 使用原核表达载体制备目的蛋白的方法和试剂为本领域所公知， 可参见 本领域常用工具书， 例如冷泉港实验室编译的 《分子克隆实验指南》 ( (美） 萨姆布鲁克等著， 黄培堂等译， 北京， 科学出版社出版）、 《细 胞实验指南》（（美） D.L.斯佩克特等著， 黄培堂等译， 北京， 科学出版 社出版） 等。
对于嵌合体的制备， 一直以来都属于一个技术难题。 如何在与融合 蛋白嵌合之前避免单体自身形成五聚体， 是影响嵌合体收率的关键。 一 般情况下， 原核表达蛋白后大部分会形成包涵体， 需要经过洗涤之后用 尿素溶液溶解包涵体。 被尿素溶液所溶解的蛋白质基本属于变性状态， 很难产生其天然构象。 而常规的复性过程就是逐步除去尿素的过程， 以 使其逐步重折叠形成正确的构象。 但是， 对于能够形成多聚体的单体蛋 白质， 其去除尿素的过程中， 很容易自发形成多聚体， 而不是跟融合蛋 白中的单体蛋白质形成多聚体。 因此， 常规方法制备嵌合体时产率会非 常低。
本发明的发明人选择在复性液中保持一定浓度的尿素和 DTT (或尿 素和巯基乙醇）， 能够有效避免形成多聚体的单体其自身聚集成为五聚 体， 促进嵌合体的形成， 提高嵌合体组装的效率。 有效克服了釆用常规 复性处理导致嵌合体组装效率较低的缺陷。
本发明提供了一种通过复性制备嵌合体的方法 ,所述嵌合体包含（ 1 ) 一种蛋白质与能形成多聚体的单体蛋白质的融合蛋白和 （2) 能形成多 聚体的单体蛋白质，通过所述（1 )融合蛋白中的单体蛋白质和所述（2) 单体蛋白质形成多聚体来形成嵌合体， 所述方法包括： 将所述 （2) 能 形成多聚体的单体蛋白质在含 6M至 9M,优选 8M的尿素，且 pH为 3.0 至 4.0的缓冲液中预复性 0.5至 3小时， 优选预复性 1小时； 和将所述 ( 1) 融合蛋白和所述 （2) 能形成多聚体的单体蛋白质在含有 1.0M至 2.5M的尿素和 0.2mM至 l.OmM的 DTT或巯基乙醇复性液中复性形成 所述嵌合体。
不同于一般的复性液， 本发明釆用含一定浓度尿素和还原剂的复性 液， 使融合蛋白和单体蛋白质缓慢复性过程中组合成为所需的嵌合体。 在优选过程中通过在一定 pH条件下进行预复性，然后在还原剂存在下， 在浓度稍低的尿素溶液中缓慢复性， 意外地得到了高收率的嵌合体。
而对于大规模生产来说， 收率的高低直接影响了生产的成本， 更直 接影响了大规模生产的可能性。 釆用本发明的方法， 嵌合体的收率可以 达到实现大规模生产的水平。 下文通过具体的实施例详细说明本发明的优选实施方式。
1.抗原的制备方法
实施例 1 CTB-UreB/4CTB制备：
1.1.1重组工程菌 PET-28a-CTB-UreB/BL21-DE3的构建
1.1.1.1融合基因 CTB-UreB的构建：
UreB DNA序列来自 HP菌株 MEL-HP27 , CTB DNA序列来自霍乱 弧菌 0395株， 在融合 DNA片段间引入富含甘氨酸 /丝氨酸具有柔性功 能的 (G4S)3连接体（linker)序列（用斜体下划线表示），并合成 CTB-UreB 基因密码子。
基因序列为 SEQ ID No. 1 :
Figure imgf000019_0001
TCTTTAGCATTTTCTAA 2067 ο
1.1.1.2表达载体 PET-28a-CTB-UreB的构建：
表达载体 PET-28a、 HP UreB-CTB融合基因片段， 分别用 Ndel和
Hmdlll酶切后， 经琼脂糖电泳凝胶中的目的 DNA回收纯化， 然后进行 连接反应， 根据目的片段和载体摩尔数比为 1:3-1:10的原则， 16°C连接 反应 16小时， 设计连接反应体系如下：
目的 DNA 15μ1
PET-28a载体 3μ1
ΙΟχ的连接缓冲液 2.5μ1
ddH20 3.5μ1
T4连接酶 Ιμΐ
总体积 25μ1 1.1.1.3 大肠杆菌感受态细胞的制备 （CaCl2法）
从 -70°C保存的 DH5a甘油菌中挑取微量菌划板， 挑取 E.coli DH5a 受体菌的单菌落于装有 5ml LB培养基的有盖试管中， 37 °C振荡（200rpm) 培养 10〜12小时， 得到种子液， 将 5ml种子液接入 100 ml新鲜 LB培 养基中振荡 （200rpm) 培养， 至菌液的 OD600为 0.4〜0.9时， 取新鲜 培养液 1.5ml转入 Eppendorf管中，冰上放置 10分钟，然后于 4°C下 4000 rpm离心 10分钟，小心弃上清，重复前两步一次， 收集菌体。加入冰预 冷的 0.1mol/L CaCl2溶液 300μ1轻轻悬浮细胞， 冰上放置 30分钟， 4°C 下 4000 rpm离心 10分钟， 弃去上清， 沉淀加入 ΙΟΟμΙ预冷的 0.1mol/L CaCl2溶液轻轻悬浮细胞， 即为感受态细胞。 如果感受态细胞需贮存以 后再使用， 则需用预冷的 30μ150%甘油与 70μ1的 0.1mol/L的 CaCl2溶 液（甘油终浓度为 15%)重悬细胞，置 -70°C保存（可保存半年），备用。
1.1.1.4用连接产物转化大肠杆菌感受态细胞
