WO2012031530A1 - 一种多靶点融合蛋白，其编码基因及应用 - Google Patents

一种多靶点融合蛋白，其编码基因及应用 Download PDF

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WO2012031530A1
WO2012031530A1 PCT/CN2011/079042 CN2011079042W WO2012031530A1 WO 2012031530 A1 WO2012031530 A1 WO 2012031530A1 CN 2011079042 W CN2011079042 W CN 2011079042W WO 2012031530 A1 WO2012031530 A1 WO 2012031530A1
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seq
fusion polypeptide
target fusion
target
amino acid
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PCT/CN2011/079042
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French (fr)
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王保宁
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四川万可泰生物技术有限责任公司
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Priority to US13/516,733 priority Critical patent/US8945585B2/en
Publication of WO2012031530A1 publication Critical patent/WO2012031530A1/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
    • C07K14/205—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
    • 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
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01005—Urease (3.5.1.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the field of the invention relates to the field of biotechnology of the present invention, and in particular to the use of a multi-target recombinant gene and a protein thereof for preventing and treating H. pylori infection. Background technique
  • He li cobacter pyl or i is an important pathogen found in 1982.
  • the current research shows that the bacteria are closely related to MALT lymphoma and gastric cancer except gastritis and gastric ulcer. It is currently the only bacterial pathogen associated with human tumorigenesis published by the WHO.
  • Recent studies have also found that Hp is also highly correlated with cardiovascular diseases such as coronary heart disease. Hp can infect the elderly, children and young adults, but different countries and regions have different infection rates due to different economic levels and living habits.
  • the infection rate of the general population in China is about 50-80%, and it increases at a rate of 1-2% per year.
  • urel gene has no relationship with the urease synthesis of Hp, but is closely related to the intragastric colonization of Hp, and is a gene essential for Hp colonization in the stomach.
  • Rektorschek used a gene mutation technique to compare studies, pointing out that ure I encodes a urea membrane channel protein of Hp.
  • the urel gene was transcribed in vitro into 6 fragments of the transmembrane protein Ure l, which is independent of the cell membrane of Hp, and protects the enzymatic activity of urease in the gastric acidic environment at pH below 4.
  • the pH of the mucosal layer of the normal human stomach is about 2-4, and the pH of the gastric juice is about 2.
  • Weeks and other studies have shown that Hp extracellular urease is inactivated at pH 4.5 and survives less than 5 minutes at pH below 4.0. Why does Hp survive in a highly acidic environment in the stomach? Weeks and Scott believe that the Hp urea channel Urel can take urea from the outside of the cell for the decomposition of intracellular urease into ammonia (NH3) and carbon dioxide (C02).
  • NH3 ammonia
  • C02 carbon dioxide
  • the ammonia cloud formed by ammonia (NH3) creates a low colonization of Hp.
  • the "comfortable" environment of oxygen weak acid, which is the necessary molecule for Hp colonization, but Urel molecular immunity There is no further report on sex and whether it can be used as a drug target for preventing Hp infection.
  • UreB is a urease activity subunit and is a gene essential for Hp colonization and proliferation under low pH conditions in the stomach.
  • the UreB protein was the main component of the "oral recombinant H. pylori vaccine".
  • it passed the approval of a new class of drugs by the China Food and Drug Administration, and entered the stage of industrial scale production and mass clinical promotion.
  • UreB is currently recognized as a Hp vaccine target, because it is a single gene target, there are many technical defects in prokaryotic expression, preparation and purification, renaturation preservation, etc., and the dominant epitopes of Urel and UreB are concatenated together to form more
  • the target recombinant gene can be used as a multi-target nucleic acid vaccine for Hp control and its corresponding recombinant protein vaccine or specific antibody preparation.
  • the multi-target recombinant gene and its protein have not been reported in the biopharmaceutical research for the prevention and treatment of Hp infection.
  • Hp infection treatment is the use of antibiotics or combined antibiotics.
  • This broad-spectrum antibiotic treatment has a certain effect on the clearance of Hp in individuals, but this treatment is likely to cause bacteria in the human body.
  • Group disorder and drug-resistant strains are produced, which is not conducive to the prevention and treatment of Hp in the stomach and in the natural environment.
  • PPI Plasma Pump Inhibitor
  • treatment can inhibit gastric acid secretion, as the pH of the stomach environment rises, the establishment of Hp in the stomach creates a neutral environment, which is conducive to an increase in the number of Hp.
  • the immune system is used to block the urea membrane channel of Hp, thereby blocking the biochemical reaction of urease to decompose urea, and using Hp urine.
  • the enzyme B subunit eliminates the activity of decomposing urea in the extracellular cells of Hp, so that Hp cannot die or colonize against the acidic environment of the stomach.
  • This key molecule using Hp colonization, multi-target fusion design has achieved the purpose of both preventing and treating Hp infection, while avoiding the clinical deficiency of antibiotics and PPI agents in preventing and treating Hp.
