WO2008040155A1 - 口服重组幽门螺杆菌疫苗及其制备方法 - Google Patents

口服重组幽门螺杆菌疫苗及其制备方法 Download PDF

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WO2008040155A1
WO2008040155A1 PCT/CN2007/002655 CN2007002655W WO2008040155A1 WO 2008040155 A1 WO2008040155 A1 WO 2008040155A1 CN 2007002655 W CN2007002655 W CN 2007002655W WO 2008040155 A1 WO2008040155 A1 WO 2008040155A1
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recombinant
subunit
ureb
lta2b
recombinant protein
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PCT/CN2007/002655
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English (en)
French (fr)
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WO2008040155A8 (zh
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Quanming Zou
Wende Tong
Xuhu Mao
Gang Guo
Dongshui Lu
Chao Wu
Hao Zeng
Yichao Wang
Jun Yang
Weijun Zhang
Kaiyun Liu
Ping Luo
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Chongqing Kang Wei Biotechnology Co., Ltd.
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Priority to EP07800872A priority Critical patent/EP2082750B1/en
Priority to US12/310,658 priority patent/US8900594B2/en
Priority to AT07800872T priority patent/ATE510560T1/de
Priority to KR1020097006936A priority patent/KR101100237B1/ko
Priority to JP2009526999A priority patent/JP5327873B2/ja
Publication of WO2008040155A1 publication Critical patent/WO2008040155A1/zh
Publication of WO2008040155A8 publication Critical patent/WO2008040155A8/zh
Priority to HK10101022.8A priority patent/HK1137337A1/xx

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    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • 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
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    • 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
    • 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)
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    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to a recombinant protein for the immunoprophylaxis of human Helicobacter pylori infection and a degradable sustained-release microsphere-encapsulated oral preparation vaccine prepared therefrom, and to a preparation method thereof.
  • Background technique
  • Hp Helicobacter pylori
  • WHO World Health Organization
  • Vaccine is the most cost-effective way to control infectious diseases. It can prevent or treat Hp infection by stimulating the body to produce a specific immune response caused by natural infection. Due to the difficulty in large-scale cultivation of Hp, harmful antigens such as potential carcinogens in crude antigens have greatly restricted the research progress of whole-bacteria vaccines. Genetically engineered vaccines are safe, effective, and inexpensive, and are easy to promote and apply. They are a major target of Hp vaccines. Hp vaccine has been studied at home and abroad, but it has not been successful so far.
  • Urease is a nickel-dependent enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. It is the most abundant protein in Helicobacter pylori, distributed in both Hp cells and cell membranes, accounting for 5%-10% of total Hp protein. . Urease produces ammonia by decomposing urea to neutralize gastric acid, helping bacteria settle in the stomach and provide ammonia for bacterial protein synthesis. Host tissues can also be directly damaged by ammonia, or indirectly stimulated by urease-induced inflammatory reactions. damage. Blocking the expression of the urease gene will inhibit the colonization of Helicobacter pylori in the host and reduce bacterial protein synthesis. And reduce the inflammatory response associated with Helicobacter pylori.
  • Hp infection in patients especially those like: patients are able to detect antibodies, antibody levels correlated with the severity of the urease B have a certain correlation.
  • Oral vaccination with Helicobacter pylori urease or recombinant urease B subunit (rUreB) protects mice from Helicobacter pylori infection and eliminates existing infections (Michetti et al., Gastroenterol. 1994) 0
  • Urease activity Hp infection plays an important role. Hp lacking urease activity does not cause infection in animal models, and antibodies that neutralize urease activity may play a key role in resisting Hp colonization. From the above findings, it can be considered that urease antibodies, particularly antibodies capable of neutralizing urease activity, play a major role in resisting Hp infection.
  • the mucosal immune system is an important part of the body's immune system, including the intestinal mucosa-associated lymphoid tissue and the bronchial mucosa-associated lymphoid tissue. It plays a unique role in the body's defense function.
  • the immune-inducing site and the immune effect site are composed of mesenteric lymph nodes, and a large number of lymphocytes dispersed in the lamina intestinal and intestinal epithelium.
  • Various immune cells on the mucosal surface such as the gastrointestinal tract produce and secrete antibodies against the antigen (mainly slgA) by ingesting, processing and presenting antigens, which can be specific to carriers carrying such antigens such as bacteria and viruses.
  • the binding reaction prevents it from colonizing the mucosal surface or invading the body, thereby exerting a certain immune defense effect.
  • the biggest drawback of mucosal immunity is that it is easy to produce immune tolerance to the antigen. Even if the antigen amount is increased, the antigen-specific slgA level produced by the body or mucosa is very low, and the microbial infection of the corresponding antigen has almost no immune defense ability or ability. , can easily lead to the occurrence of immune tolerance.
  • Heat labile toxin is a heat-labile enterotoxin produced by Enterotoxigenic Escherichiacoli (ETC) that causes severe diarrhea in humans and certain livestock.
  • the LT consists of one A subunit (LTA) and five B sub-single (LTB) bits. Five identical LTBs form a cyclic pentamer in space, one LTA is located in the center of the cyclic pentamer, and its C-terminus is bound to LTB by a non-covalent bond.
  • the A subunit consists of two subunits, A1 and A2. A1 is the toxic part of the toxin, A2 is combined with the B subunit, and there is a disulfide bond between A1 and A2.
  • the A and B subunits in the cytosol are present as precursors carrying the signal peptide and assemble into intact LT when traversing the cell membrane.
  • the role of the B subunit is to specifically bind to the GM1-ganglioside receptor on the surface of eukaryotic cells, causing the LT molecule to be allosteric,
  • a The subunit leaves the B subunit and enters the cell membrane, followed by degradation of the disulfide bond, activation of the A1 peptide chain, and the A1 subunit has GTP-dependent ADP-ribosyltransferase activity, which is destroyed by G protein-mediated ADP-ribosylation.
  • the degradation and balance of cAMP within the stimuli stimulates the increase of cAMP levels, thereby triggering the toxin effect.
  • LT has been considered a very promising mucosal immune adjuvant. Rollwagen and other studies have shown that LT can enhance the mucosal immune response of Campylobacter and accelerate the clearance of the bacteria in the intestine, and LT will not be immune to the antigen for a long time after initial immunization with the antigen.
  • LT acts as a mucosal immune adjuvant
  • LT has a strong intestinal toxicity, so it is currently mainly non-toxic and low-toxic with its B subunit or LT.
  • Mutant as adjuvant Yamamoto M, et al. J. Immunol, 1999, 162:7015-7021; Martin M, et al.
  • LT mutants lacking ADP-ribosylase activity are still adjuvant, and these results indicate that LT adjuvant is independent of ADP-ribosyl active subunit A1, while A2 subunit is LT of The activity of the agent has contributed.
  • De Haan experiments also showed that: LT mutants without toxic activity immunized mice through the nasal mucosa, still retaining the immunological properties of wild-type toxins, but using recombinant LTB alone than the immunogen of non-toxic active LT mutants Sexual weakness (De Haan L, et al.
  • LTA's ADP ribosylase activity is not directly related to the immunity of the toxin.
  • LT holotoxin produces high levels of systemic IgG and mucosal S-IgA
  • the A1 subunit has GTP-dependent ADP-ribosyltransferase Activity is related to the toxin effect, suggesting that the adjuvant effect of LTA is mainly exerted by the LTA2 subunit.
  • LTB has been used as an adjuvant.
  • LTB and UreB were fused to form recombinant protein LTB-UreB (Wu Chao, Zou Quanming et al. Fusion and expression of Helicobacter pylori UreB and E. coli LTB gene. Chinese Journal of Microbiology and Immunology, 2002, 22(2): 175 -179), and an in-component adjuvant vaccine can be prepared.
  • LTB-UreB Wang Chao, Zou Quanming et al. Fusion and expression of Helicobacter pylori UreB and E. coli LTB gene. Chinese Journal of Microbiology and Immunology, 2002, 22(2): 175 -179
  • an in-component adjuvant vaccine can be prepared.
  • the immune protection rate of this recombinant protein is not very satisfactory.
  • An object of the present invention is to provide a recombinant protein for use in immunoprophylaxis and treatment of human Helicobacter pylori infection, from which a recombinant H. pylori vaccine for vaccination against human Helicobacter pylori infection can be prepared.
  • Another object of the present invention is to provide a method of preparing the recombinant protein for use in the immunoprophylaxis and treatment of human Helicobacter pylori infection.
  • Another object of the present invention is to provide a method of preparing the above-described recombinant H. pylori vaccine for use in immunoprevention and treatment of human Helicobacter pylori infection.
  • the present invention provides a recombinant protein for the prevention and treatment of human Helicobacter pylori infection, which comprises an A2 subunit and a B subunit of Enterotoxigenic Escherichia coli LT and a Helicobacter pylori urease.
  • the B subunit is fused (ie, LTA2B-UreB, abbreviated as LU).
  • LTA2B acts as an intramolecular mucosal immune adjuvant
  • UreB acts as an immunogen.
  • LTA2B lacks the A1 moiety with ADP ribosylase activity in LT molecule, overcoming the toxic side effects of LT adjuvant.
  • LTA2B increases the A2 part of LT for the first time, which can improve adjuvant activity and immunogen. Sex.
  • the LTA2B-UreB fusion protein has a similar biological activity and immunogenicity and reactogenicity as full-length UreB, and has a function of enhancing mucosal adjuvant activity.
  • the present invention also provides a sustained-release microsphere-encapsulated oral preparation for the immunoprophylaxis and treatment of human Helicobacter pylori infection, which comprises the above-mentioned recombinant protein for the prevention and treatment of human Helicobacter pylori infection.
  • a degradable sustained-release microsphere (MS) is used to encapsulate the above recombinant fusion protein.
  • sustained-release microspheres can effectively prevent the decomposition and destruction of antigens by gastric acid and digestive enzymes, and maintain the overall stability and antigenic activity of antigenic substances.
  • the size of MS determines the distribution of various organs in the body.
  • the particle size is controlled within a certain range and induced to be captured by the target organ, thereby maximizing the immune efficacy; when encapsulating the antigen, by changing the ratio of the carrier material to the antigen, The biodegradation of the carrier material is adjusted to change the particle size of the MS and the adhesion of the antigenic substance and the MS to achieve long-term sustained release of the antigen.
  • the wrap comprises sodium alginate, vegetable oil, CaCl 2 and chitosan.
  • the t-ball wrap formulation has a particle size of 3.33 ⁇ m.
  • the sustained-release microsphere-encapsulated oral preparation is finally developed into an oral immuno-lyophilized preparation
  • the lyophilized product has an excipient of 8% mannitol and a stabilizer of 0.05% EDTA-Na 2 , which is optimal.