从 -70°C冰箱中取出感受态细胞悬液，室温下使其解冻，解冻后立即 置冰上， 加入连接反应液 （含量不超过 50ng, 体积不超过 10μ1)， 轻轻 弹匀， 冰上放置 30分钟， 42 °C水浴中热冲击 90后， 迅速置于冰上冷却 5分钟，向管中加入 800μ1 Ι^Β液体培养基，混匀后 37°C 150 rpm振荡培 养 1.5小时， 涂布于含卡那霉素 （Kan) 的筛选平板， 37°C培养过夜。
1.1.1.5 融合蛋白克隆菌的构建及筛选：
挑取经转化的 DH5a单菌落， 提取质粒， 经双酶切后片段大小为
2067bp (见图 5) ,酶切后片段大小与设计一致，证明重组质粒构建成功。
1.1.1.6融合蛋白工程菌的构建及筛选：
大肠杆菌 BL21-DE3的感受态菌制备、 转化及重组菌的质粒抽提方 法同融合蛋白克隆菌的构建及筛选。
从含 Kan的筛选平板上挑取单克隆菌落， 加入 5ml的 LB试管中， 37 °C 150 rpm振荡培养 4小时， 加入 ImM的 IPTG诱导表达， 其表达 结果见图 6。
1.1.2 重组工程菌 PET-28a-CTB-UreB/BL21 (DE3) 的发酵
1.1.2.1 种子活化
种子活化釆用 LB (胰蛋白胨 10g/L;酵母提取物 5g/L ; NaCl 10g/L) 培养基 ,配制体积为 50mL， 121 °C灭菌 20分钟，冷却后加入 Kan( 50mg/L) 和工作种子批菌液各 50μ1, 37°C±1 °C , 培养 15~16小时。
1.1.2.2 种子制备
种子釆用 2 YT培养基（胰蛋白胨 16g/L;酵母提取物 10g/L ; NaCl 5g/L) , 配制体积为 3L , 121 °C灭菌 20分钟后加入活化好的种子液 3ml， 37°C±1 °C , 培养 4~6小时。
1.1.2.3 发酵罐发酵
1.1.2.3.1 基础培养基
胰蛋白胨 500g
酵母提取物 250g
(丽 4)2S04 75g
甘油 250g
MgS04.7H20 50g
KH2P04 100g
K2HP04.3H20 200g
Na2HP04.12H20 350g
上述基础培养基用纯化水溶解后， 加纯化水至 50L。 1.1.2.3.2补料 甘油 2400g
胰蛋白胨 600g
酵母提取物 600g
MgS04.7H20 24g
按以上补料配方进行配制， 先用纯化水溶解后， 再加纯化水至 4L , 121 °C灭菌 20分钟后使用。
1.1.2.3.3补碱
4M的氢氧化钠 1L。
1.1.2.3.4 IPTG溶液
用已灭菌纯化水溶解 5.0g IPTG, 经 0.22μπι滤膜过滤后置 2~8°C冰 箱备用， 保存期为 24h。
1丄 2.3.5培养参数
在发酵罐上设置培养参数：
培养温度 37。C
搅拌速度 200rpm
补碱控制 pH7.0
转子流量计 3m3/h
罐压 0.03-0.05 Mpa
溶氧 当溶氧浓度低于 30%时， 通过提
升转速和增加纯氧比例维持溶氧
不低于 30%。 OD 补碳速度 (ml/L-h) 补氮速度 (ml/L-h)
4-5 5 5
9-11 10 10
20-25 12 12 当培养至 OD600在 20~30之间时加入 IPTG溶液，继续培养 3小时 后放罐。
1.1.2.3.4离心
培养结束， 发酵液转移到预冷的贮罐中， 待发酵液冷却至 15°C以下 后， 使用管式离心机离心收集菌体。
1.1.3 CTB-UreB/4CTB的制备
1.1.3.1 CTB-UreB的制备
( 1) 包涵体制备
细菌培养结束， 将发酵液转移到预冷的贮罐中， 待发酵液冷却至 15°C以下后， 使用管式离心机离心收集菌体。。
菌体用 50mM Tris-HCl , 0.5%Triton X-100 , 150mM NaCl , ImM EDTA pH 8.0缓冲液以 1 : 10 (W/V) 比例悬浮， 4°C预冷后， 高压匀浆破菌 900bar, 3次， 14000g离心 20min, 收集沉淀。
将收集的包涵体沉淀以 1 : 10 (W/V)的比例分别用 50mM Tris-HCl, 0.5%Triton X-100 , 2M尿素， ImM EDTA H 8.0缓冲液洗涤沉淀 3次， 50mM Tris-HCl, 5mM DTT, pH8.0缓冲液洗涤 1次。 洗涤条件为： 室 温搅拌 30min, 6000g离心 20min。
(2) CTB-UreB蛋白变性 用 50mM Tris-HCl , 8M尿素， lOmM DTT, 10%甘油， pH8.0的包 涵体溶解液充分溶解包涵体， 4°C搅拌过夜， 12000rpm离心 30min, 取 上清备用。
(3) CTB-UreB蛋白纯化
经过 DEAE和 SPHP层析得到纯化的 CTB-UreB。
DEAE柱纯化
平衡液： 20mM Tris-HCl, 8M尿素， lOmM DTT, 10%甘油 pH8.0 洗脱液： 20mM Tirs-HCl, 8M尿素， 250mM NaCl, lOmM DTT 10%甘 油 pH8.0
SPHP纯化