  • This multi-gene multi-target dominant epitope The combined application of biotechnology is the ideal method to control Hp. Summary of the invention
  • the technical problem to be solved by the present invention is to provide a multi-target fusion polypeptide having the amino acid sequence of SEQ ID NO: 2, and a multi-target fusion polypeptide having the nucleotide sequence shown by SEQ ID NO:
  • the target recombinant gene, and the above-described multi-target recombinant gene or the above-described multi-target fusion polypeptide or the specific antibody of the above multi-target fusion polypeptide are used as a biological product for preventing and treating Helicobacter pylori infection.
  • the multi-target fusion polypeptide is obtained by fusing a Helicobacter pylori urea membrane channel protein and a B cell and T cell epitope peptide of a Helicobacter pylori urease B subunit, and the multi-target fusion polypeptide can be expressed by prokaryotic or eukaryotic expression or Prepared by chemical synthesis.
  • the multi-target fusion polypeptide is a reference Helicobacter pylori urea membrane channel protein (Urel) and urease B subunit (UreB) amino acid sequence, bioinformatics predicts its B cell antigen epitope and T cell antigen epitope, screening optimized splicing Made. Experiments have shown that it can better stimulate humoral and cellular immune responses in humans and animals.
  • the nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 2 is a multi-target recombinant gene of the nucleotide sequence shown by SEQ ID NO: 1.
  • a nucleotide sequence having the same coding product as SEQ ID NO: 1 may be mutated, substituted or the like on the basis of the sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence shown in SEQ ID NO: 1 above is composed of Helicobacter pylori urea membrane channel protein and seclu
  • the nucleotide sequence corresponding to the B cell of the Helicobacter pylori urease B subunit and the T cell dominant epitope peptide is recombined, and the recombinant sequence is prepared by PCR or artificial synthesis.
  • the multi-target fusion polypeptide encoded by the nucleotide sequence can better stimulate humoral and cellular immune responses in humans and animals.
  • a prokaryotic expression vector or eukaryotic expression vector comprising the above nucleotide sequence (i.e., the sequence of SEQ ID NO: 1) according to the claims.
  • a multi-target nucleic acid vaccine comprising an eukaryotic expression vector comprising the above nucleotide sequence (ie, the sequence of SEQ ID NO: 1) in the prevention or treatment of a Helicobacter pylori-infected biological product application.
  • a multi-target fusion polypeptide vaccine for use in the prevention or treatment of a H. pylori-infected biological product comprising a multi-target fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • An antibody preparation which is a specific antibody comprising a multi-target fusion polypeptide epitope against the amino acid sequence shown in SEQ ID NO: 2, which is useful for preventing or treating a Helicobacter pylori-infected biological product.
  • the present invention has been intensively studied, and computer-predicted and epitope-expressed Hur's urel and ureB genes and encoded proteins are selectively combined to form a nucleotide sequence encoding a multi-target fusion polypeptide that may induce an immune response in the body. .
  • the multi-target fusion polypeptide immunizes the animal with serum titer, immunoblot assay and serum neutralization assay, CD4 + lymphocyte proliferation and other experiments to prove that the multi-target fusion polypeptide has prevention and treatment of pylorus
  • the role of Helicobacter infection The details are as follows:
  • the present invention provides a B cell and T cell epitope multi-target fusion polypeptide vaccine component from a Helicobacter pylori urea membrane channel protein and a Helicobacter pylori urease B subunit, comprising The amino acid sequence shown in SEQ ID NO: 2 or a derivative thereof.
  • the nucleic acid sequence containing SEQ ID NO: 1 may also be cloned into a plant expression vector, and the multi-target fusion polypeptide comprising the amino acid sequence shown by SEQ ID NO: 2 or the like may be expressed in a plant.
  • the multi-target fusion polypeptide can be prepared into various biological products such as vaccines, diagnostic reagents or health care products.
  • the present invention provides a multi-target nucleic acid vaccine for preventing and treating Helicobacter pylori infection, the multi-target nucleic acid vaccine comprising the nucleotide sequence shown in SEQ ID NO: 1 or each derivative derived from the sequence A nucleic acid preparation.
  • the preparation method of the nucleic acid vaccine comprises: artificially synthesizing or obtaining a nucleic acid sequence represented by SEQ ID NO: 1 or a derivative thereof by PCR, and ligating and ligating into a eukaryotic expression vector, such as pCDNA or various viral vectors, to prepare Various nucleic acid preparations.
  • the present invention provides an antibody preparation for preventing and treating Helicobacter pylori infection.
  • the antibody is characterized by comprising a monoclonal or polyclonal antibody against an epitope of a multi-target fusion polypeptide based on the amino acid sequence set forth in SEQ ID NO: 2.
  • the antibody preparation method can be carried out by immunizing various experimental animals such as chickens, cows, mice, and the like with a multi-target fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 2 to prepare a monoclonal or polyclonal antibody.
  • the antibody may be purified by methods such as salting out or affinity chromatography, or may be directly prepared into various biological products such as therapeutic antibodies, diagnostic reagents or health care products without purification.
  • the beneficial effects of the invention are: effective integration of antigen targets of ⁇ intragastric key target proteins Urel and UreB to create an optimal drug target.
  • the multi-target fusion polypeptides predicted by the bioinformatics method for predicting the dominant epitopes of urel and ureB are not only important for the pathogenesis of Hp, but also for the research of Hp prevention and therapeutic preparations.