  • the pH value is 10.0, because oral administration has the following advantages: First, the vaccine antigen component directly stimulates the lymphocytes associated with the gastrointestinal mucosa, and through the recognition, presentation and immune effects on the intestinal antigen, a related mucosal immune response is produced, and effective prevention is achieved. The purpose of the disease caused by Helicobacter pylori infection; Second, oral immunization is painless, non-invasive therapy, convenient, low cost, and easy to be accepted by the subject.
  • the present invention also provides a nucleotide sequence encoding the above recombinant protein, which is a gene encoding an A2 subunit of enterotoxic Escherichia coli heat labile enterotoxin, a B subunit coding gene, and a Helicobacter pylori urea.
  • the enzyme B subunit encodes a nucleoside formed by fusion of the gene ltA2B-ureB (abbreviated as lu).
  • the present invention also provides a recombinant plasmid which is obtained by linking the nucleotide sequence of claim 8 and the plasmid pET-llc.
  • the present invention also provides a method of producing the recombinant protein of claim 1, which comprises the steps of:
  • step (1) the nucleotide sequence cloned in step (1) is ligated into the fusion gene ltA2B-ureB;
  • the ltA2B-ureB fusion gene is constructed on the vector pET-llc, transformed into E. coli BL21 (DE3), and the recombinant plasmid pET-llc-LU/BL21(DE3) 0 is constructed.
  • a coding nucleoside sequence comprising at least the above-described LU amino acid sequence or an amino acid sequence homologous to more than 95% thereof and having its protein activity;
  • the recombinant protein LTA2B-UreB is fermented from the above recombinant engineered bacteria in an 80 L fermentor.
  • the invention also provides a method of preparing an oral recombinant H. pylori vaccine comprising the steps of:
  • the sustained release microsphere wrapping preparation is made into a lyophilized product.
  • the obtained recombinant protein is mixed with the sodium alginate solution, added to the vegetable oil, and after emulsification, the reverse titration method is dropped into the CaCl 2 solution to prepare a sodium alginate-coated protein microsphere;
  • the obtained microspheres are solidified and washed, centrifuged to take a precipitate and then resuspended; then added to the chitosan solution to form a re-encapsulation to obtain chitosan alginate double-encapsulated protein microspheres, and then the microsphere suspension Slowly pour into the vial, the liquid level is less than 1cm.
  • the refrigerator is directly placed in a pre-cooled vacuum dryer to dry, and slowly raises the temperature to quickly sublimate the water.
  • the gas pressure indicator shows no gas generation, the lyophilized product is taken out for inspection.
  • the resulting vaccine is subjected to an animal test to test the safety and immunogenicity of the recombinant H. pylori vaccine. In another embodiment of the invention, the vaccine is subjected to human clinical trials to verify the immune response of the recombinant H. pylori vaccine.
  • the present invention comprises LTA2B fused with the A2 subunit of the enterotoxigenic Escherichia coli LT as a mucosal immune intramolecular adjuvant, and a urease B subunit as an immunogen to constitute a recombinant Helicobacter pylori vaccine.
  • a urease B subunit as an immunogen to constitute a recombinant Helicobacter pylori vaccine.
  • Figure 1 is an agarose gel electrophoresis map of the lu ( ltA2B-ureB ) fusion gene obtained by the overlap extension method, wherein lane 1 is a PCR amplification product of the ureB gene; lane 2 is a nucleic acid (DNA) molecular weight standard (Marker ); 3 is a PCR amplification product of the ltA2B fusion gene.
  • lane 1 is a PCR amplification product of the ureB gene
  • lane 2 is a nucleic acid (DNA) molecular weight standard (Marker )
  • 3 is a PCR amplification product of the ltA2B fusion gene.
  • Figure 2 shows the restriction enzyme digestion of the pET-llc-lu recombinant plasmid, wherein lane M1. is PCR Marker; lane 1. is It A2B PCR product (about 0.4 kb); lane 2. is ureB PCR product (1.7 kb); lane 3. is the lu PCR product (about 2.1 kb); lane 4. is pET-llc Ndel digestion (5.7 kb); lane 5. is pET-llc-lu Ndel digestion (5.7 kb + 2.0 kb); lane 6. Is pET-llc-lu forward recombinant BamHI digestion (6.0kb+1.0kb+0.7kb); Lane 7. is pET-llc-lu reverse recombinant BamHI digestion (6.4kb+1.0kb+0.3kb) ; Lane M2. is DNA/Hindlll Marker.,
  • Figure 3 is a PAGE electrophoresis map of the fusion gene recombinant strain, in which lane 1: protein molecular weight standard (Marker); lane 2: empty plasmid induction for 1 hour; lane 3: gene recombinant induction for 5 hours; lane 4: recombinant strain Induction for 4 hours; Lane 5: induction of recombinant bacteria for 3 hours; Lane 6: induction of recombinant bacteria for 2 hours; Lane 7: induction of recombinant bacteria for 1 hour. It can be seen that the gene recombinant strain has an increased protein expression band at a molecular weight of 72 kDa after induction, which is consistent with the molecular weight of the target protein. After UVP image scanning analysis, the expression of the target protein was induced by about 26% for 5 hours.
  • Figure 4 is a PAGE electrophoresis diagram of the purification effect of the target protein LU, wherein, lane 1: inclusion body lysate (pre-purification sample); lane 2, 3: impurity protein elution peak sample; lane 4: target egg White eluting peak sample; Lane 5: Protein molecular weight standard (Marker). The results showed that the purity of the target protein was significantly improved after the purification step, and the purity of the harvested protein peak was greater than 85% by UVP scanning.
  • Figure 5 shows the lyophilization curve of rHp (recombinant Helicobacter pylori vaccine) vaccine.
  • Genomic DNA of wild-type enterotoxin-producing Escherichia coli H44815 (purchased from China National Institute for the Control of Pharmaceutical and Biological Products) and genomic DNA of Helicobacter pylori NCTC11637 (purchased from the American Culture Collection ATCC) were used as templates, using P1 and Ure2, ⁇ 3, and ⁇ 4 amplify the ureB and ltA2B genes, respectively, and bacterial genomic extraction was performed according to conventional methods (Yan Ziying, Wang Hailin, Trans. Molecular Biology Experimental Guide. Science Press, 1998, P39).
  • the PCR amplification system is: 10 ⁇ Magnesium-free amplification buffer 10nL, MgCl 2 (25mmol/L) ⁇ , dNTPs (2.5mmol/L each) 8 L, upstream and downstream primers (PI and P2 or P3 and P4) 2 ⁇ of the above bacterial genome 2 L, Ex-Taq DNA polymerase (3 units / ) ⁇ plus sterile water to ⁇
  • the PCR amplification reaction was predenatured at 94 ° C for 5 minutes, denatured at 94 ° C for 50 seconds, annealed at 60 ° C for 50 seconds, extended at 72 ° C for 50 seconds, 35 cycles, and fully extended at 72 ° C for 10 minutes. After the agarose gel electrophoresis, the target fragment was recovered. (The underlined part is the recognition sequence of the corresponding enzyme)
  • the PCR products of ureB and ltA2B were recovered separately.
  • the recovered ureB and ltA2B genes were used as templates, and P1 and P4 were primers for overlap extension PCR reaction.
  • the PCR amplification system is: 10 ⁇ Magnesium-free amplification buffer 5 L, MgCl 2 (25 mmol/L) 4 ⁇ dNTPs (25 mmol/L) 4 ⁇ , upstream and downstream primers (PI and P4) lnL, the above ureB and ltA2B genes 2 ⁇ Ex-Taq DNA polymerase (5 units 1 ) O ⁇ , add sterile water to 50 ⁇ .
  • Overlap extension PCR amplification reaction pre-denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 60 seconds, annealing at 60 °C for 60 seconds, extension at 72 °C for 60 seconds, 35 cycles, and complete extension at 72 °C for 10 minutes. After agarose gel electrophoresis, the target fragment was recovered.
  • FIG. 1 1% agarose gel electrophoresis analysis (see Figure 1), the size of the fragment shown in the figure is as expected (about 2.1 kb), initially identified as the target gene fragment, and named ltA2B-ureB, as SEQ ID NO: 1 Show.
  • Figure 2 shows: Fusion gene fragment The size is consistent with the prediction, indicating that the overlap extends to obtain the fusion gene.
  • the lu (ltA 2 B-ureB) fusion gene amplification (PCR) product was purified by 1.0% agarose electrophoresis, gel recovery, and then ligated with the vector pMD-18T (purchased from TaKaRa) to transform Escherichia coli DH5a, and the shield particles were extracted. It was digested with Ndel and identified by 1.0% agarose gel electrophoresis.
  • DNA was extracted from pMD-18-lu/DH5a-positive recombinant strain, Ndel was digested, 2.0 kb lu DNA fragment was recovered, and pDe-llc vector (purchased from Novagen, USA) which was digested with Ndel and dephosphorylated. Ligation, transformation of E. coli DH5a, screening of ampicillin-positive LB plates, picking up suspected colonies to extract plasmids, Ndel digestion to identify positive recombinants, BamHI digestion to identify positive and negative. The results of enzyme digestion identification are shown in Figure 2.
  • the positive recombinant plasmid DNA was digested by Ndel to generate a 5.7 kb vector fragment and a 2.1 kb lu gene fragment (lane 5). After BamHI digestion, the reverse recombinant produced 6.4 kb+1.0 kb+0.3 kb three fragments (the first fragment). Lane 7), the forward recombinant produced three fragments of 6.0 kb + 1.0 kb + 0.7 kb (lane 6).
  • the reaction mixture includes: l g plasmid DNA; ⁇ 10 ⁇ buffer (see Shanghai Biotech Co., Ltd. product description); ⁇ restriction enzyme Nde I
  • the target DNA electrophoresis band on the agarose gel was observed and cut under a UV lamp, and transferred into a 1.5 ml EP tube.
  • Centrifuge at 12000g for 1 minute dispose the tube to another clean 1.5ml EP tube, add a certain volume of TE buffer, incubate at 65 °C for 10 minutes, centrifuge at 12000g for 1 minute, take a certain amount of electrophoresis, and detect the purification effect by UVP UV scanner. .
  • the concentration of the target DNA fragment and the vector fragment was determined by an ultraviolet spectrophotometer. According to the principle that the molar ratio of the exogenous fragment to the carrier was generally 1:2 to 10, the ligation reaction system was designed as follows: target DNA ⁇ ; shield particle carrier 1 ⁇ 2 ⁇ 1 ; ligation solution 5 ⁇ 1; ddH 2 0 2 ⁇ 3 ⁇ 1; total volume 10 ⁇ 1. The reaction was carried out at 22 ° C for 12-16 hours.
  • Sterile inoculating loops were used to extract the bacterial stock solution frozen at -70 °C, and inoculated on LB plates by three-line method, and cultured at 37 ° C for 12-16 hours. A single colony was picked and inoculated into 2 ml of LB medium and cultured at 37 ° C for 12 to 16 hours on a shaker. The overnight cultured DH5a was transferred to the LB medium at a ratio of 1%, and cultured to OD 6 at 37 ° C on a shaker. . The bacteria were collected by centrifugation at 8000 g for 5 minutes for 0.2 to 0.4 hours.