平衡液： 20mM PB+8M尿素 +5mM DTT 10%甘油 pH7.2
洗涤液： 20mM PB, 8M尿素， 30mM NaCl, 5mM DTT, 10%甘油 pH7.2 , 电导小于 3.7ms/m
洗脱液： 20mM PB, 8M尿素， 0.3M NaCl, 5mM DTT, 10%甘油 pH7.2
洗杂液： 20mM PB, 8M尿素， 1M NaCl, 5mM DTT, 10%甘油 pH7.2 CTB-UreB纯化电泳见图 8。
1.1.3.2. CTB-UreB/4CTB的制备
将 CTB原液用 50mM Tris-HCl, 8M尿素， pH8.0缓冲液稀释调 pH至 3.0-4.0 , 室温放置 1小时， 然后调 pH到 8.0 , 4°C放置备用。
将两种蛋白 CTB-UreB与 CTB单体以摩尔数比为 1 : 4混合后， 5 倍体积稀释到最终复性液中 （50mM Tris-HCl pH8.0+10%甘油 +150mM NaCl) , 控制 DTT 的浓度范围在 0.2-1.0mM之间， 尿素浓度范围在 1.0M-2.5M之间。 室温放置过夜。
上述溶液经过螯合 FF和 QHP纯化， 得到纯化的 CTB-UreB/4CTB。 螯合 FF
平衡液： 20mM Tris-HCl +5%甘油 +20mM咪唑 pH8.0 ,
洗脱液： 20mM Tris-HCl +5%甘油 +200~300mM咪唑洗脱目的蛋白， 收全峰；
洗杂液： 20mM Tris-HCl +5%甘油 +500mM咪唑 pH8.0
Q HP纯化
缓冲液 A: 20mM Tris-HCl +5%甘油 pH7.5
缓冲液 B： 20mM Tris-HCl +5%甘油 +0.3M NaCl pH7.5
0-100%B 10CV洗脱目的蛋白。
CTB-UreB/4CTB纯化电泳见图 9A。
1.1.3.3.稳定性研究
将纯化后的 CTB-UreB/4CTB抗原嵌合体原液加稀盐酸调整至不同 pH值， 室温放置 1小时后取样品进行 SDS-PAGE电泳， 电泳结果见图 9B , 其中各个泳道的样品如下： M: Fermentas蛋白预染； 泳道 1-7样品 分别为 pH9.0、 8.0、 7.0、 6.0、 5.0、 4.0、 3.0条件下处理后的样品电泳 结果。
由图 9B结果可见， 随着 pH降低，在电泳图上显示位于嵌合体位置 的蛋白浓度逐渐降低， 且出现了多条分子量小于本嵌合体的蛋白条带， 说明产生了小分子量的降解产物。 而在大于 pH7.0 , 特别是 pH8.0下本 嵌合体蛋白稳定， 未出现分子量小于本嵌合体的蛋白条带。 这说明 CTB-UreB/4CTB抗原嵌合体在中性偏碱性环境中稳定，在偏酸性环境下 容易解离。 具体而言， 在 pH>7.0的情况下比较稳定，优选 pH为 8.0。
1.2 CTB-CagA/4CTB的制备方法
1.2.1重组工程菌 PET-28a-CTB-CagA/BL21-DE3的构建 釆用与实施例 1相同的步骤制备 CTB-CagA,区别仅在于釆用 CagA 基因代替 UreB基因。 经构建的工程菌质粒经双酶切后片段大小为 1266 bp (见图 5) , 其融合蛋白的表达见图 7。
基因序列为 SEQ IDNo.:2 , 其中 linker用斜体下划线表示：
Figure imgf000027_0001
CAAAAATACTACAAAATAA 1269
1.2.2 重组工程菌 PET-28a-CTB-CagA/BL21 (DE3) 的发酵 釆用 与 实 施例 1 相 同 的 方法进行重组工程菌 PET-28a-CTB-CagA/BL21 (DE3) 的发酵。 1.2.3 CTB-CagA/4CTB的制备
1.2.3.1 CTB-CagA的制备
(1)釆用与实施例 1相同的方法进行 CTB-CagA包涵体的提取。
(2)包涵体用包涵体溶解液以 1 : 20 (W/V) 的比例混合， 4 °C搅拌 3 小时， 过夜， 12000rpm离心 30min收集上清。
(3) Q FF柱纯化
缓冲液 A : 20mM Tirs-HCl +8M尿素 +5mM DTT 5%甘油 H7.5 缓冲液 B： 20mM Tirs-HCl, 8M尿素， l .OM NaCl, 5mMDTT 5%甘油, pH7.5
用 A液平衡、 淋洗， B液洗脱。
(4) SP Big beads柱纯化
缓冲液 A： 20mM Tris-HCl, 8M尿素， 5mM DTT,5%甘油 ,pH7.5 缓冲液 B： 20mM Tirs-HCl, 8M尿素， 150mM NaCl, 5mM DTT 5%甘 油，pH7.5
用 A液平衡、 淋洗， B液洗脱。
CTB-CagA纯化电泳见图 10。
1.2.3.2 CTB-CagA/4CTB的制备
将 CTB原液用 50mM Tris-HCl, 8M尿素， pH8.0缓冲液稀释调 pH至 3.0-4.0 , 室温放置 1小时， 然后调 pH到 8.0 , 4°C放置备用。