  • the obtained multi-target fusion polypeptide vaccine, nucleic acid vaccine, and antibody preparation showed good characteristics for controlling H. pylori infection in vitro and in vivo experiments, and the invention has a good application prospect.
  • FIG. 1 is a schematic diagram of double restriction enzyme digestion of eukaryotic expression vector
  • FIG. 2 is a schematic diagram of SDS-PAGE identification of multi-target fusion polypeptide induced expression
  • FIG. 3 is a schematic diagram of SDS-PAGE identification of recombinant multi-target fusion polypeptide purification
  • Figure 4 is a schematic representation of the specific reactivity of a recombinant multi-target fusion polypeptide Western Blot
  • Figure 5 is a schematic representation of the identification of specific immunogenicity after immunization of an animal with a recombinant multi-target fusion polypeptide.
  • Multi-target fusion polypeptide (Urel and UreB epitope peptide) predictive synthesis
  • the nucleotide sequence corresponding to the amino acid of the B cell and T cell dominant epitope containing Urel and UreB was named ureI- ⁇ , ie the DNA sequence shown in SEQ ID NO: 1
  • the above amino acid is named Urel- ⁇ , which is the amino acid sequence shown by SEQ ID NO: 2.
  • the online prediction website URL is as follows: www, imtech. res, in/ raghava/ propred www. epipredict. de/ index, html is not limited to the URLs listed above.
  • Example 2 Multi-target fusion polypeptide nucleic acid vaccine design and construction selection
  • pIRES2-DsRed2 is a nucleic acid vaccine vector, and the DNA sequence encoding the multi-target fusion polypeptide synthesized in Example 1 was digested with Nhel and Kpnl. The ligation transforms DH5a competent cells. The positive clones identified by PCR and double restriction enzyme digestion were concentrated to prepare transfected cells. The results of double enzyme digestion were shown in Figure 1.
  • Lane 1 DNA MARKER DL2000
  • Lane 2 pIRES2-DsRed2-ureI-B recombinant plasmid
  • Lane 3 Recombinant plasmid Nhel and Kpnl double enzyme digestion
  • Lane 4 pIRES2-DsRed2 empty vector Nhel and Kpnl double digestion; the results showed that: pIRES2-DsRed2-ureI-B recombinant plasmid contains a gene fragment of the size of the expected multi-target nucleotide gene sequence.
  • Example 3 Verification of transfection, transfection rate and expression of nucleic acid vaccine in Example 2
  • HEK293T cells were trypsinized and counted. Cells were seeded in IX 107 wells in 6-well plates, 2 ml of 10% bovine serum basal medium was added, cultured at 37 ° C, 5% C0 2 incubator, and the cells were grown to 60-80. % density (about 24h), then transfection can be performed.
  • Solution A 25 plasmid +375 ⁇ ⁇ serum-free medium, mix.
  • Recombinant plasmids pIRES2-DsRed2, pIRES2-DsRed2-urel, pIRES2-DsRed2-c-i/re/transfected HEK293T cells and mouse skeletal muscle cells were observed under fluorescent inverted microscope for 48 h and 72 h, respectively. Express the situation. Fluorescent The ratio of cells to non-fluorescent cells was analyzed, and the transfection rate of HEK293T cells was about 80%.
  • Hp Helicobacter pylori
  • ⁇ mainly causes gastric mucosal hemorrhage, degeneration and necrosis in mice, accompanied by inflammatory reaction of lymphocytic infiltration, which is similar to the symptoms of ⁇ -induced gastric disease. Therefore, this experiment selected BALB/c Mice were used to evaluate nucleic acid vaccines using HP-infected animal models.
  • mice in each group were intramuscularly injected with the endotoxin nucleic acid vaccine plasmid, and the control group was also injected with the empty vector plasmid. After the initial immunization, the rats were injected again on the 7th and 14th day and injected a total of three times.
  • Hp infection experiments were performed one week after the last immunization.
  • the Hp of the Brucella agar culture was quickly aseptically eluted into a sterile test tube with 0.02 mol/l Ph 7. 4 PBS, and the bacterial concentration was adjusted to 10 9 CFU/ml for the infected bacterial solution.
  • Two groups of mice were infected by circulating drip.
  • mice were sacrificed in the fourth week after the last drip, and serum and gastric mucosa were separated. Gastric mucosa tissue was subjected to rapid urease experiments and direct smear, Gram stain microscopy.
  • Infection rate (number of infections / number of survivors) X 100%
  • mice in each group were set up, and 30 mice in each group.
  • the Hp dyeing experiment was carried out in the same manner as above. After 14 days of infection, serum was measured for anti-Hp antibody titer to confirm the infection effect. The mice in each group were confirmed to be infected. The muscles of the experimental group were injected with the endotoxin nucleic acid vaccine plasmid, and the control group was injected with the empty vector. Granules. After the initial injection, the rats were injected again on the 7th and 14th days, and a total of three injections were made.