  • the pellet was resuspended by adding 1 ml of pre-cooled 0.1 M CaCl 2 and ice-bathed for 3 hours. Centrifuge at 8000 g for 5 minutes at 4 ° C and discard the supernatant. The precipitate was suspended by adding ⁇ pre-cooled 0.1 M CaCl 2 , and ice-bathed for 1 hour, and set aside.
  • the recombinant expression plasmid containing the LU fusion gene was transformed into E. coli BL21 and the plasmid was extracted and identified by enzyme digestion.
  • 2 is a gel electrophoresis pattern of pET-llc-LU/BL21(DE3) recombinant expression plasmid, wherein lane 5 is pET-llc-LU/BL21(DE3) recombinant plasmid Ndel digestion product; lane M1 M2 is a nucleic acid (DNA) molecular weight standard (Marker); Lane 4 is an empty vector plasmid Me / single digestion product (5700 bp). The size of the fragment was consistent with the design, preliminary proof The recombinant plasmid was successfully constructed. Preparation and transformation of competent bacteria of genetically engineered Escherichia coli BL21 and plasmid extraction and restriction enzyme digestion of recombinant bacteria.
  • the unidentified recombinant strain was inoculated into 3 ml of kanamycin-containing LB medium and cultured overnight at 37 °C on a shaker. On the next day, the recombinant engineering bacteria cultured overnight were transferred to 20 mL of LB medium containing kanamycin at a ratio of 1%, cultured at 37 ° C for 2.5 hours on a shaker, induced by IPTG for 5 hours, and detected by SDS-PAGE. Expression patterns and expression levels were screened for highly expressed strains, Figure 3.
  • the present invention also verified the stability of UreB and LTA2B and whether it can replace the urease B full-length protein.
  • the LTA2B and UreB and LTA2B fragment genes were amplified by PCR and constructed into the prokaryotic expression vector pET-llc(+), which was transformed into the host Escherichia coli BL21(DE3). IPTG induced the stable expression of LTA2B-UreB fusion protein.
  • ELISA and immunoblot analysis confirmed that recombinant LTA2B-UreB has good immunogenicity and reactogenicity.
  • the fermentation process is as follows: using German B.Bron 80L fermenter, 10% of the seed bacteria in the fermentation process; temperature 37 ° C; pH 7.0, the pH is kept constant by adding 30% ammonia water automatically; At 500 rpm, as the cell density increases and the oxygen consumption increases, the number of revolutions is changed to cascade dissolved oxygen control, that is, the P0 2 controller is the main controller, and the stirring controller is the servo controller; dissolved oxygen concentration Cascade control and negative oxygen bypass control to make it end The concentration is controlled at 45%-50%; when A600 does not reach 2, no feeding is added, and then feeding is added every 0.5 hours, so that the final concentration of glucose, trypsin and 8% yeast extract is 0.5% respectively. , 0.2% and 0.2%. After the fourth feeding, when the glucose concentration was decreased to 0.1%, IPTG 500 ⁇ 1 ⁇ was added to induce the bacteria for 4 hours; the fermentation process was fed on the basis of the batch culture of the cascade dissolved oxygen control.
  • the medium used in the fermentation process was modified M9-CAA medium, and 0.6% yeast extract and 2 mg/L ZnCl 2 .4H 2 0, 2 mg/LCoCl 2 .4H 2 0, 4 mg/L FeS0 4 were added to the M9-CAA. 16H 2 0, 5 mg/LH 3 B0 3 , 1.6 mg/LMnCl 2 '4H 2 0, 4 mg/L CuS0 4 was formed.
  • the bacterial solution was recovered and centrifuged (8000 g) at 4 ° C for 15 minutes. Aspirate the supernatant, collect the bacteria, weigh it and store it for later use.
  • Results The results showed that the bacterial yield was above 75g/L.
  • the expression of the target protein was stable at around 30%. It proves that this pilot fermentation process is advanced, and the process repeatability and stability have reached a high level.
  • Inclusion body extraction 5000g of highly expressed cells were suspended in a ratio of 1:10 (W/V) in TE buffer. After pre-cooling at 4 °C, the cells were homogenized by a cell homogenizer. Using a high-pressure homogenizer, the bacteria are sterilized under the pressure of 40 - 70Mpa (4 to 6 times). After the bacteria is broken, a small amount of smear is stained, and the integrity of the cells is observed under the microscope to ensure the cells. The fragmentation was completed, followed by centrifugation at 500 g for 25 minutes, the pellet was discarded, and the mixture was centrifuged at 15,000 g for 40 minutes, and the supernatant was discarded to collect the precipitate.
  • the washing conditions were as follows: stirring at 4 ° C for 20 minutes, centrifugation at 15,000 g for 40 minutes, collecting the inclusion body precipitate; finally, the inclusion body was mixed with the inclusion body solution at a ratio of 1:10 (W/V), and stirred at 4 ° C. After an hour, centrifuge at 15,000 g for 45 minutes, and take the supernatant as a raw material for the next purification.
  • Buffer for inclusion body extraction 1) TE buffer: 20 mmol/L Tris, 5 mmol/L EDTA, pH 8.0; 2) Inclusion body wash A: 5 mmol/L EDTA, 20 mmol/L Tris, 1 % Triton X-100 , pH 8.0; 3) Inclusion body wash B: 20 mmol/L Tris, 2 mol/L Urea, pH 8.0; 4) Inclusion body solution: 1 mmol/L EDTA, 20 mmol/L Tris .8 mol/L urea (pH 8.0).
  • Chromatographic purification This step is purified as Q Sepharose HP anion exchange column and Purified by Sephadex G-25 column chromatography, and the intermediate purification was performed on an AKTA explorer 100 system using an XK50/30 column. Due to the precise and automated operation of the AKTA explorer 100 system, two sets of AKTA explorer 1()() were used for continuous automated chromatography, and the yield of each batch of rHp vaccine was 40 g. The protein of interest was purified using 20 mmol/L Tris, 5 mmol/L EDTA, pH 8.0, eluting with a gradient of NaCl.
  • Purified target protein was subjected to SDS-PAGE, and the purity of the target protein finally obtained was determined to be >80%, and the yield was >79%.
  • the high-pressure bacteria-breaking technology used in the production or pilot purification is used in step 1, and the bacteria-breaking rate is more than 98%, and the inclusion body precipitate is obtained by differential centrifugation.
  • the affinity chromatography purification packing in step 2 is selected from the group consisting of Chelating Sepharose Fast Flow.
  • the anion purification filler of step 3 is selected from the group consisting of Q Sepharose HP, Q Sepharose FF and Q Sepharose XL.
  • the recombinant protein prepared in Example 4 was mixed with a 2% sodium alginate solution at room temperature, and vegetable oil was added, and the ratio of vegetable oil to sodium alginate AGS emulsion was 2:8. After emulsification for 10 minutes at 8000 r/min, it was dropwise added to the CaCl 2 solution, stirred at 800 r/min for 30 minutes, and then an OAV emulsion was formed, and the precipitate was centrifuged, washed, and resuspended.
  • the suspension was added to a 1% concentration of chitosan solution, stirred at 800 rpm for 30 minutes, and then the chitosan-alginate-coated recombinant LU protein microspheres were prepared and collected by centrifugation for 3 times.
  • the above microsphere suspension was slowly poured into a glass dish at a height of less than 1 cm. - After pre-freezing for 12 hours in the refrigerator at 40 °C, it is directly placed in a pre-cooled vacuum dryer to dry, and slowly raise the temperature to quickly sublimate the water. When the gas pressure indicator shows no gas generation, take out the freeze-dried ⁇ mouth. Check. MS, lyophilized powder and lyophilized powder reconstituted material were applied to the slides after washing and centrifugation respectively. The morphology of the microspheres before and after lyophilization was observed under light microscope.
  • the size and shape of the microspheres after centrifugation were regular; It is irregularly distributed, and its shape exhibits many irregular shapes such as rod shape, fusiform shape and oblate shape.
  • the number of microspheres and the morphology are basically restored to the original characteristics before lyophilization.
  • the prepared MS surface was full and uniform in size, and the average particle diameter was 3.33 ⁇ m.
  • the highly purified recombinant LU protein was prepared by appropriate dilution and then lyophilized into lyophilized bottles.
  • a stable and mature rHp vaccine freeze-drying curve was obtained: the product pre-freezing temperature was -45 ° C, time 4 hours; the first sublimation temperature was 20 ° C, time 30 hours; the second sublimation temperature was 30 ° C At 8 hours, the entire freeze-drying process took 42 hours.
  • the moisture content is ⁇ 3%, which is in line with the requirements of the Pharmacopoeia of the People's Republic of China (2000 edition) for the moisture content of freeze-dried products.
  • the excipient of the rHp vaccine lyophilized product was 8% mannitol
  • the stabilizer was 0.05% EDTA-Na 2
  • the optimum pH was 10.0
  • the mature was obtained.
  • Parameters such as freeze-drying curve. After repeated verification of more than 20 batches, the performance of the pilot freeze-drying process is stable and can meet the requirements of large-scale production. Based on 15 mg per lyophilized bottle, 40 g of vaccine protein can hold about 2,600.
  • the 5 m 2 lyophilizer used can be lyophilized to prepare about 5,000 lyophilized bottles of 32 mm diameter at one time, which can meet the needs of lyophilization of large-volume products.
  • the present invention also investigates the effect of artificial gastric juice on the stability of rHp vaccine.
  • the rHp vaccine is an oral immunoprecipitated preparation that must pass through the acidic pH environment of the stomach during its passage through the digestive tract to the target organ. Since the rHp vaccine itself is a proteinaceous substance, it is susceptible to acidification and denaturation or enzymatic degradation, thereby reducing its immunological activity.
  • the study found that rHp vaccine protein has a significant decrease in antigenic reactivity and artificial protein degradation in artificial gastric juices with pH values of 1 and 2, but maintains high antigen reactivity and protein stability in a pH of 3-7 environment.
  • the immunogenicity of the rHp vaccine was observed using rabbits, BALB/c mice and rhesus monkeys, respectively.
  • the vaccine was induced by two-way immunodiffusion test and ELISA.
  • the vaccine induced rabbits to produce high titers of anti-UreB antiserum, which confirmed that rHp vaccine protein has good immunogenicity.
  • the gerbils of each group were simultaneously fed with the prepared Hp bacteria solution 10 days after the last immunization. All experimental animals were fasted and watered 24 hours in advance, each time feeding 0.3 ml of bacterial solution, about 10 8 cf / ml (number of colonies per ml); 1 time in the morning and afternoon, 6 hours apart, 2 after the last feeding Drink water for hours. In the 4th week after the challenge, all the experimental animals were sacrificed and the specimens were collected. The water was stopped 24 hours before the execution, the gerbils were dissected, the stomach of the rats was taken out, and the stomach was cut along the stomach. The stomach residue was gently washed away with normal saline. Half of the gastric mucosa tissue was coated with Hp medium, three-line method was used for streaking, and micro-aerobic culture. The colonization of Hp in mice after immunization was as shown in Table 1.