将两种蛋白 CTB-CagA与 CTB单体以摩尔数比为 1： 4混合后， 5 倍体积稀释到最终复性液中 （50mM Tris-HCl pH8.0+10%甘油 +150mM NaCl) , 控制 DTT 的浓度范围在 0.2-1.0mM之间， 尿素浓度范围在 1.0M-2.5M之间。 室温放置过夜。
上述溶液经过螯合 FF和 SP HP纯化，得到纯化的 CTB-CagA/4CTB。 螯合 FF柱纯化 缓冲液 A: 20mM Tris-HCl +5%甘油 +20mM咪唑 pH8.0
缓冲液 B： 20mM Tris-HCl +5%甘油 +200mM咪唑洗脱目的蛋白 100%B洗杂
SP HP柱纯化
缓冲液 A: 20mM Tris-HCl +5%甘油 pH7.5
缓冲液 B： 20mM Tris-HCl +5%甘油 +0.3M NaCl pH7.5
CTB-CagA/4CTB嵌合体蛋白纯化电泳图见图 11A。
1.2.3.3 稳定性研究
将纯化后的 CTB-CagA/4CTB抗原嵌合体原液加稀盐酸调整至不同 pH值， 室温放置 1小时后取样品进行 SDS-PAGE电泳， 电泳结果见图 11B ,其中各个泳道的样品如下： M: Fermentas蛋白预染；泳道 1-7样品 分别为 pH9.0、 8.0、 7.0、 6.0、 5.0、 4.0、 3.0条件下处理后的样品电泳 结果。
由图 11B结果可见， 随着 pH降低， 在电泳图上显示位于嵌合体位 置的蛋白浓度逐渐降低， 且出现了多条分子量小于本嵌合体的蛋白条 带， 说明产生了小分子量的降解产物， 而在大于 pH 7.0 , 特别是 pH8.0 下本嵌合体蛋白稳定， 未出现分子量小于本嵌合体的蛋白条带。 这说明 CTB-CagA/4CTB抗原嵌合体在中性偏碱性环境中稳定， 在偏酸性环境 下容易解离。 具体而言， 在 pH>7.0的情况下比较稳定，优选 pH为 8.0。
1.3 CTB-NAP/4CTB的制备方法
1.3.1重组工程菌 PET-28a-CTB-NAP/BL21-DE3的构建
釆用与实施例 1相同的步骤制备 CTB-NAP, 区别仅在于釆用 NAP 基因代替 UreB基因。经构建的工程菌质粒经双酶切后片段大小为 789bp (见图 5) , 其融合蛋白的表达见图 7。 基因序列为 SEQ ID No.:3 , 其中 linker用斜体下划
Figure imgf000030_0001
TTGCAAAAATCCATTTGGATGCTGCAAGCCCATTTAGCTTAA 792。
1.3.2 重组工程菌 PET-28a-CTB-NAP/BL21 (DE3) 的发酵
釆用 与 实 施例 1 相 同 的 方法进行重组工程菌 PET-28a-CTB-NAP/BL21 (DE3) 的发酵。
1.3.3 CTB-NAP/4CTB的制备
1.3.3.1 CTB-NAP的制备
(1)釆用与实施例 1相同的方法进行 CTB-NAP包涵体的提取。
(2) CTB-NAP包涵体经洗涤后用 20mM Tris-HCl, 8M尿素， pH8.0 的缓冲液溶解过夜， 12,000rpm离心 30分钟取上清。
(3) QFF层析：
缓冲液 A: 20mM Tris-HCl +5%甘油 pH7.5
缓冲液 B： 20mM Tris-HCl +5%甘油 +0.3M NaCl pH7.5
用 A液平衡、 淋洗， B液洗脱。 CTB-NAP融合蛋白纯化电泳图见 图 12。 11..33..33..22 CCTTBB--NNAAPP//44CCTTBB的的制制备备
((11)) CCTTBB--NNAAPP的的处处理理：： 用用 2200mmMM TTrriiss--HHCCll,, 88MM尿尿素素，， ppHH88..00 的的缓缓 冲冲液液将将上上述述 CCTTBB--NNAAPP样样品品稀稀释释，， 并并调调 ppHH至至 88..00 ,, 并并补补加加巯巯基基乙乙醇醇使使其其 终终浓浓度度为为 33mmMM 。。
55 ((22)) CCTTBB的的处处理理：： 用用 2200mmMM TTrriiss--HHCCll,, 88MM尿尿素素，， ppHH88..00的的缓缓冲冲液液将将
CCTTBB原原液液稀稀释释，，并并调调 ppHH到到 33..55~~33..77，，放放置置 11小小时时后后，， 调调 ppHH到到 88..00，，并并补补 加加巯巯基基乙乙醇醇使使其其终终浓浓度度为为 33mmMM。。
((33))复复性性：： 取取上上述述处处理理好好的的两两种种蛋蛋白白 CCTTBB--NNAAPP与与 CCTTBB单单体体以以摩摩尔尔 数数比比为为 11 :: 44混混合合，， 用用 5500mmMM TTrriiss--HHCCll ,, 55%%甘甘油油，， 00..0033mmMM GGSSSSGG,, ppHH88..00 iioo 缓缓冲冲液液以以 11::66稀稀释释，， 并并控控制制巯巯基基乙乙醇醇的的浓浓度度范范围围在在 00..22--11.. OOmmMM之之间间，， 尿尿 素素浓浓度度范范围围在在 11..00MM--22..55MM之之间间。。 2211 °°CC放放置置过过夜夜。。