  • mice were observed daily, and after intramuscular injection of the recombinant nucleic acid vaccine plasmid and the empty plasmid, the clinical symptoms of the mice were observed by observation on 14, 39, and 69 days (the hair was scored 0 times, and the symptoms disappeared -1 ELISA detection of Hp antibody IgG in peripheral blood of model mice (decline -1, increase or not 0), urease test (negative -1, positive 0), bacterial culture colony count to determine Hp colonization Quantity (number decreased by -1 point, increased or unchanged by 0 points); pathological examination of gastric tissue glandular inflammation (reduced -1 point, aggravated or no change 0 points), hemorrhage (reduced -1 point, aggravated or no change) 0 points), edema (-1 point reduction, aggravation or no change 0 points), atrophy (-1 point reduction, aggravation or no change 0 points), necrosis (-1 point reduction, aggravation or no change 0 points),
  • the effect of treatment was determined based on the bacterial colonization amount, antibody IgG level, gastric tissue disease damage, and IFN-Y change score.
  • the calculation method is as follows: During the onset period, the sum of the highest scores of animals in a group divided by the number of animals is the average clinical score of the group of animals, and the clinical scores of SD and the peripheral blood antibody IgG between the groups. Cytokine levels in taxis were compared using the Kruskal-Wallis test, when? ⁇ 0. 05, the Mann-Whitney U test was used for comparison between groups.
  • Example 5 Design, construction and identification of prokaryotic expression vector of multi-target fusion polypeptide Select PET28a(+) prokaryotic expression vector, and encode the DNA sequence of the synthetic fusion polypeptide.
  • the vector was digested with EcoRI and Xhol and ligated into DH5a competent cells.
  • the positive clones identified by PCR and double digestion were transformed into plasmids expressing the host strain Rosseta garni II, and clones positive by PCR were sent for sequencing. Preparation to induce expression of the epitope peptide of interest.
  • Example 6 Induction expression, purification and immunoblotting identification of multi-target fusion polypeptide According to the induction process of PET prokaryotic expression system, induced by IPTG induction, sampling before and after induction
  • Lane 3 Protein MARKER; The results show a significant protein band at 30 KD after induction, which is comparable to the predicted protein molecular weight.
  • the multi-target fusion polypeptide was purified by the His tag on the PET28a(+) vector, and the purification result is shown in Fig. 3, in which: lane 1: Protein MARKER;
  • Lane 2 Precipitation after sonication of the cells
  • Lane 3 supernatant after sonication of the cells
  • Lane 4 Ni column elution; The results showed that the target protein was present in the cytoplasm in a soluble form, and the purity of the electrophoresis after purification was over 90%.
  • the purified multi-target fusion polypeptide was electroporated onto the NC membrane, and the anti-Hp human serum and the anti-UreB monoclonal antibody were used as primary antibodies to verify the immunospecificity of the multi-target fusion polypeptide.
  • Fig. 4 in which: Lane 1: Prote in MARKER;
  • Lane 2 Multi-target fusion polypeptide, primary antibody is anti-human Hp positive serum; Lane 3: multi-target fusion polypeptide, primary antibody is anti-UreB mAb; Lane 4: multi-target fusion polypeptide, primary antibody is normal human serum Lane 5: empty expression lysate, the primary antibody is anti-UreB monoclonal antibody;
  • Example 7 Multi-target fusion polypeptide immunization Animal effect verification Multi-target fusion polypeptide immunized rabbits, 500 ug/head, and 5 co-immunizations. Immunize every 14 days after the first immunization. Blood was collected one week after each immunization and serum was separated. Multi-target fusion polypeptide package prepared
  • Example 8 Specific antibody preparation against multi-target fusion polypeptides
  • the rabbit immune titer reached 10 5
  • cardiac blood was sacrificed, serum was collected, and affinity chromatography was prepared.
  • the rabbit serum collected in the previous step was diluted 5 times with Loading Buffer, and the sample was loaded 4-5 times. Elution Buffer elutes and collects eluted peaks.
  • the antibody concentration was analyzed by 0D260/280, and the antibody purity was determined by SDS-PAGE. The results showed that the purity of the antibody was above 95%.
  • the antibody was dialyzed into 10 mM PBS and concentrated to more than 2 mg/ml for use.
  • the experimental group and the control group were set up, and 30 mice in each group.
  • the Hp infection experiment was carried out in the same way as above. After 14 days of infection, serum was measured for anti-Hp antibody to confirm the effect of infection.
  • the mice in each group were confirmed to be infected, the experimental group was orally administered with anti-multi-target fusion polypeptide-specific antibody, and the control group was orally administered with PBS buffer. Oral daily, for 14 days.
  • mice were observed daily, and after oral administration of the antibody, the clinical symptoms of the mice were observed by observation on 14, 39, and 69 days (the score of the hair was 0, the symptoms disappeared -1 point), and the model mice were detected by ELISA.
  • Peripheral blood antibody IgG decline -1, increase or not 0
  • urease test negative -1, positive 0
  • colony count by bacterial culture colony count number decreased by -1 point, increased Or unchanged 0 points
  • pathological examination of gastric tissue glandular inflammation (reduced -1 points, aggravated or no change 0 points), bleeding (reduced -1 points, aggravated or no change 0 points), edema (alleviation -1 Points, aggravation or no change 0 points), atrophy (-1 point reduction, aggravation or no change 0 points), necrosis (-1 point reduction, aggravation or no change 0 points), comprehensive evaluation of treatment effect.