  • mice immunized with the vaccine antigen LU can produce an effective protective effect against Hp whole bacterial challenge compared with the non-immunized group.
  • the BALB/c mouse model of Hp infection was confirmed by intramuscular injection of the fusion protein and the two subunits in vitro.
  • the immunization was performed every 0, 2, and 4 weeks, and the immunization dose was 100 g (100 uL) / only, etc.
  • a volume of aluminum adjuvant is mixed.
  • Four weeks after the last immunization the mice were sacrificed and samples were collected. The mice were treated with different experimental methods.
  • mice after treatment mice after treatment, untreated, treated mice, Hp colonization
  • the number of Hp is measured, the number is significantly reduced.
  • the rHp vaccine can effectively induce the mucosal immune response of the body and has good immunogenicity.
  • the clinical research data of the present invention are as follows:
  • the subjects were observed for 30 minutes immediately after taking the vaccine, and the whole body (body temperature) and local (gastrointestinal) reactions and the occurrence of abnormal reactions (including fever and stool shape) were observed in detail at 6, 24, 48 and 72 hours. And the number of abnormalities, diarrhea, vomiting, etc.).
  • body temperature is below 37 ° C;
  • body temperature is 37.6 ° C ⁇ 38.5 ° C ;
  • Severe reaction body temperature is 38.6 ° C or above;
  • the immunogenicity was evaluated by antibody status 14 days after the whole immunization, and the antibody growth and decline was observed 60 days after the whole immunization.
  • Serum-specific IgG was ⁇ 1:100 before immunization, and ⁇ 1:100 was positive after immunization; serum-specific total Ig, saliva-specific sIgA, and ratio of post-immunization to pre-immune titer ⁇ 4 times were transgenic.
  • phase I clinical study was an uncontrolled study of 30 healthy children who received oral recombinant H. pylori vaccine at a dose of 45 mg/time, and observed systemic (body temperature) and local (gastrointestinal) responses.
  • the clinical study of the flood season continued when no serious abnormal reactions were observed.
  • Phase II clinical studies were randomized, double-blind, controlled studies. The study was divided into four groups (see Table 4). Immunization procedure: Oral immunization once every two weeks for three consecutive times, ie 0, 14 and 28 days.
  • Phase I clinical studies 30 subjects received 45 mg/dose of oral recombinant H. pylori vaccine immunization, and 30 of the three full-course immunizations did not observe any immediate response, systemic or local response, delayed response, and Other abnormal reactions, coupling reactions, and any clinically significant diseases and events (Tables 5, 6). Tips The 45 mg/dose oral recombinant H. pylori vaccine has good safety in humans. Based on the results of Phase I clinical studies, Phase II clinical studies were conducted to further expand the safety of population studies and focus on the study of their immune effects.

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Description

口服重组幽门螺杆菌疫苗及其制备方法 技术领域
本发明涉及生物制药领域， 尤其涉及一种用于人幽门螺杆菌感染免 疫预防的重组蛋白和由其制得的可降解的緩释微球包裹口服制剂疫苗， 还涉及其制备方法。 背景技术
2005 年度诺贝尔医学 /生理学奖授予了两位发现幽门螺杆菌的科学 家， 因为幽门螺杆菌 (Helicobacter pylori, Hp)是人类上消化道疾病的重要 致病菌， 为慢性胃炎、 胃溃疡及十二指肠溃疡的主要病因。 世界卫生组 织 (WHO)确认其是与胃癌密切相关的病原菌， 同时将其列为第一级致癌 因子。 Hp是世界上感染率最高的细菌之一， 世界人口的平均感染率达 50%, 发展中国家的感染率更高， 中国 Hp感染者达 6亿， 每年因为胃癌 死亡者达 20万人。 人类健康正面临着 Hp的严重威胁与危害。 目前抗生 素治疗 Hp感染虽具有积极的临床意义，但仍存在明显不足： 1)毒副作用； 2)耐药菌株产生； 3)费用高； 4)疗程长， 患者依顺性差； 5)疗效欠稳定等。 疫苗是控制感染性疾病最为经济有效的办法， 通过疫苗刺激机体产生不 同于自然感染导致的特异性免疫应答，可以达到预防或者治疗 Hp感染的 目的。 由于 Hp大规模培养困难， 粗制抗原中有潜在致癌物等有害成分， 大大制约了全菌疫苗的研究进展。 基因工程疫苗具有安全、 有效、 价格 低廉等特点， 易于推广和应用， 是 Hp疫苗的一个主攻方向。 国内外竟相 研究 Hp疫苗， 但迄今尚未成功。
尿素酶是一种催化尿素水解成氨和碳酸的镍依赖性酶， 为幽门螺杆 菌中表达最丰富的蛋白质， 在 Hp细胞内和细胞膜上都有分布， 占 Hp总 蛋白的 5%-10%。 尿素酶通过分解尿素产生氨进而中和胃酸， 帮助细菌在 胃内定居并为细菌蛋白质合成提供氨， 宿主组织还可以直接受到氨的损 伤， 或间接受到尿素酶诱导的炎性反应的刺激作用的损伤。 阻断尿素酶 基因的表达将抑制幽门螺杆菌在宿主内的定居， 减少细菌蛋白质合成， 并降低与幽门螺杆菌相关的炎性反应。在 Hp感染患者中， 尤其是有 状: 的患者中都能够检测到尿素酶 B的抗体， 抗体水平与病情的严重程度有 一定的相关性。 口服接种幽门螺杆菌尿素酶或重组尿素酶 B 亚单位 (rUreB)可保护小鼠免受幽门螺杆菌的感染并可消除已存在的感染 (Michetti et al.， Gastroenterol.1994) 0 尿素酶活性在 Hp感染中具有重要作 用，缺失尿素酶活性的 Hp在动物模型中不能导致感染， 中和尿素酶活性 的抗体可能在抵抗 Hp定植中起关键作用。 从以上的研究结果可以认为， 尿素酶抗体、尤其是能够中和尿素酶活性的抗体， 在抵抗 Hp感染中发挥 着主要作用。
粘膜免疫系统是机体免疫系统的重要组成部分，主要包括肠粘膜相关 淋巴组织和支气管粘膜相关淋巴组织等， 它在机体防御功能方面发挥着 独特的作用。 由肠系膜淋巴结， 以及分散在粘膜固有层和肠上皮中的大 量淋巴细胞组成免疫诱导部位和免疫效应部位。 胃肠道等粘膜表面各种 免疫细胞通过摄取、 加工和提呈抗原， 产生并分泌针对该抗原的抗体 (主 要为 slgA), 后者能与携带此抗原的载体如细菌、 病毒等发生特异性结合 反应， 阻止其定植粘膜表面或侵袭机体， 从而起到一定的免疫防御作用。
但是， 粘膜免疫的最大缺陷是易对抗原产生免疫耐受， 即使提高抗 原量， 机体或粘膜产生的抗原特异性 slgA水平却很低， 对相应抗原的微 生物感染几乎没有免疫防御能力或能力极弱， 极易导致免疫耐受性的发 生。