((44)) NNII FFFF纯纯化化：：
缓缓冲冲液液 AA:: 5500mmMM TTrriiss--HHCCll ,, 55%%甘甘油油，， 2200mmMM咪咪唑唑，， PPHH88..00 缓缓冲冲液液 BB:: 5500mmMM TTrriiss--HHCCll ,, 55%%甘甘油油，， 330000mmMM咪咪唑唑，， PPHH88..00
1155 复复性性样样品品补补加加终终浓浓度度为为 2200mmMM咪咪唑唑，， 上上 NNII FFFF ,, 流流速速 88mmll//mmiinn,, 用用
AA液液淋淋洗洗，， 用用 BB液液洗洗脱脱。。
((55)) QQHHPP纯纯化化：：
缓缓冲冲液液 AA::5500mmMM TTrriiss--HHCCll ,, 55%%甘甘油油
缓缓冲冲液液 BB:: 5500mmMM TTrriiss--HHCCll ,, 55%%甘甘油油，， 11ΜΜ NNaaCCll PPHH88..00
2200 过过 NNII FFFF洗洗脱脱样样 （（电电导导在在 44~~55之之间间）） 上上 QQHHPP,, 上上样样速速度度 33mmll//mmiinn,, 用用 AA液液平平衡衡、、 淋淋洗洗，， 3300%%缓缓冲冲液液 BB洗洗脱脱 （（流流速速为为 llmmll//mmmm))。。
CCTTBB--NNAAPP//44CCTTBB嵌嵌合合体体蛋蛋白白纯纯化化电电泳泳图图见见图图 1133。。
22..重重组组幽幽门门螺螺杆杆菌菌疫疫苗苗 （（rrHHPP)) 小小鼠鼠免免疫疫原原性性研研究究
22..11 CCTTBB--UUrreeBB//44CCTTBB嵌嵌合合体体蛋蛋白白的的免免疫疫原原性性研研究究
25 * 2.1.1.1 试验动物： 8-10周，体重 18-20g, SPF级雌性 B ALB/c小鼠，
2.1.1.2试验药物： UreB , CTB-UreB融合体蛋白， CTB-UreB /4CTB 嵌合体蛋白均用 Tris-HCl缓冲液（pH 8.0)稀释， UreB蛋白免疫剂量为 20( g/只， CTB-UreB融合蛋白和 CTB-UreB/4CTB嵌合体蛋白均以与 UreB蛋白等摩尔数给药， 抗酸剂用 PBS稀释至 50mg/ml。
2.1.1.3 试验分 4组： UreB IS, CTB-UreB组， CTB-UreB /4CTB组 和生理盐水组 （或称， 空白对照组 （CON, Control) )。
2.1.1.4小鼠的免疫程序： 小鼠共免疫 4次， 每隔 7天免疫 1次。每 只动物灌胃给予 0.2ml试验药物， 给药前 10mm给予 0.2ml的抗酸剂。 所有动物给药前禁食 12h, 不禁水， 给药后 lh再进水进食。
2.1.1.5 样品釆集及检测：
各组小鼠在免疫前一天和首次免疫后的 2周、 4周、 6周、 8周、 10 周需釆血用于检测血清 IgG和 IgA抗体水平。除最后一次眼球釆血之外， 其余均为鼠尾釆血，所有的血清离心前需 4 °C过夜，次日以 4 °C， 5000rpm， 15min离心， 取上层血清， -20°C保存。
各组动物在免疫后 10周经眼球釆血后被处死，剖腹，取近回盲部末 端的回肠 10cm, 用 PBS冲洗肠道 3次后， 无菌刀片刮取肠黏膜， 溶于 lml O.Olmol/L PBS中 （pH7.4 , 含 50mmol/L EDTA、 20nmol/L蛋白酶抑 制剂亮肽素和胃酶抑素）。 lOOOOrpm离心 lOmin,取上清并加入 20μ1 100 mmol/L PMSF(Sigma)。以 ELISA检测血清 IgG和肠黏膜 slgA抗体水平。
96孔酶标板每孔加入 ΙΟΟμΙ Ure B抗原 （1.25 g/ml) , 4°C包被过夜， 次 日弃去孔内溶液，用 PBST洗涤 3次；每孔加封闭液 200 μΐ (含 2%BSA) , 37。C封闭 2h, PBST洗涤 3次；每孔加入 ΙΟΟμΙ血清 ( 1:300稀释）， 37 °C 反应 lh后 PBST洗涤三次， 每孔加入 100 μΐ HRP标记的羊抗鼠 IgG ( 1:2000-1:50000) /HRP标记的羊抗鼠 IgA ( 1:4000-1:8000) , 37。C孵 育 l h， PBST洗涤 6次； 立即加入新鲜配制的底物液， 每孔 ΙΟΟ μΙ , 室 温反应 15 min, 加终止液 50 μΐ; 酶标检测仪测定每孔的吸光度 OD 450 值。
2丄 2结果
BALB/C小鼠经 HP疫苗口服免疫后， 其血清 IgG和肠黏膜 slgA抗 体反应见图 14、 15。 空白对照组和 UreB蛋白组小鼠的抗体在给药后没 有增加，相反， CTB-UreB融合蛋白和 CTB- UreB/4CTB嵌合体蛋白组 的小鼠其血清 IgG抗体在首次给药后 4周明显增加， 至 8周达高峰， 与 空白对照组比较均存在显著性差异（PO.001 ) ;肠黏膜 slgA抗体也明显 升高， 与空白对照组比较差异显著 （P<0.001)。 其中 CTB-UreB融合蛋 白组小鼠的抗 -UreB抗体的阳转率为 60%, CTB-UreB/4CTB嵌合体蛋白 组小鼠的抗 -UreB抗体的阳转率为 95%, 表明 CTB-UreB/4CTB嵌合体 蛋白疫苗可诱导机体产生显著的免疫应答反应。