  • the effect of treatment was determined based on the bacterial colonization amount, antibody IgG level, gastric tissue disease damage, and IFN-Y change score.
  • the calculation method is as follows: During the onset period, the sum of the highest scores of animals in a group divided by the number of animals is the average clinical score of the group of animals, and the clinical scores of SD and the peripheral blood antibody IgG between the groups. Cytokine level taxi SD using Kruskal-Wallis Test to compare, when? ⁇ 0.05, the Mann-Whitney U test was used for comparison between groups. Assume that the clinical comprehensive score is a total of 8 points, and the relative clinical score is 100%.
  • ⁇ 5 ⁇ fruit shows that the specific antibody has excellent treatment for oral treatment of Helicobacter pylori infection.
  • the present invention can be preferably carried out.

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Description

说 明 书
一种多靶点融合蛋白， 其编码基因及应用 技术领域 本发明生物技术领域， 具体地说， 涉及一种多靶点重组基因及其蛋白在 防治幽门螺旋杆菌感染中的应用。 背景技术
幽门螺杆菌 ( He l i cobacter pyl or i , Hp ) 是 1982年发现的一种 重要的致病菌，目前研究表明该细菌除引起胃炎、胃溃疡外，其与 MALT 淋巴瘤和胃癌的关系非常密切， 是目前唯一被 WHO公布的与人肿瘤发 生相关的细菌性病原。 最近研究还发现 Hp 与人冠心病等心血管疾病 也有高度的相关性。 Hp可感染老人、 小孩和青壮年， 但不同国家和地 区， 因经济水平和生活习惯不同感染率差异很大。 我国的普通人群的 感染率约 50-80% , 且每年以 1-2%的速度增加。
1989年 Hp的尿素酶基因被克隆出， 1997年 Hp的全基因序列测定 完成。 Agnes等利用 shutte克隆技术对尿素酶进行分析， 证实编码尿 素酶一组基因位于 4. 2kb DNA片段中， 并确定这组基因中有 4个开放 性阅读框架（0RF) , 分别称为 ureA、 ureB、 ureC、 ureD。 进一步研究 发现在 ureA、 ureB下段的 ureC、 ureD阅读框架分别是 urel、 ureE、 ureF、 ureG、 ureH , 整个尿素酶基因长度约为 8kb。 1998法国巴士德 研究所 St 6phane研究认为 urel基因对 Hp的尿素酶的合成无关系， 但与 Hp 的胃内定植非常密切， 是 Hp 在胃定植所必需的基因。 2000 年 Rektorschek用基因突变技术比较研究指出， ure I 编码 Hp的尿 素膜通道蛋白。 urel基因体外转录翻译成 6个片段的跨膜蛋白 Ure l , 该蛋白独立于 Hp的细胞膜上， 保护尿素酶在 pH低于 4时的胃酸性环 境的酶活性，实验发现在 pH值大于 4时，胞内尿素酶可以不依赖 Ure l 蛋白而维持尿素酶活性，但在 pH低于 4时 Urel是维护尿素酶活性必 需基因。 2001年 David L. Weeks研究认为 Urel是 Hp联系尿素酶和 在胃内定植的重要通道蛋白， Urel对 Hp在胃定植起关键作用。 有研 究用非洲爪蛙卵细胞 （Xenopus oocytes) 作为转基因细胞模型， 观 察表达的 Urel 能在酸性条件下促进尿素吸收， 而细胞周质第 123位 组氨酸 （histidinel23) 缺失的 i/re/突变体无促进尿素吸收作用， 同时 Urel 介导的尿素转运是尿素特异的、 被动的、 非饱和的、 非极 性的和非温度依赖性的。 