不耐热肠毒素 （heat labile toxin, LT ) 是由肠产毒性大肠杆菌 (Enterotoxigenic Escherichiacoli, ETEC)产生、 能引起人和某些家畜严重 腹泻的一种不耐热肠毒素。 LT由 1个 A亚单位（ LTA )和 5个 B亚单 (LTB) 位组成。 5个完全相同的 LTB在空间上形成环状五聚体， 1个 LTA位于 环状五聚体中央， 其 C-端以非共价键与 LTB结合。 A亚单位由 A1和 A2 两个亚单位组成， A1是毒素的毒性部分， A2与 B亚单位结合在一起， A1与 A2间有二硫键连结。 胞浆中的 A、 B亚单位均以携带信号肽的前 体形式存在， 当穿越细胞膜后才组装成完整的 LT。 B亚单位的作用是与 真核细胞表面的 GM1-神经节苷脂受体特异性结合， 使 LT分子变构， A 亚单位脱离 B亚单位进入细胞膜， 随后二硫键降解， A1 肽链活化， A1 亚单位具有 GTP依赖的 ADP-核糖基化转移酶活性， 通过 G蛋白介导的 ADP-核糖基化反应破坏胞内 cAMP的降解与平衡，刺激 cAMP水平增高， 从而引发毒素效应。
近年来 LT被认为是非常有前景的粘膜免疫佐剂。 Rollwagen等研究 表明， LT可以加强弯曲菌的粘膜免疫应答， 并加速肠道对该菌的清除， 而且 LT 与抗原一起初次免疫动物后， 将长期不会对该抗原产生免疫耐 受。
LT作为粘膜免疫佐剂的观点已获公认 (Lycke N, et al. Immunology, 1986, 59(2):301-308;Clements JD, et al. Vaccine, 1988, 6(3):269-277; Giuliani MM, et al. J. Exp. Med., 1998, 187(7):1123-1132 ),但 LT具有艮强 的肠毒性，所以目前主要以其 B亚单位或构建 LT无毒和低毒突变体作佐 剂 （ Yamamoto M, et al. J. Immunol, 1999， 162:7015-7021; Martin M， et al. Immunol" 2002, 169(4): 1744- 1752; Tamura SI, et al. Vaccine, 1994,12:1238-1240 但是， LT的 A亚单位除了 ADP核糖基酶活性外， 还与佐剂活性相有关 （ Giuliani MM, et al. J. Exp. Med., 1998 )。 LTA链刺 激粘膜相关 B细胞的同型转换，同时 LTA参与粘膜免疫系统的初始阶段， 使定型的 IgA B 细胞从产生部位迁移到远端效应位点 (DeHann L, et al. Vaccine, 1996;(4):260-266)o De Haan等发现， 缺乏 ADP-核糖基酶活性的 LT突变体仍存在辅佐性，这些结果表示 LT的辅佐性不依赖于 ADP-核糖 基活性亚基 A1， 而 A2亚单位对 LT的佐剂活性具有贡献。 De Haan实验 还表明： 没有毒性活性的 LT突变体通过鼻腔粘膜免疫小鼠， 仍然保留野 生型毒素的免疫特性， 但单独使用重组 LTB比无毒性活性的 LT突变体 的免疫原性弱 ( De Haan L, et al. Infect Immun, 1996 )， 说明 LTA的 ADP 核糖基酶活性与毒素的免疫性没有直接联系。 此外， LT全毒素产生高水 平的全身的 IgG和粘膜 S-IgA反应， 而等量 LTB仅产生低水平的 IgG反 应， LT比 LTB的免疫原性强， 说明 LTA在毒素的免疫反应中起重要作 用。而 A1亚单位具有 GTP依赖的 ADP-核糖基化转移酶活性与毒素效应 有关， 提示 LTA的佐剂效应主要是 LTA2 亚单位发挥的。 目前 LTB已经被作为佐剂使用。 例如将 LTB 与 UreB融合形成重组 蛋白 LTB-UreB (吴超， 邹全明等. 幽门螺杆菌 UreB与大肠杆菌 LTB基 因融合及表达的研究.中华微生物学和免疫学杂志， 2002,22(2):175-179 )， 并可制备成分子内佐剂疫苗。 但是这种重组蛋白的免疫保护率还不是十 分理想。
综上所述，本领域特别需要提供一种对幽门螺杆菌感染具有更强、更 完全的保护效果， 并使用方便的重组 Hp亚单位分子内佐剂疫苗。 发明内容
本发明的一个目的是， 提供一种用于人幽门螺杆菌感染免疫预防和 治疗的重组蛋白， 由该重组蛋白可制得用于人幽门螺杆菌感染免疫预防 的重组幽门螺杆菌疫苗。
本发明的另一个目的是， 提供一种制备所述用于人幽门螺杆菌感染 免疫预防和治疗的的重组蛋白的方法。
本发明的再一个目的是， 提供一种用于人幽门螺杆菌感染免疫预防 和治疗的的重组幽门螺杆菌疫苗， 它包含上述的用于人幽门螺杆菌感染 免疫预防和治疗的的重组蛋白。
本发明的另一个目的是,提供制备上述用于人幽门螺杆菌感染免疫预 防和治疗的的重组幽门螺杆菌疫苗的方法。
为达到上述目的， 本发明提供了一种用于人幽门螺杆菌感染预防和 治疗的重组蛋白， 所述重组蛋白由肠产毒性大肠杆菌 LT的 A2亚单位与 B亚单位和幽门螺杆菌尿素酶 B亚单位融合而成（即 LTA2B-UreB，简称 为 LU )。 在所述的重组蛋白中， LTA2B作为分子内粘膜免疫佐剂， UreB 作为免疫原。 LTA2B缺少 LT分子中具有 ADP核糖基酶活性的 A1部分， 克服 LT佐剂的毒副作用， 同时在当前常用佐剂 LTB的基础上， LTA2B 首次增加 LT 的 A2部分， 可以提高佐剂活性和免疫原性。
在本发明一个实施例中， 证实了该 LTA2B-UreB 融合蛋白具有全长 UreB相似的生物学活性和免疫原性及反应原性， 同时具有增强粘膜佐剂 活性的功能。 本发明还提供了用于人幽门螺杆菌感染免疫预防和治疗的緩释微球 包裹口服制剂， 所述制剂包裹有上述的用于人幽门螺杆菌感染预防和治 疗的重组蛋白。
本发明选用了可降解的緩释微球（MS ) 包裹上述重组融合蛋白。 首 先， 緩释微球可以有效防止胃酸以及消化道酶类物质对抗原的分解及破 坏作用， 保持抗原物质的整体稳定性及抗原活性； 其次， MS粒径大小决 定了其在体内各器官的分布， 人为把握 MS制作工艺， 将其粒径控制在 一定范围之内并定向诱导其被靶器官俘获， 从而最大限度的发挥其免疫 效力； 包裹抗原时， 通过改变载体材料与抗原的配成比例、 调节载体材 料的生物降解作用， 从而改变 MS的制作粒径以及抗原物质和 MS的粘 附作用， 达到长期緩释抗原的功效。
在一个优选实施方案中， 所述包裹物包括海藻酸钠、 植物油、 CaCl2 和壳聚糖。在一个优选实施方案中，所述 t球包裹制剂的粒径为 3.33μπι。
在一个优选实施方案中， 上述緩释微球包裹口服制剂最后开发为口 服免疫冻干制剂， 其冻干制品的赋形剂为 8%甘露醇， 稳定剂为 0.05% EDTA-Na2， 最适 pH值为 10.0, 因为口服具有如下优点： 一是疫苗抗原 成分直接刺激胃肠道粘膜相关的淋巴细胞， 通过对肠道抗原的识别、 呈 递和免疫效应，产生相关的粘膜免疫应答，达到有效预防幽门螺杆菌感染 所致相关疾病的目的； 二是口服免疫属于无痛、 无创疗法， 便利、 成本 较低、 易于被受试者接受。
本发明还提供了编码上述重组蛋白的核苷酸序列， 上述核苷酸序列 为由肠产毒性大肠杆菌不耐热肠毒素的 A2亚单位的编码基因、 B亚单位 编码基因和幽门螺杆菌尿素酶 B亚单位编码基因融合形成的核苷 ^^列 ltA2B-ureB (简称为 lu )。
本发明还提供了重组质粒， 所述质粒由权利要求 8所述核苷酸序列 和质粒 pET-llc连接而成。
本发明还提供了一种制备权利要求 1 所述的重组蛋白的方法， 其包 括以下步骤：
( 1 )分别克隆幽门螺杆菌尿素酶 B亚单位 UreB的和肠产毒性大肠 杆菌不耐热肠毒素 LTA2B的编码核苷酸序列，或克隆与其 95 %以上同源 且具有其蛋白活性的氨基酸序列的编码核苷酸序列；
( 2 )采用重叠 PCR的方法， 将步骤（1 ) 中克隆得到的核苷酸序列 连接成融合基因 ltA2B-ureB;
( 3 ) 将 ltA2B-ureB 融合基因构建到载体上， 转化宿主， 表达重组 蛋白 LTA2B-UreB;
( 4 )分离纯化由步骤（3 )得到的重组蛋白。
在一个优选的实施方案中， 将 ltA2B-ureB 融合基因构建到载体 pET-llc 上， 转化大肠杆菌 BL21(DE3) , 构建重组工程菌 pET-llc-LU/BL21(DE3)0 所得到的重组质粒中至少包含了上述 LU氨基 酸序列的或与其 95 %以上同源且具有其蛋白活性的氨基酸序列的编码核 苷^ 列；
在一个优选的实施方案中， 从上述重组工程菌用 80L发酵罐发酵表 达重组蛋白 LTA2B-UreB。
本发明还提供了一种制备口服重组幽门螺杆菌疫苗的方法， 其包括 以下步骤：
( 1 )将权利要求 9所得的重组蛋白与海藻酸钠、 植物油、 CaCl2和 壳聚糖配制， 制成可降解的緩释微球包裹制剂； 以及
( 2 )任选地将緩释微球包裹制剂制成冻干品。
在一个优选的实施方案中， 将所得重组蛋白与海藻酸钠溶液混匀， 加入植物油， 乳化后， 反向滴定方法滴入到 CaCl2溶液中， 制成海藻酸钠 包裹蛋白微球剂； 将所得微球固化后洗涤， 离心取沉淀物后重悬； 再将 其加入到壳聚糖溶液中， 形成再包裹， 得到壳聚糖海藻酸钠双重包裹蛋 白微球，再将微球混悬液緩慢倒入西林瓶中， 液面高度低于 lcm。 - 40°C 冰箱预冻 12小时后直接置于已预冷的真空干燥机内干燥，并緩慢提升温 度使水分迅速升华， 待气体压力指示计显示无气体生成时取出冻干品待 检。
在本发明一实施例中， 将所得疫苗进行动物实验， 以检测该重组幽 门螺杆菌疫苗的安全性和免疫原性。 本发明另一实施例中， 对该疫苗进行人体临床实验， 以验证重组幽 门螺杆菌疫苗的免疫效果。
综上所述， 本发明以肠产毒性大肠杆菌 LT的 A2亚单位与 B亚单位 融合成的 LTA2B作为粘膜免疫分子内佐剂、以尿素酶 B亚单位为免疫原 组成重组幽门螺杆菌疫苗， 用于人幽门螺杆菌感染免疫预防， -使用方便， 安全有效。
为让本发明的上述目的、 特征和优点能更明显易懂， 下文特举以下 实施例， 并配合附图， 作详细说明如下。 附图说明
图 1 为重叠延伸方法得到的 lu ( ltA2B-ureB ) 融合基因的琼脂糖凝 胶电泳图，其中泳道 1为 ureB基因的 PCR扩增产物； 泳道 2为核酸 (DNA) 分子量标准（ Marker ); 泳道 3为 ltA2B融合基因的 PCR扩增产物。