2.1.3 结论
口服给予重组 CTB-UreB融合蛋白和 CTB-UreB/4CTB嵌合体蛋白， 能刺激机体产生抗 UreB抗原的特异性抗体。但 CTB-UreB/4CTB嵌合体 蛋白引起的免疫应答明显优于 CTB-UreB 融合蛋白， 表明 CTB-UreB/4CTB嵌合体蛋白具有更好的免疫反应性和抗原性 ,可以作为 预防幽门螺杆菌的候选疫苗。
2.2 CTB-CagA/4CTB嵌合体蛋白的免疫原性研究
2.2.1 BALB/c小鼠免疫方法
免疫方法基本同 2.1.1。 其中试验药物 CagA蛋白， CTB-CagA融合 蛋白， CTB-CagA/4CTB嵌合体蛋白均用 Tris-HCl缓冲液 （pH 8.0) 稀 释， CagA 蛋白免疫剂量为 200μ^只， CTB-CagA 融合蛋白和 CTB-CagA/4CTB嵌合体蛋白均以与 CagA蛋白等摩尔数给药，给药容量 为 0.2ml/只。ELISA检测时用 CagA抗原包板。其血清 IgG和肠黏膜 slgA 抗体反应见图 16、 17。
2.2.3 结论
口服给予重组 CTB-CagA/4CTB 嵌合体蛋白， 能刺激机体产生抗 CagA抗原的特异性抗体。表明 CTB-CagA/4CTB嵌合体蛋白具有更好的 免疫反应性和抗原性， 可以作为预防幽门螺杆菌的候选疫苗。
2.3 CTB-NAP/4CTB嵌合体蛋白的免疫原性研究
2.3.1 BALB/c小鼠免疫方法
免疫方法基本同 2.1.1。其中试验药物 NAP蛋白， CTB-NAP融合蛋 白， CTB-NAP/4CTB嵌合体蛋白均用 Tris-HCl缓冲液 （pH 8.0) 稀释， NAP 融合蛋白免疫剂量为 200μ^只 , CTB-NAP 融合蛋白和 CTB-NAP/4CTB嵌合体蛋白均以与 UreB蛋白等摩尔数给药， 给药容量 为 0.2ml/只。 ELISA检测时用 CagA抗原包板。
2.3.2结果
BALB/C小鼠经 HP疫苗口服免疫后， 其血清 IgG和肠黏膜 slgA抗 体反应见图 18、 19。
2.3.3 结论
口服给予重组 CTB-NAP/4CTB嵌合体蛋白，能刺激机体产生抗 NAP 抗原的特异性抗体。 表明 CTB-NAP/4CTB嵌合体蛋白具有更好的免疫 反应性和抗原性， 可以作为预防幽门螺杆菌的候选疫苗。
3.重组幽门螺杆菌疫苗对幽门螺杆菌 SS1株感染 BALB/c小鼠的免 疫保护作用
3.1 试验方法
3.1.1 同 2.1.1。 3.1.2 HP SSI菌攻击：
试验组和阴性对照组动物在末次免疫后 2周，连续 3天灌胃给予 HP SSI菌 0.2ml ( Ι χΙΟ9 CFU/ml) , 每日 1次， 给予 HP SSI菌前 lOmin灌 胃给予 0.2ml抗酸剂。
3.1.3样品釆集及检测
血清及肠黏膜釆集及检测方法同 2丄 1.5。
所有动物用 HPSS1菌攻击 4W后全部处死，剖腹取胃，沿纵轴将胃 组织分为三部分， 每部分均包括胃底、 胃体和胃窦三部分。 一份做定量 细菌培养： 用无菌镊夹取胃窦部组织， 黏膜面向下， 涂布于 HP选择性 培养平板， 37°C , 在微需氧条件下培养 1天左右， 见透明、 光滑、 针尖 状小菌落， 即为阳性。
一份置 96孔板行快速尿素酶试验:将尿素酶试剂加入含鼠胃组织的 96孔板各孔中， 每孔定量至 ΙΟΟμΙ, 密闭下室温静置 0.5h, 试剂由黄色 变为红色判定位阳性， 即样本有 Hp感染。
另一份置 4%多聚甲醛固定， 经石蜡包埋、 切片、 HE染色、 Gmisa 染色后用显微镜观察 HP SSI菌的定植情况和组织的炎症反应。
3.2结果
3.2.1免疫 /攻击后小鼠毒副反应观察
各组小鼠精神状态良好， 未发现精神萎靡、 腹泻等不良反应。
3.2.2血清 /肠黏膜 IgG/sIgA抗体结果
空白对照组、 UreB蛋白组以及 UreB+CTB小鼠的抗体在给药后没 有增加，而 CTB-UreB融合蛋白和 CTB- UreB/4CTB嵌合体蛋白组的小 鼠其血清 IgG抗体和肠黏膜 slgA抗体在首次给药后 9.5周明显增加，与 空白对照组比较均存在显著性差异。 结果见图 20、 21。
3.2.3组织涂片及快速尿素酶检测 各组小鼠在攻击后 4周做快速尿素酶检测及组织涂片结果显示： 模 型组的 8只动物均被感染了 HPSS1 , 尿素酶及组织涂片均为阳性； 空白 对照组 （未给予 HPSS攻击） 所有动物其尿素酶及组织涂片均为阴性结 果； UreB组有 7只动物显示尿素酶及组织涂片结果为阳性； CTB-UreB 组显示有 5只动物尿素酶结果为阳性， 其中 4只动物涂片结果为阳性； CTB-UreB/4CTB组仅有 1只动物显示尿素酶检测阳性，而所有动物涂片 结果均为阴性。 结果详见表 1。
表 1： 根据本发明的实施方式 rHP疫苗口服给予小鼠， 再用 HPSS1 攻击后 HP检测结果