Weeks指出 Urel是 的 H+控尿素通道， 调 控细胞内尿素酶代谢， 对 ^ 在胃的定植和生存非常必要。 David R. Scott研究认为在有尿素的情况下， Urel在 pH 5时刺激胞内氨 产生， 但用乙酰胺代替尿素却无此现象。 因此， Urel协助尿素酶分解 尿素的作用是特异的。此外研究发现 Urel有四个保守组氨酸残基（H71， H123, H131, H193)； 有三个胞内多肽环中有保守区（L165， G166, K167, F168). 远端的 H193 (非 H123)对 Hp在低 pH环境中刺激产生氨起决 定性作用； Urel的第三个胞内环对 Urel的膜通活性很重要。 这些说 明了 Hp在胃环境定植的新分子机制。 国内外的大量研究和临床治疗实践表明 Hp 感染并定植是其致病 的首要条件， 如果清除 Hp, 胃炎和胃溃疡即好转。 正常人体胃的黏膜 层 pH值为 2-4左右， 胃液 pH值为 2左右。 Weeks等研究表明， Hp细 胞外尿素酶在 pH值 4.5时失活，在 pH值低于 4.0时存活不到 5分钟， 那么 Hp为何能在胃内高酸性环境生存？ Weeks 和 Scott 等认为 Hp 尿素通道 Urel可从细胞外摄取尿素， 供胞内尿素酶分解成氨（NH3)和 二氧化碳（C02) , 氨（NH3)形成的 "氨云" 为 Hp的定植创造了低氧弱 酸的" 舒适" 环境，这就是 Hp定植的必要分子， 但 Urel分子免疫特 性和是否其可以作为防止 Hp感染的药物靶点没有进一步的报道。
2000年， Scott等研究发现 Urel在酸性环境下对 Hp的细胞内尿素酶激 活很重要。 2001年 Week等通过基因突变研究发现 Urel对 Hp在胃定植和致 病非常重要。 Skouloubris等通过 RNAi等基因沉默技术研究证实 Urel 与尿 素酶的活性无关， 但却是 Hp在低的 PH值条件下定植和增殖所必需的。 2002 年， Mol lenhauer等用沙鼠试验证明 Urel对 Hp在胃内的定植和定植后的生 存均具有重要意义 。
1998年， Skouloubris等研究证实 UreB是尿素酶活性亚单位， 是 Hp在 胃低 pH值环境条件下定植和增殖必需的基因。 2009年重组 UreB蛋白为主要 成分的 "口服重组幽门螺旋杆菌疫苗" 2009年 4月通过中国食品药品监督管 理局的一类新药审批， 进入产业化规模生产和大量临床推广应有阶段。 UreB 虽然是目前公认的 Hp疫苗靶点， 但因为其为单基因靶点， 原核表达、制备纯 化、 复性保存等有很多技术缺陷， 将 Urel和 UreB的优势抗原表位集结串联 起来，形成多靶点重组基因可以作为 Hp防治的多靶点核酸疫苗及其对应的重 组蛋白疫苗或特异抗体制品。 目前， 该多靶点重组基因及其蛋白在预防和治 疗 Hp感染的生物药物研究中还没有研究报道。
目前， 从对 Hp的临床治疗现状来看， 常规 Hp感染治疗是使用抗生素或 用联合抗生素，这个广谱抗生素治疗的医疗措施虽然对个体清除 Hp有一定作 用，但这种治疗易引起人体内菌群紊乱和耐药菌株产生， 同时不利于 Hp在胃 内和自然环境中的防治。 虽然 PPI (Proton Pump Inhibitor ) 治疗能抑制胃 酸分泌， 但随着胃环境 pH值回升， 给 Hp在胃内定植创造了中性环境， 反有 利于 Hp数量增加。 从 Hp 生理代谢所涉及的分子作用和生化机制出发， 利用免疫技术阻断 Hp的尿素膜通道， 从而阻断尿素酶分解尿素的生化反应， 同时利用 Hp的尿 素酶 B亚单位作为靶点，使 Hp细胞外细胞内的分解尿素的活性消失，从而使 Hp不能对抗胃酸性环境而死亡或不定植。 这种利用 Hp定植的关键分子， 多 靶点融合设计达到了既预防又治疗 Hp感染的目的， 同时避免了抗生素和 PPI 试剂防治 Hp在临床上的不足，这种多基因多靶点优势抗原表位联合应用的生 物技术是理想的防治 Hp方法。 发明内容
本发明所要解决的技术问题是提供一种具有 SEQ ID NO : 2 所示氨基酸 序列的多靶点融合多肽、 具有 SEQ ID N0 : 1所示的核苷酸序列的编码多靶点 融合多肽的多靶点重组基因、 以及上述多靶点重组基因或上述多靶点融合多 肽或上述多靶点融合多肽的特异性抗体作为生物制品在预防和治疗幽门螺旋 杆菌感染中的应用。
本发明解决上述技术问题所采用的技术方案是： 一种具有 SEQ ID NO : 2 所示氨基酸序列的多靶点融合多肽或其衍生物。
上述多靶点融合多肽由幽门螺旋杆菌尿素膜通道蛋白及幽门螺旋杆菌尿 素酶 B亚单位的 B细胞及 T细胞表位肽融合而成， 该多靶点融合多肽可采用 原核或真核表达或化学合成方法制备。 该多靶点融合多肽是参考幽门螺旋杆 菌尿素膜通道蛋白 （Urel ) 和尿素酶 B亚单位 （UreB) 氨基酸序列， 生物信 息学预测其 B细胞抗原表位和 T细胞抗原表位， 筛选优化拼接而成。 实验证 明能较好的刺激人体和动物体的体液免疫反应和细胞免疫反应。