图 2表示 pET-llc-lu重组质粒的酶切鉴定， 其中泳道 Ml.是 PCR Marker; 泳道 1. 是 It A2B PCR产物（约 0.4kb ); 泳道 2. 是 ureB PCR产 物 (1.7kb); 泳道 3. 是 lu PCR产物 (约 2.1kb); 泳道 4. 是 pET-llc Ndel酶 切 (5.7kb); 泳道 5. 是 pET-llc-lu Ndel酶切 (5.7kb+2.0kb); 泳道 6. 是 pET-llc-lu 正向重组子 BamHI 酶切 (6.0kb+1.0kb+0.7kb); 泳道 7. 是 pET-llc-lu反向重组子 BamHI 酶切 (6.4kb+1.0kb+0.3kb); 泳道 M2. 是 DNA/Hindlll Marker.,
图 3 是融合基因 lu重组菌表达 PAGE电泳图，其中泳道 1: 蛋白质 分子量标准（ Marker ); 泳道 2: 空质粒菌诱导 1小时； 泳道 3:基因重组 菌诱导 5小时； 泳道 4: 基因重组菌诱导 4小时； 泳道 5: 基因重组菌诱 导 3小时； 泳道 6: 基因重组菌诱导 2小时； 泳道 7: 基因重组菌诱导 1 小时。 从中可见， 基因重组菌经过诱导后在分子量 72KDa处有增加的蛋 白表达条带， 与目的蛋白分子量一致。 经过 UVP图像扫描分析， 诱导 5 小时目的蛋白表达量约 26%。
图 4 为目的蛋白 LU纯化效果 PAGE电泳图， 其中， 泳道 1: 包涵体 溶解液（纯化前样品）； 泳道 2,3: 杂质蛋白洗脱峰样品； 泳道 4: 目的蛋 白洗脱峰样品； 泳道 5: 蛋白质分子量标准（ Marker )。 结果显示经过纯 化步骤， 目的蛋白的纯度得到明显改善， 收获的目的蛋白峰经 UVP扫描 分析纯度大于 85 %。
图 5 为 rHp (重组幽门螺杆菌疫苗)疫苗冻干曲线。 具体实施方式
实施例 1 融合基因 lu(ltA2B-ureB)的构建
一、 幽门螺杆菌的 UreB和产毒;^杆菌 LTA2B编码基因的克隆
分别以野生型肠产毒大肠杆菌 H44815 (购自中国药品生物制品检定 所）的基因组 DNA,及幽门螺杆菌 NCTC11637(购自美 Ί¾典型培养物保藏 中心 ATCC)的基因组 DNA为模板， 用 P1和 Ρ2、 Ρ3和 Ρ4分别扩增 ureB 和 ltA2B基因， 细菌基因组抽提按常规方法（颜子颖， 王海林译.精编分 子生物学实验指南. 科学出版社， 1998, P39 )进行。 PCR扩增体系为： 10χ不含镁离子扩增緩冲液 10nL， MgCl2(25mmol/L) ΙΟμί , dNTPs (2.5mmol/L each) 8 L， 上下游引物（ PI和 P2或 P3和 P4 )各 2μ 上述 细菌基因组 2 L， Ex-Taq DNA聚合酶（ 3单位 / ) Ιμ 加灭菌水至 ΙΟΟμΙ^
PCR扩增反应： 94°C预变性 5分钟， 94°C变性 50秒， 60 °C退火 50 秒， 72°C延伸 50秒， 35个循环， 72°C完全延伸 10分钟。 琼脂糖凝胶电 泳后， 回收目的片段。 （下划线的部分为相应的酶的识别序列）
PI: cat atg get cct cag tot att aca gaa cta tgt tc
Ndel
P2: tga tat egg ate ctg agg gta gttttc cat act gat tgc c
BamHI
P3： tac cct cag gat ccg ata tea atg aaa aag att age ag
BamHI
P4: cat atg cta gaa aat get aaa gag ttg tgc caa gc
Ndel
按常规方法（ J.Sambrook, 分子克隆， 冷泉港实验室出版社 1989 聚 丙烯酰胺凝胶电泳 1.21-1.32 )提取 TA克隆转化菌株的质粒 DNA, 采用 双脱氧末端终止法， 对插入片段进行序列测定。
二、 融合基因 ltA2B-ureB的构建
分别回收 ureB和 ltA2B的 PCR产物。 以回收的 ureB和 ltA2B基因 为模板， P1和 P4为引物进行重叠延伸 PCR反应。 PCR扩增体系为： 10χ 不含镁离子扩增緩沖液 5 L， MgCl2(25mmol/L) 4μ dNTPs(25mmol/L ) 4μί, 上下游引物 （PI和 P4 )各 lnL, 上述 ureB和 ltA2B基因各 2μ Ex-Taq DNA聚合酶（5单位 1 ) O^ , 加灭菌水至 50μ 。
重叠延伸 PCR扩增反应： 94°C预变性 5分钟， 94°C变性 60秒， 60 °C 退火 60秒， 72°C延伸 60秒， 35个循环， 72°C完全延伸 10分钟。 琼脂糖 凝胶电泳后， 回收目的片段。
PCR产物的克隆及序列分析同前。
重叠延伸 PCR反应体系
回收的 ureB DNA片段 Ι μΐ
回收的 ltA2B DNA片段 Ιμΐ
P1 (5ρπιο1/μ1) 2μ1
Figure imgf000011_0001
lOxPCR緩冲液 ΙΟμΙ dNTPs(5mmol/L) 4μ1
Taq plus DNA聚合酶 (3υ/μ1) Ι μΐ
ddH20 79μ1 总体积 ΙΟΟμΙ 将反应体系振荡混匀， 离心处理后， 加入 20μ1石蜡油。 94°C预变性 10分钟， 94°C变性 30秒， 58°C退火 45秒， 72°C延伸 1分钟， 35个循环， 72°C完全延伸 10分钟。反应完毕后取 3μ1反应产物， 1%琼脂糖凝胶电泳 检测 PCR扩增产物并回收目的基因片段 ltA2B-ureB。 采用 PCR重叠延 伸技术得到 ltA2B-ureB融合基因片段。 1%琼脂糖凝胶电泳分析 (见图 1)， 图中所示片段大小与预计相符 (约 2.1kb), 初步判定为目的基因片段， 并 命名为 ltA2B-ureB, 如 SEQ ID NO: 1所示。 图 2中显示： 融合基因片段 大小与预计一致， 表明重叠延伸得到融合基因。
实施例 2 重组工程菌 pET-llc-LU/BL21(DE3)的构建
一、 重组工程菌 pET-llc-LU/BL21(DE3)的构建
将 lu(ltA2B-ureB )融合基因扩增 （PCR )产物经 1.0%琼脂糖电泳、 胶回收纯化后与载体 pMD-18T (购自 TaKaRa公司）连接， 转化大肠杆 菌 DH5a, 提取盾粒， 用 Ndel酶切， 1.0%琼脂糖凝胶电泳鉴定。
从 pMD-18-lu/DH5a阳性重组菌中抽 ^粒 DNA， Ndel酶切， 回收 2.0kb lu DNA片段，与 Ndel酶切且经过去磷酸化处理的 pET-llc载体（购 自美国 Novagen公司）连接， 转化大肠杆菌 DH5a, 氨苄阳性 LB平板筛 选， 挑取可疑菌落抽提质粒， Ndel酶切鉴定阳性重组子， BamHI酶切鉴 定正反向。 酶切鉴定结果如图 2所示。 阳性重组子质粒 DNA经 Ndel 酶切后均产生 5.7kb载体片段和 2.1kb lu基因片段（第 5泳道）， BamHI 酶切后反向重组子产生 6.4kb+1.0kb+0.3kb三个片段（第 7泳道）， 正向 重组子产生 6.0kb+1.0kb+0.7kb三个片段（第 6泳道）。
有关操作具体步骤如下：
1 )使用 Omega公司提供的质粒抽提试剂盒， 按照试剂盒使用说明 书推荐的方法提取质粒 DNA。
2 ) 以常规琼脂糖凝胶电泳法： （1.0%琼脂糖凝胶， ΙχΤΑΕ緩冲液， 120-150mA, 电泳 20-40分钟。 50χΤΑΕ储存液配方： 2.0mol/L Tris碱， LOmol/LNaAc, 0.1mol/LNa2EDTA;用冰醋酸调节 pH8.3 )分离质粒 DNA。
3 )质粒 DNA的酶切鉴定： 反应混合物包括： l g质粒 DNA; Ιμΐ 10χ緩沖液（见上海生工公司产品说明书）； Ιμΐ 限制性内切酶 Nde I
( 10υ/μ1 ); 用双蒸水补齐至 10μ1。 混合后 37°C温育 1-2小时。
4 )琼脂糖电泳凝胶中的目的 DNA的回收纯化：
在紫外灯下观察并切下琼脂糖凝胶上的目的 DNA电泳带，移入 1.5ml EP管。
加入 Omega公司胶回收试剂盒的 DNA结合緩冲液， 65 V水浴，使凝 胶完全溶化并保持溶液 pH在 5.0~6.0之间。将溶胶液移入分离管， 12000g 离心 1分钟， 弃去收集管中的液体。 加入配套的洗涂緩沖液， 12000g离心 1分钟， 弃去收集管中的液体。 重复洗涤 1次。
12000g离心 1分钟， 分离管移置另一干净 1.5mlEP管， 加入一定体 积的 TE緩冲液， 65°C孵育 10分钟， 12000g离心 1分钟， 取一定量电 泳， UVP紫外扫描仪检测回收纯化效果。
5 )连接反应 (使用上海生工公司连接试剂盒）
通过紫外分光光度计检测目的 DNA片段和载体片段的浓度，根据外 源片段与载体摩尔数比一般为 1:2 ~ 10的原则， 设计连接反应体系如下： 目的 DNA Ιμΐ;盾粒载体 1 ~ 2μ1;连接溶液 5μ1; ddH20 2 ~ 3μ1; 总体积 10μ1。 22°C连接反应 12-16小时。
6 )感受态菌的制备 (CaCl2法）
无菌接种环蘸取 -70°C冻存的细菌保种液， 三线法划线接种于 LB平 板， 37°C培养 12 ~ 16小时。挑取单个菌落接种于 2ml LB培养液中， 37°C 摇床培养 12 ~ 16小时。将过夜培养的 DH5a按 1%比例转种至 LB培养液 中， 37°C摇床培养至 OD6。。为 0.2 ~ 0.4小时， 8000g离心 5分钟收集细菌。 加入 lml预冷的 0.1M CaCl2重悬沉淀， 冰水浴 3小时。 4°C 8000g离心 5 分钟， 弃上清。 加入 ΙΟΟμΙ预冷的 0.1M CaCl2悬浮沉淀， 冰水浴 1小时， 备用。
7 )用连接产物转化大肠杆菌宿主细胞
取感受态菌液 100μ1， 加入连接反应产物； 冰水浴 60分钟， 42°C水 浴热休克 100秒， 迅速放置冰水浴 1 ~ 2分钟。 加 lOO lLB培养液， 37°C 摇床培养 1小时。 以 8000g离心 10分钟， 吸弃 ΙΟΟμΙ上清后混匀沉淀， 各取 50μ1涂布平板， 37°C孵箱培养过夜。
二、 高^^达融^ ^白工程菌的构建及筛选
将含 LU融合基因的重组表达质粒转化大肠杆菌 BL21并提取质粒， 酶切鉴定。 图 2为 pET-llc-LU/BL21(DE3)重组表达质粒的酶切鉴定琼脂 糖凝胶电泳图，其中泳道 5为 pET-llc-LU/BL21(DE3)重组质粒 Ndel酶切 产物； 泳道 Ml、 M2为核酸 (DNA)分子量标准（Marker ); 泳道 4为空 载体质粒 M e/单酶切产物 (5700bp)。 酶切片段大小与设计一致， 初步证 明重组质粒构建成功。基因工程大肠杆菌 BL21的感受态菌制备、转化及 重组菌的质粒抽提、 酶切鉴定同前。