Figure imgf000036_0001
3.2.4病理组织切片染色结果
对照组小鼠胃小凹有大量成团的幽门螺杆菌定植， 胃黏膜固有层深 部至中部有大量的巨噬细胞和中性粒细胞浸润， CTB-UreB 融合蛋白组 小鼠胃小凹有少量的幽门螺杆菌定植， 胃黏膜固有层深部有中等程度的 巨噬细胞和中性粒细胞浸润， 炎症反应较轻； 而 CTB-UreB/4CTB嵌合 体蛋白组胃小凹有极少量的幽门螺杆菌定植， 组织切片上未见巨噬细胞 和中性粒细胞浸润 （见图 22)。
3.3 结论
在整个实验过程中， 给予 CTB-UreB/4CTB嵌合体蛋白免疫的小鼠 未发现不良反应， 病理学检查显示免疫小鼠胃内无明显炎症改变， 表明 以 CTB-UreB/4CTB嵌合体蛋白为抗原成分的 HP疫苗以试验剂量进行免 疫时具有良好的安全性。
CTB-UreB/4CTB嵌合体蛋白经口服免疫可有效诱导免疫应答反应， 对 HP SSI菌攻击的小鼠具有良好的保护作用，作为抗 HP感染的口服疫 苗具有良好的应用前景。 上述检测例的结果表明， 口服给予重组 CTB-UreB 融合蛋白和 CTB-UreB/4CTB嵌合蛋白，能刺激机体产生抗 UreB抗原的特异性抗体； 其中 CTB-UreB/4CTB嵌合蛋白免疫小鼠的血清抗 UreB抗原的特异性 IgG和胃黏液 s-IgA抗体滴度和阳转率均明显高于 CTB-UreB融合蛋白， 与空白对照组比较有显著性差异， 表明上述抗原具有良好的免疫反应性 和抗原性， 可以作为 HP的候选疫苗。
结果表明， CTB-UreB/4CTB嵌合蛋白产生的抗体水平显著高于仅仅 用 UreB产生的抗体。 而且无论是针对在血清中的抗体还是分泌型的抗 体， 都观察到 CTB-UreB/4CTB嵌合蛋白诱导产生了显著升高的抗体。 这表明本发明的抗原组合物明显提高了原抗原的免疫原性水平， 增强了 机体的免疫反应， 有助于更加有效的疫苗的制备。 而且表 2和图 14分 泌型 IgA检测的结果还表明， 本发明的重组抗原构成的嵌合蛋白利于抗 原穿透肠黏膜， 并刺激黏膜产生分泌型 IgA免疫。 对比例 1
发明人构建载体并表达幽门螺杆菌抗原如 UreB与霍乱毒素的入2亚 单位 （CTA2) 的融合蛋白， 并将其在体外与表达的 CTB蛋白组合， 使 其形成 UreB-CTA2/5CTB嵌合蛋白。
根据现有技术理解， CTA2与 CTB的结合与 CTB之间的结合同为非 共价结合， 用同样的方法应该有相似的产率。
然而，实验结果表明 UreB-CTA2/5CTB嵌合蛋白组装效率 <5 % (见 图 23A) , 而 CTB-UreB/4CTB嵌合蛋白组装效率 >90 % (见图 23B) , 本 发明 CTB-UreB/4CTB的组装效率远远高于 UreB-CTA2/5CTB的组装效 率。 CTB-UreB/4CTB嵌合蛋白可明显克服 UreB-CTA2/5CTB由于组装效 率低导致产业化可能性低的缺陷。
对比例 2
发明人尝试利用酵母为代表的真核表达系统同时表达 CTB-抗原融 合蛋白和 CTB ,希望得到自然形成的有预期效果的嵌合体结构。发明人 发现， 釆用酵母系统表达时， CTB能正确形成由 5个相同 CTB单体组 成的五聚体结构， 但是表达的 CTB分子量（约为 65KD) 明显大于天然 状态时（55KD)。提示釆用真核系统表达时，可能发生了 CTB的糖基化 翻译后修饰， 致使表达的 CTB与天然的 CTB分子存在较大差异， 进而 影响与 GM1受体的结合力。同时，釆用这种方式制备嵌合体时对与 CTB 融合的抗原大小有严格要求，并且利用此表达方式时，融合蛋白和 CTB 单体自然组装效率较低， 致使整个表达体系的得率很低， 不适用于大规 模疫苗的制备。
对比例 3
发明人尝试在以大肠杆菌为代表的原核表达系统中，同时表达 CTB- 抗原融合蛋白和 CTB , 希望能得到自然形成的嵌合体。 由于霍乱毒素 B 亚单位的五聚体组装机理不明， 研究一致认为嵌合体的组装在穿膜过程 中发生。 在大肠杆菌表达系统中， 带信号肽的 CTB-抗原融合蛋白无法 正确穿膜， 形成预期的嵌合体结构。 并且， 在胞内表达的融合蛋白大都 形成包涵体， CTB 表达也因起始氨基酸甲硫氨酸的存在无法形成天然 CTB五聚体， 因此也无法形成预期的嵌合体结构。 以上仅仅以幽门螺杆菌相关抗原为例， 说明了本发明的 CTB-X-4CTB系统在增强抗原的免疫原性方面的应用和效果。基于本领 域技术人员的经验和理解，可以推测出本发明的 CTB-X-4CTB系统还可 以应用于多种其他黏膜免疫抗原， 包括但不限于幽门螺杆菌抗原、 伤寒 抗原、 流感 HA抗原， 提高这些抗原的免疫原性， 并制备高效价疫苗。 其相应的载体选择和制备、 蛋白质的表达和纯化均为本领域公知技术， 本领域普通技术人员在了解了本发明的发明点之后， 均可以利用本发明 的 CTB-X-4CTB系统制备相应的融合蛋白和抗原组合物，并检测其相应 的免疫原性的提高。
所以， 以上所述仅为本发明的较佳实施例而已， 并不用以限制本发 明， 凡在本发明的精神和原则之内， 所做的任何修改、 等同替换、 改进 等， 均应包含在本发明保护的范围之内。