编码 SEQ ID NO : 2所示氨基酸序列的核苷酸序列， 为 SEQ ID NO : 1所 示核苷酸序列的多靶点重组基因。 或者在 SEQ ID N0 : 1所示序列的基础上有 个别碱基的变异、 替换等与 SEQ ID NO : 1具有相同编码产物的核苷酸序列。
上述 SEQ ID N0： 1所示核苷酸序列由幽门螺旋杆菌尿素膜通道蛋白及幽 门螺旋杆菌尿素酶 B亚单位的 B细胞及 T细胞优势表位肽所对应的核苷酸序 列重组而成， 该重组序列采用 PCR或人工合成的方法制备。 该核苷酸序列所 编码的多靶点融合多肽能够较好的刺激人体和动物体的体液免疫反应和细胞 免疫反应。
包含权利要求上述核苷酸序列 （即 SEQ ID NO : 1所示序列） 的原核表达 载体或真核表达载体。
一种多靶点核酸疫苗， 为包含上述核苷酸序列 （即 SEQ ID NO ： 1所示序 列） 的真核表达载体， 该真核表达载体在预防或治疗幽门螺旋杆菌感染的生 物制品中的应用。
一种多靶点融合多肽疫苗， 为包含 SEQ ID N0 : 2所示氨基酸序列的多靶 点融合多肽在预防或治疗幽门螺旋杆菌感染的生物制品中的应用。
一种抗体制品， 为包含抗 SEQ ID N0 : 2所示氨基酸序列的多靶点融合多 肽表位的特异性抗体， 该特异性抗体在预防或治疗幽门螺旋杆菌感染的生物 制品中的应用。
本发明经过深入研究，对 Hp的 urel及 ureB基因和编码蛋白进行计算机 预测和抗原表位分析， 选择性组合成了可能诱导机体产生免疫应答的 1条多 靶点融合多肽的编码核苷酸序列。 经过原核表达， 纯化后， 该多靶点融合多 肽免疫动物后的血清效价、 免疫印迹实验和血清中和试验、 CD4+淋巴细胞增 殖等实验证明了该多靶点融合多肽具有预防和治疗幽门螺旋杆菌感染的作 用。 具体内容如下：
第一方面： 本发明提供了来自幽门螺旋杆菌尿素膜通道蛋白及幽门螺旋 杆菌尿素酶 B亚单位的 B细胞及 T细胞表位多靶点融合多肽疫苗组分， 含有 SEQ ID NO : 2所示的氨基酸序列或其衍生物。
1： 基于 SEQ ID NO : 2所示氨基酸序列。
2： 人工合成基于 SEQ ID NO : 2所示氨基酸序列或用 PCR方法获得含有 SEQ ID NO : 1所示的核苷酸序列或其衍生物， 酶切连接该基因入原核表达载 体或真核表达载体， 如 PET22b (+)或 pIRES2-DsRed2或其他类型的表达载体 中。 转化重组表达载体进入宿主菌， 如： Rosseta garni II 、 BL21 、 酵母等， 构建基因表达工程菌。 也可将含有 SEQ ID NO ： 1所示的核酸序列克隆至植物 表达载体， 在植物中表达该含 SEQ ID NO : 2所示氨基酸序列的多靶点融合多 肽等。
3： 诱导表达， 通过各种介质纯化， 获得 SDS-PAGE纯度大于 90%的多靶 点融合多肽。
4： 该多靶点融合多肽可以制备成各种生物制品， 如疫苗、诊断试剂或保 健品等。
第二方面： 本发明提供了一种用于预防和治疗幽门螺旋杆菌感染的多靶 点核酸疫苗， 该多靶点核酸疫苗含有 SEQ ID N0 ： 1所示核苷酸序列或该序列 衍生的各种核酸制剂。 该核酸疫苗的制备方法： 人工合成或用 PCR方法获得 含有 SEQ ID NO : 1所示的核酸序列或其衍生物， 酶切连接入真核表达载体， 如 pCDNA或各种病毒载体上， 配制成各种核酸制剂。
第三方面： 本发明提供了一种用于预防和治疗幽门螺旋杆菌感染的抗体 制剂。
1： 该抗体其特征在于含有抗基于 SEQ ID NO : 2所示氨基酸序列多靶点 融合多肽表位的单克隆或多克隆抗体。 2： 该抗体制备方法可以用含 SEQ ID NO : 2所示氨基酸序列的多靶点融 合多肽免疫各种实验动物， 如鸡、 牛、 小白鼠等制备单克隆或多克隆抗体。
3：该抗体可以用盐析或亲和层析等方法进行纯化，也可以不纯化直接制 备成各种生物制品， 如治疗性抗体、 诊断试剂或保健品。
本发明的有益效果是：对 Φ胃内定值关键靶蛋白 Urel及 UreB的抗原靶 点进行有效整合， 创造出最佳药物靶点。 通过生物信息学的方法对 urel及 ureB 的优势表位进行预测获得的多靶点融合多肽不仅对 Hp发病机制研究， 而且对 Hp预防和治疗制剂的研究均有重要意义。 获得的多靶点融合多肽疫 苗、 核酸疫苗、 抗体制品在体外实验和动物体内实验中显示了良好的防治幽 门螺旋杆菌感染的特性， 该发明具有良好的应用前景。
附图说明 图 1是真核表达载体双酶切鉴定的示意图； 图 2 是多靶点融合多肽诱导表达 SDS-PAGE鉴定的示意图； 图 3是重组多靶点融合多肽纯化 SDS-PAGE鉴定的示意图； 图 4是重组多靶点融合多肽 Western Blot鉴定特异反应原性的示意图； 图 5 是重组多靶点融合多肽免疫动物后鉴定特异免疫原性的示意图。 具体实施方式