取鉴定无误的重组菌接种于 3ml含卡那霉素的 LB培养液中， 37°C摇 床培养过夜。 次日将过夜培养的重组工程菌按 1%的比例转种于 20mL含 卡那霉素的 LB培养液中， 37°C摇床培养 2.5小时， 以 IPTG诱导 5小时， SDS-PAGE检测融合蛋白的表达形式和表达量，筛选高效表达菌株，图 3。
本发明还通过实验验证了 UreB和 LTA2B 的稳定性及能否代替尿素 酶 B全长蛋白。 采用 PCR方法扩增目的 LTA2B和 UreB和 LTA2B片段 基因， 并构建到原核表达载体 pET-llc(+)上， 转化宿主菌大肠杆菌 BL21(DE3)中， IPTG诱导稳定表达 LTA2B - UreB融合蛋白。首先， ELISA 及免疫印迹分析，证实重组 LTA2B - UreB具有良好的免疫原性和反应原 性； 其次， 纯化后的 LTA2B - UreB免疫家兔， 产生的抗体在体外能够中 和尿素酶活性， 表现出与天然 HP的 UreB相似的生物学活性； 再者， 重 组 LTA2B - UreB 口服免疫 BB/c小鼠， 能有效激发机体产生 Thl、 Th2 平衡的免疫应答反应， 与重组 UreB (吴超， 邹全明等. 幽门螺杆菌 UreB 与大肠杆菌 LTB基因融合及表达的研究， 中华微生物学和免疫学杂志, 2002，22(2):175-179 )—致； 最后， 小鼠攻毒保护试验表明， LTA2B - UreB 的保护率为 91.6 % , 明显高于全长 UreB 68 %的保护率。 GM1-ELISA试 验表明， 融合 LTA2B - UreB无 ADP核糖基酶活性， 但保留了与神经节 苷脂 GM1相结合的生物学特性， 小鼠攻毒保护试验表明， LTA2B - UreB 的保护率明显高于 UreB-LTB 74.1 %和 UreB与 LTB物理混合组合 78.6 % 的保护率。 首次证实了 LTA2B - UreB具有全长 UreB相似的生物学活性 和免疫原性及反应原性， 同时具有增强粘膜佐剂活性的功能。
实施例 3 重组工程菌 pET-llc-LU/BL21(DE3)的发酵
发酵工艺如下：采用德国 B.Bron 80L发酵罐，发酵过程中种子菌 10% 比例接种； 温度 37°C ; pH7.0， 通过自动加入 30%氨水使 pH值保持恒 定； 转数起始设定为 500rpm, 随着菌体密度的增大、 耗氧的升高， 转数改为级联溶氧控制， 即以 P02控制器为主控制器， 搅拌控制器 为伺服控制器； 溶解氧浓度采用级联控制及负氧旁路控制， 使其终 浓度控制在 45%-50%; 在 A600未达到 2时不加补料， 之后每 0.5小时 流加补料一次， 使葡萄糖、 胰化蛋白胨和 8%酵母抽提物的终浓度分别为 0.5%、 0.2% 和 0.2%。 在第 4次补料后， 待葡萄糖浓度降为 0.1%时加入 IPTG 500μπιο1^诱导 4小时收菌； 发酵过程在级联溶氧控制的分批培养 基础上， 流加补料。
发酵过程所用培养基为改良 M9-CAA培养基，在 M9-CAA的基础上 添加 0.6 %酵母浸出液和 2mg/L ZnCl2.4H20、 2mg/LCoCl2.4H20、 4mg/L FeS04 16H20、 5mg/LH3B03、 1.6mg/LMnCl2'4H20、 4mg/L CuS04而成。
发酵结束后回收菌液， 4°C离心 （8000g ) 15分钟。 吸弃上清， 收集 细菌， 称重后冻存备用。
结果： 结果显示菌体产量均在 75g/L 以上。 目的蛋白表达量均稳定 在 30%左右。 证明此中试发酵工艺先进， 工艺重复性及稳定性均达了较 高水平。
实施例 4 重组蛋白 LU ( LTA2B-UreB ) 的纯化
1. 包涵体提取：将高效表达的菌体 5000g以 TE緩冲液 1: 10 ( W/V ) 比例悬浮， 4°C预冷后， 采用细胞匀浆机使其混合均匀。 采用高压均质机 在压力为 40 - 70Mpa的条件下进行破菌（共破菌 4 ~ 6次），破菌完毕后， 取少量菌液涂片染色， 显微镜下观察细胞的完整性， 确保细胞破碎完全， 随后以 500g离心 25分钟， 弃沉淀， 再以 15,000g离心 40分钟， 弃上清 收集沉淀。 以 1: 10 ( W/V ) 的比例分别用洗涤液 A和 B各洗涤 2次。 洗涤条件为： 4°C搅拌 20分钟， 15，000g离心 40分钟， 收集包涵体沉淀； 最后将包涵体用包涵体溶解液以 1: 10 ( W/V ) 的比例混合， 4°C搅拌 3 小时， 15,000g离心 45分钟， 取上清作为下一步纯化的原料。
包涵体提取所用緩冲液： 1) TE緩冲液： 20 mmol/L Tris, 5 mmol/L EDTA, pH 8.0; 2) 包涵体洗涤液 A: 5 mmol/L EDTA、 20 mmol/L Tris, 1 % Triton X-100 ， pH 8.0; 3) 包涵体洗涤液 B: 20 mmol/L Tris、 2mol/L Urea, pH 8.0; 4) 包涵体溶解液： 1 mmol/L EDTA, 20 mmol/L Tris.8 mol/L 尿素 (pH 8.0)。
2. 层析纯化： 该步纯化确定为 Q Sepharose HP 阴离子交换柱及 Sephadex G-25 柱层析纯化， 中试纯化采用 XK50/30 柱， 在 AKTA explorer100系统上进行纯化。 由于 AKTA explorer100系统具有精确的自动 化操作特点， 通过编制程序， 采用 2套 AKTA explorer1()()进行连续自动化 层析操作， 每批 rHp疫苗的中试产量可达 40 g。 使用 20 mmol/L Tris, 5 mmol/L EDTA, pH 8.0对目的蛋白进行纯化，采用 NaCl梯度洗脱。
3. 经过 Q Sepharose High Performance层析纯化的重组蛋白纯度达到 80%以上， 但有大量的盐和尿素存在， 居分子量的差异， 故选用分子筛 层析方法来脱盐和去除尿素， 使用传统的分子筛填料 Sephadex G-25 medium
4. 纯化后的目的蛋白进行 SDS-PAGE,检定其纯度最终获得的目的 蛋白纯度>80%， 收率 >79%。
5. Lowry法检测蛋白浓度。
其中， 步骤 1 所述采用生产或中试纯化中使用的高压破菌技术， 破 菌至细菌破解率大于 98 % , 差速离心获得包涵体沉淀物。
步骤 2所述亲和层析纯化填料选自 Chelating Sepharose Fast Flow。 步骤 3所述阴离子纯化填料选自 Q Sepharose HP、 Q Sepharose FF和 Q Sepharose XL。
实施例 5 口服重组 HP疫苗的制备
一、 重组 LU蛋白^的制备
在室温下， 将实施例 4 中制备的重组蛋白与 2 %的海藻酸钠溶液混 匀， 加入植物油， 植物油与海藻酸钠 AGS乳液的比例为 2: 8。 8000 r/ 分钟乳化 10分钟后， 逐滴滴入到 CaCl2溶液中， 800 r/分钟搅拌 30分 钟后， 形成 OAV乳液， 离心取沉淀物， 洗涤后重悬。 将悬液加入到 1 % 浓度的壳聚糖溶液中， 800rpm χ30分钟搅拌完成， 再包裹制备出壳聚糖 -海藻酸钠包裹重组 LU蛋白微球， 离心洗涤 3次后收集 。
将上述微球混悬液緩慢倒入玻璃平皿中，谈面高度低于 lcm。 - 40°C 冰箱预冻 12小时后， 直接置于已预冷的真空干燥机内干燥， 并緩慢提升 温度使水分迅速升华， 待气体压力指示计显示无气体生成时， 取出冻干 σ口待检。 分别取洗涤离心后 MS、 冻干粉以及冻干粉复溶物微量涂于载玻片 上， 光镜下观察其冻干前后微球形态变化， 离心洗涤后的微球大小形态 规则； 冻干后则呈不规则分布， 形态呈现出杆状、 梭状以及扁圆状等诸 多不规则形状； 而用等量蒸馏水复溶后镜下可见微球数量以及形态基本 恢复冻干前原有特征。 所制备 MS 表面饱满、 大小均一， 平均粒径为 3.33μιη。
二、 重组 LU蛋白微球溶液的冻干
经高度纯化的重组 LU蛋白，经适当稀释配制后，再分装入冻干瓶进 行冻干。 通过研究获得了稳定成熟的 rHp疫苗冻干曲线： 制品预冻温度 为 -45°C， 时间 4小时； 第一次升华温度为 20°C , 时间 30小时； 第二次 升华温度为 30°C， 时间 8小时， 整个冻干过程共耗时 42小时。 水分含量 均<3%， 符合《中华人民共和国药典》（2000年版)二部对冻干制品水分含 量的检测要求。
通过较系统的冻干中试工艺研究， 确定了 rHp疫苗冻干制品的赋形 剂为 8%甘露醇， 稳定剂为 0.05% EDTA-Na2, 最适 pH值为 10.0， 并获得 了成熟的冻干曲线等参数。 经 20余批次的反复验证表明， 该中试冻干工 艺性能稳定， 能满足规模化生产的要求。 以每支冻干瓶装量 15 mg计算， 40 g疫苗蛋白可装 2 600支左右。 所采用的 5 m2冻干机一次可冻干制备 32 mm直径的冻干瓶 5 000支左右， 能满足大批量制品冻干的需要。
本发明还研究了人工胃液对 rHp疫苗稳定性的影响。 rHp疫苗为口服 免疫冻干制剂， 在通过消化道到达靶器官的过程中， 必须经过胃内酸性 pH环境。由于 rHp疫苗本身为蛋白质物质，易受酸作用而变性或酶降解， 从而降低其免疫活性。 研究发现， rHp疫苗蛋白在 pH值为 1和 2的人工 胃液中其抗原反应活性降低明显， 蛋白降解显著， 但在 pH 3~7的环境中 能够保持较高的抗原反应性及蛋白稳定性。 该研究结果提示， 在疫苗使 用过程中，可以采取对胃酸进行中和的方法，适当提高胃内环境的 pH值， 以保持 rHp疫苗蛋白的生物活性， 从而获得良好的免疫效果。 在口服重 组疫苗的剂型上， 建立了先进实用的抗胃酸、 抗胃蛋白酶的多聚微嚢包 裹制剂工艺， 克服了胃酸和胃蛋白酶的破坏， 提高了疫苗的有效性和稳 定性。
实施例 6 口服重组 HP疫苗的动物实验
一、 动物安全性评价
LD5Q测定组各试验小鼠均未见异常反应， 无中毒症状， 无死亡发生， 未测出 LD50, 同时经最大耐受剂量试^ r测出 rHp疫苗口服的最大耐受剂 量（MTD ) 大于 150mg/kg (相当于推荐人体正常用药量的 600倍）。 在 腹腔注射三批 rHp疫苗后的观察期内，三批豚鼠均健存，未见异常毒性反应 : 至第 7天体重均有正常增加。 分别进行了以下试验， 试验结果提示 rHp 疫苗安全性良好。
二、 免^^性研究
分别采用家兔、 BALB/c小鼠和恒河猴观察了 rHp疫苗的免疫原性。
1. rHp疫苗免疫接种家兔的免疫原性研究
将 rHp疫苗免疫接种家兔后， 经双向免疫扩散试验和 ELISA检测， 结果该疫苗可诱生家兔产生高效价的抗 UreB的抗血清，证实 rHp疫苗蛋 白具有良好的免疫原性。