序列表
<110> 上海联合赛尔生物工程有限公司
<120> 抗原嵌合体、 抗原组合物、 疫苗及其制备方法和试剂盒
<130> OP60-120072
<160> 3
<210> 1
<211> 2067
<212> DNA
<213> 人工合成
<400>
Figure imgf000040_0001
Figure imgf000041_0001
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Figure imgf000042_0001

Claims

权利要求书
1、 一种抗原嵌合体， 包括：
抗原与能形成多聚体的黏膜免疫佐剂蛋白质单体的融合蛋白， 和 所述能形成多聚体的黏膜免疫佐剂蛋白质单体， 其中，
所述能形成多聚体的黏膜免疫佐剂蛋白质单体选自霍乱毒素 B亚单 位和大肠杆菌不耐热肠毒素 B亚单位中的一种，
所述多聚体为五聚体， 而且
在所述嵌合体中所述融合蛋白与所述能形成多聚体的黏膜免疫佐剂 蛋白质单体的摩尔比为 1:4 ,
其中所述抗原嵌合体稳定存在的 PH大于 7.0， 优选 pH为 8.0。
2、 如权利要求 1所述的抗原嵌合体， 其中所述霍乱毒素 B亚单位 和大肠杆菌不耐热肠毒素 B亚单位为其天然组成结构或其能够形成五聚 体结构的突变体。
3、如权利要求 1或 2所述的抗原嵌合体，其中所述抗原的分子量在 10至 lOOkD范围内， 优选 16kD至 65kD。
4、如权利要求 1至 3中任一项所述的抗原嵌合体，其中所述抗原为 适用于黏膜免疫的抗原。
5、如权利要求 1至 4中任一项所述的抗原嵌合体，其中所述抗原选 自经由黏膜途径感染人体或动物体的病原菌的抗原， 包括但不限于幽门 螺杆菌抗原、 伤寒抗原、 流感血凝素 （HA) 抗原。
6、如权利要求 5所述的抗原嵌合体，其中所述幽门螺杆菌抗原选自 幽门螺杆菌尿素酶 B亚单位 （UreB)、 幽门螺杆菌细胞毒素相关基因 A
(CagA)蛋白和幽门螺杆菌中性粒细胞激活蛋白（NAP)中的至少一种。
7、如权利要求 1至 6中任一项所述的抗原嵌合体，其中所述融合蛋 白包含位于所述抗原与所述黏膜免疫佐剂蛋白质单体之间的 3个 G4S连 接体。
8、一种抗原组合物，包含如权利要求 1至 7中任一项所述的抗原嵌 合体， 其中所述抗原嵌合体稳定存在的 PH大于 7.0， 优选 pH为 8.0。
9、一种疫苗，包含如权利要求 8所述的抗原组合物和适用于疫苗的 赋形剂。
10、 如权利要求 9所述的疫苗， 为口服疫苗、 经鼻给药的疫苗或经 直肠给药的疫苗。
11、 一种用于制备抗原组合物的试剂盒， 包括表达如权利要求 1-7 中任一项中所定义的融合蛋白的载体和表达如权利要求 1-7中任一项中 所定义的能形成多聚体的黏膜免疫佐剂蛋白质单体的载体。
12、如权利要求 11所述的试剂盒，其中所述载体均为原核表达载体。
13、 制备如权利要求 1至 7中任一项所述抗原嵌合体的方法， 包括 用权利要求 10所述的试剂盒中的载体分别表达所述融合蛋白和所述能 形成多聚体的黏膜免疫佐剂蛋白质单体， 然后通过复性方法使这两种蛋 白结合形成所述嵌合体， 其中所述复性方法包括将所述两种蛋白共同放 置在含有尿素和二硫苏糖醇 （DTT) 或尿素和巯基乙醇的复性液中进行 复性的步骤， 优选所述复性液的 pH大于 7.0， 更优选所述复性液的 pH 为 8.0。
14、 如权利要求 13 所述的方法， 其中所述复性液中尿素的浓度为 1.0M至 2.5M。
15、如权利要求 13或 14所述的方法，其中所述 DTT或巯基乙醇的 浓度为 0.2mM至 1.0mM。
16、 如权利要求 13至 15中任一项所述的方法， 其中在所述复性液 中进行复性步骤之前，先将所述蛋白质单体在含 6M至 9M，优选 8M的 尿素， 且 pH为 3.0至 4.0的缓冲液中预复性 0.5至 3小时， 优选预复性 1小时。
17、一种通过复性制备嵌合体的方法，所述嵌合体包含（1 )一种蛋 白质与能形成多聚体的单体蛋白质的融合蛋白和 （2) 能形成多聚体的 单体蛋白质， 通过所述 （1) 融合蛋白中的单体蛋白质和所述 （2) 单体 蛋白质形成多聚体来形成嵌合体， 所述方法包括：
将所述 （2) 能形成多聚体的单体蛋白质在含 6M至 9M, 优选 8M 的尿素， 且 pH为 3.0至 4.0的缓冲液中预复性 0.5至 3小时， 优选预复 性 1小时； 和
将所述 （1 ) 融合蛋白和所述 （2) 能形成多聚体的单体蛋白质在含 有 1.0M至 2.5M的尿素和 0.2mM至 l.OmM的 DTT或巯基乙醇的复性 液中复性形成所述嵌合体，优选所述复性液的 pH大于 7.0 ,更优选所述 复性液的 pH为 8.0。
PCT/CN2014/083291 2013-12-06 2014-07-30 抗原嵌合体、抗原组合物、疫苗及其制备方法和试剂盒 WO2015081711A1 (zh)

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