2. rHp疫苗免疫接种沙鼠的免疫原性研究
各组沙鼠于末次免疫后 10天同时灌喂制备好的 Hp 菌液。 所有实验 动物提前 24小时断食、断水，每次每只灌喂菌液 0.3ml, 约 108cf /ml (每 毫升菌落形成数）； 上、 下午各一次， 间隔 6小时， 末次灌喂后 2小时供 食水。 攻毒后第 4周所有实验动物均处死并采集标本， 处死前 24小时断 食水， 解剖沙鼠， 取出鼠胃， 沿胃大弯剖开， 用生理盐水轻轻沖掉胃内 残留物， 将一半胃粘膜组织涂布 Hp培养基， 三线法划线接种， 微需氧培 养， 本观察免疫后小鼠 Hp的定植情况如表 1。
表 1. 融合蛋白口服免疫小鼠后攻毒免疫保护效果
, , 攻毒后存 未感染数 保护率
组别
活数 (只） （只） (%)
PBS组（磷酸盐緩冲
1 28 27
液 pH7.0 ) 2 UreB组 30 20 66.7
3 UreB + LTB组 29 19 65.5
4 UreB + LTA2B组 27 21 71.3
5 UreB-LTB组 30 24 72.8
6 LTA2B - UreB组 29 27 91.6
"UreB-LTB"、 "LTA2B - UreB"表示为融合蛋白， "UreB + LTB"、 "UreB + LTA2B"表示为两种重组蛋白的物理混合。
结果显示， LU免疫组（第 6组） 小鼠的保护率达到 90%以上。
结论： 融合疫苗抗原 LU免疫小鼠， 与未免疫组相比， 可产生针对 Hp全菌攻击有效的保护作用。
以融合蛋白与两个亚单位体外混合后肌肉注射免疫已经确定 Hp感 染的 BALB/c小鼠模型， 每 0, 2， 4周各免疫一次， 免疫剂量为 lOO g ( lOOuL ) /只， 与等体积铝佐剂混合。 末次免疫后 4周， 处死小鼠， 采 集样本， 以不同实验方法观察治疗后小鼠带菌情况。
表 2. 多亚单位蛋白疫苗治疗 Hp感染小鼠观察
组别 治疗后小鼠未检 治疗后小鼠 Hp定植
测到 Hp只数 数量明显减少只数
LU治疗组（30只） 21 9
两个亚单位混合组（ 30只） 23 7
对照组（ 30只） 0 1
两组结果经统计学分析， p < 0.0010 说明疫苗抗原 LU或者两个亚单 位抗原体外混合后， 对已经感染小鼠进行免疫治疗后可有效减少小鼠带 菌量或者能够清楚小鼠的感染。 多亚单位疫苗抗原包括融合抗原 LU对 Hp感染有一定治疗作用。
3. rHp疫苗口服免疫恒河猴的实验研究
研究发现， 恒河猴经 rHp疫苗口服免疫后， 血清中抗 UreB IgG抗体 和唾液中 slgA抗体水平均有明显升高， 且维持到免疫后 15周， 表明该 疫苗可诱导恒河猴产生显著的系统免疫反应和粘膜免疫反应。 为探讨疫苗剂量与免疫应答水平的关系， 采用了三个不同的剂量进 行口服免疫，结果显示 0.5、 2.0 mg/kg剂量组诱导的系统及粘膜免疫应答 水平均明显高于 0.2 mg/kg剂量组， 而两个高剂量组间未见明显差异， 提 示该疫苗口服免疫恒河猴的最佳剂量为 0.5 mg/kg体重。
通过以上家兔、 BALB/c小鼠及恒河猴动物试验， 证实 rHp疫苗可有 效诱导机体的粘膜免疫应答， 具有良好的免疫原性。
实施例 7 口服重组 HP疫苗的人体实验
为表明本发明的治疗效果， 本发明作了临床研究。 本发明临床研究 资料如下：
一、 入选标准：
1. 自愿参加。
2. 经查问病史、 体检和临床判定健康者。
3. 近期无幽门螺杆菌感染史。
4. 无同类疫苗接种史。
5. 无疫苗接种禁忌症。
二、 疗效和安全性判定
(一） 安全性判定
受试者服苗后进行 30分钟的即时观察， 并于 6、 24、 48及 72小时 详细观察全身 （体温）和局部 （胃肠道）反应及它异常反应的发生情况 (包括发热、 大便形状及次数异常、 腹泻、 呕吐等）。
1.全身 （体温）反应：
无反应： 体温在 37°C及以下；
轻度反应： 体温在 37.1 °C ~ 37.5°C ;
中度反应： 体温在 37.6°C ~ 38.5°C ;
重度反应： 体温在 38.6°C或以上；
2.局部（胃肠道）反应：
无反应： 无胃肠道反应；
轻度反应： 经一般处理， 胃肠道症状消失； 中、 重度反应： 需经多次处理或住院治疗；
(二）免疫原性研究
以全程免疫后 14天抗体状态评价免疫原性， 以全程免疫后 60天观 察抗体消长情况。 血清特异性 IgG以免疫前<1： 100, 而免疫后≥1： 100 为阳转；血清特异性总 Ig、唾液特异性 sIgA、免疫后与免疫前滴度比值≥4 倍者为转阳。
三、 研究方法
分 I 、 II期两个阶段进行。 I期临床研究为非对照研究， 30名健康 儿童，按 45mg/次的剂量的免疫程序口服重组幽门螺杆菌疫苗，服后观察 全身 （体温）和局部 （胃肠道）反应。 未见严重的异常反应时继续开展 Π期临床研究。 II期临床研究为随机、 双盲、 对照研究。 研究分为四个 组（见表 4 )。 免疫程序： 每两周口服免疫一次， 连续三次， 即 0、 14、 28天各一次。
四、 研究对象
口服重组幽门螺杆菌疫苗 1 期临床研究受试对象的基本情况和临床 反应详见表 3-5
表 3. I期临床研究受试者基本情况
年龄（岁） 男 （人数) 女（人数） 合计
10 0 1 1
11 6 6 12
12 6 7 13
13 4 0 4
合计 16 14 30 表 4. II期临床研究受试对象基本情况表
组别 受试人数 性别构成 平均年龄（岁）
男 女
安慰剂 151 77 74 10.1 15mg/次 148 69 79 10.3
30mg/次 171 82 89 10.4
45mg/次 153 74 79 10.8
合计 623 317 306 10.2
五、 结果
1、.疫苗的安全性
在 I期临床研究中， 有 30名受试对象接受了 45mg/次剂量的口服重 组幽门螺杆菌疫苗免疫，三次全程免疫中 30人均未观察到任何即时反应、 全身或局部反应、 迟发反应以及其他的异常反应、 偶合反应和任何有临 床意义的疾病和事件（表 5、 6 )。 提示 45mg/次剂量的口服重组幽门螺杆 菌疫苗对人体具有良好的安全性。 根据 I期临床研究的结果， 进行 II期 临床研究， 进一步扩大人群研究其安全性， 并着重对其免疫效果进行研 咒。
表 5. 口服重组幽门螺杆菌疫苗 I期临床研究受试者胃肠道反应情况
Figure imgf000022_0001
表 6. 口服重组幽门螺杆菌疫苗 I期临床研究受试者全身反应情况
观 无反应 轻度反应 中度反应 重度反应 服苗次 察 ( <37.0 ) (37.1-37.5) (37.6-38.5) ( >=38.6 ) 数 人
数 人数 % 人数 % 人数 % 人数 % 第一次 30 30 100 0 0.00 0 0.00 0 0.00 第二次 30 30 100 0 0.00 0 0.00 0 0.00 第三次 30 30 100 0 0.00 0 0.00 0 0.00
2、 疫苗的免疫原性
II期临床研究结果显示： 15mg/次、 30mg/次和 45mg/次剂量的口服 重组幽门螺杆菌疫苗对人体均能刺激人体产生较好的血清特异性 IgG抗 体、血清特异性总 Ig抗体、唾液特异性 slgA抗体应答，且持续时间较长， 在全程免疫后两个月仍处于较高的水平 （表 7 - 9 )。
表 7. 口服重组幽门螺杆菌疫苗 II期临床研究受试者血清特异性 IgG抗体 阳转情况
Figure imgf000023_0001
表 8 口服重组幽门螺杆菌疫苗 II期临床研究受试者血清特异性 IgG抗体 水平情况
Figure imgf000023_0002
GMT: 几何平均滴度 表 9、 口服重组幽门螺杆菌疫苗 II期临床研究受试者唾液特异性 slgA抗 体水平情况
Figure imgf000024_0001
GMT: 几何平均滴度

Claims

权 利 要 求
1.一种重组蛋白，其特征在于，所述重组蛋白为由肠产毒性大肠杆菌 不耐热肠毒素的 A2亚单位、 B亚单位和幽门螺杆菌尿素酶 B亚单位融合 形成的重组蛋白。
2. 一种緩释微球包裹口服制剂， 其特征在于， 所述制剂包裹有权利 要求 1所述的重组蛋白。
3. 根据权利要求 2所述的微球包裹口服制剂， 其特征在于， 所述包 裹物包括海藻酸钠、 植物油、 CaCl2和壳聚糖。
4. 根据权利要求 2或 3所述的微球包裹口服制剂， 其特征在于， 所 述微球包裹制剂的粒径为 3.33μηι。
5.根据权利要求 2或 3所述的微球包裹口服制剂， 其特征在于， 所 述包裹制剂为冻干制剂。
6. 根据权利要求 5所述的冻干制剂， 其中所述冻干制剂的赋形剂为 8%甘露醇， 稳定剂为 0.05% EDTA-Na2， 最适 pH值为 10.0。
7.—种编码权利要求 1所述的重组蛋白的核苷酸序列，其特征在于， 上述核苷酸序列为由肠产毒性大肠杆菌不耐热肠毒素的 A2 亚单位的编 码基因、 B亚单位编码基因和幽门螺杆菌尿素酶 B亚单位编码基因融合 形成的核苷酸序列 ltA2B-ureB。
8. —种重组质粒， 其特征在于， 所述质粒由权利要求 7所述核苷酸 序列和质粒 pET-llc连接而成。
9. 一种制备权利要求 1所述的重组蛋白的方法， 其包括以下步骤：
( 1 )分别克隆幽门螺杆菌尿素酶 B亚单位 UreB的和肠产毒性大肠 杆菌不耐热肠毒素 LTA2B的编码核苷酸序列，或克隆与其 95 %以上同源 且具有其蛋白活性的氨基酸序列的编码核苷酸序列；
( 2 )采用重叠 PCR的方法， 将步骤（1 ) 中克隆得到的核苷酸序列 连接成融合基因 ltA2B-ureB;
( 3 ) 将 ltA2B-ureB 融合基因构建到载体上， 转化宿主， 表达重组 蛋白 LTA2B-UreB; (4)分离纯化由步骤 (3)得到的重组蛋白。
10. 一种制备口服重组幽门螺杆菌疫苗的方法， 其包括以下步骤：
( 1 )将权利要求 9所得的重组蛋白与海藻酸钠、 植物油、 CaCl2和 壳聚糖配制， 制成可降解的緩释微球包裹制剂； 以及
( 2 )任选地将緩释微球包裹制剂制成冻千品。
PCT/CN2007/002655 2006-09-05 2007-09-05 口服重组幽门螺杆菌疫苗及其制备方法 WO2008040155A1 (zh)

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AT07800872T ATE510560T1 (de) 2006-09-05 2007-09-05 Oraler rekombinanter helicobacter-pylori-impstoff und sein herstellungsverfahren
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