下面结合实施例及附图， 对本发明作进一步的详细说明， 但本发明的实 施方式不限于此。
实施例 1：
多靶点融合多肽 （Urel及 UreB表位肽） 预测合成
在 NCBI蛋白质数据库里查找 Urel及 UreB氨基酸序列，采用多种在线预 测软件及 DNAstar软件同时分析该两目标序列的 B细胞及 T细胞优势抗原表 位， 在实验实施过程中， 将含 Urel及 UreB的 B细胞及 T细胞优势抗原表位 的氨基酸对应的核苷酸序列命名为 ureI-Β , 即 SEQ ID NO : 1所示的 DNA序 歹 将上述氨基酸命名为 Urel-Β , 即 SEQ ID NO : 2所示的氨基酸序列。 在线预测网站网址如下： www, imtech. res, in/ raghava/ propred www. epipredict. de/ index, html 不限于以上所列举网址。 实施例 2： 多靶点融合多肽核酸疫苗设计、 构建 选择 pIRES2-DsRed2为核酸疫苗载体， 将实施例 1中合成的编码多靶点 融合多肽的 DNA序列与该载体用 Nhel及 Kpnl双酶切后连接转化 DH5a感受态 细胞。 通过对 PCR和双酶切鉴定的阳性克隆提质粒浓缩准备转染细胞， 双酶 切鉴定结果如图 1所示， 图中：
泳道 1： DNA MARKER DL2000 ;
泳道 2 : pIRES2-DsRed2-ureI-B重组质粒；
泳道 3 : 重组质粒 Nhel及 Kpnl双酶切鉴定；
泳道 4: pIRES2-DsRed2空载体 Nhel及 Kpnl双酶切； 结果表明： pIRES2-DsRed2-ureI-B 重组质粒含有与预期的多靶点 核苷酸基因序列大小一致的基因片段。 实施例 3 : 实施例 2中核酸疫苗转染、 转染率及表达情况验证
3. 1转染 293T细胞和小鼠肌原代细胞过程 (DHEK293T细胞制备 胰酶消化 HEK293T细胞并计数， 将细胞以 I X 107孔接种于 6孔板， 加 2ml 含 10%牛血清基础培养基， 37°C、 5% C02孵箱培养， 待细胞生长至 60〜80%密 度 (约需 24h)， 即可进行转染。
(2)小鼠肌肉细胞制备
无菌取 7日龄 BALB/c小鼠四肢骨骼肌， 去筋腱， 剪成小块， 用 0. 125%胰酶 消化后， 纱网过滤， 20%牛血清的 DMEM培养基 37°C， 5%C02培养 48h后备用。 ( 脂质体化的转染质粒的制备
A液： 25 质粒 +375 μ ΐ无血清的培养液， 混匀。
Β液： 12 μ 1 Polyfect Transfection加入 375 μ 1无血清的培养液， 混匀。 (4)室温下静置 5min后， 将 A、 B两液轻轻混合， 室温下放置 20min。
(5)用 2ml无血清的培养液洗 6孔板中的细胞 1次。
(6)每管中加入 750 μ 1无血清的 RPMI 1640培养液到脂质体 -DNA复合物中，混 合均匀， 并用吸头反复吹打数次， 将其完全覆盖于洗过的细胞上。
(7)孵箱培养 5h。
(8)加入 1. 5ml 20%血清的 RPMI 1640, 继续培养。 转染细胞继续培养 18〜24h， 换完全 1640培养基（10%的小牛血清， 含 100U/ml青霉素 G、 100 μ g/ml链霉 素的 1640 )。 当培养至 48h， 荧光显微镜观察细胞的红色荧光。
3. 2重组质粒转染率及表达情况验证
(1)重组质粒转染效率验证
重 组 质 粒 pIRES2-DsRed2 、 pIRES2-DsRed2 - urel 、 pIRES2-DsRed2-c -i/re/转染 HEK293T细胞和小鼠骨骼肌细胞分别于 48h和 72h 后， 置于荧光倒置显微镜下观察红色荧光蛋白的表达情况。 从发荧光的 细胞与不发荧光的细胞的比例分析， HEK293T细胞的转染率为 80 %左右。
(2)重组质粒细胞内表达情况验证
转染 72h后， 0. 01M的 PBS (PH7. 2 ) 洗涤 3次后， 4%的多聚甲醛室温作 用 20min固定细胞， PBS洗涤 3次后， 用 500 μ 1正常山羊血清工作液 4°C封 闭过夜， 分别用 500 μ 1人 Hp阳性抗体（1 : 500 )作一抗， 37°C作用 2小时， 洗涤 3次后，用 500 μ 1羊抗人荧光抗体（1 : 200 )作二抗， 37°C作用 1小时， 洗涤 3次， 50%的甘油 PBS封闭后， 荧光倒置显微镜下观察绿色荧光蛋白（仅 pIRES2-DsRed2-i/re/质粒转染 HEK293T细胞组，空质粒转染 HEK293T细胞组 设对照。（肌肉细胞因贴壁无法固定而没做荧光免疫组化检测，用转染质粒上 的红色荧光标记间接计算目标基因的转染。）细胞原位免疫组化显示有棕色颗 粒在细胞质靠近细胞膜附近表达。
实施例 4:
核酸疫苗预防和治疗效果验证
4. 1实验动物品系筛选和动物模型的建立
为探索何种品系的小鼠最能模拟人 ^感染的临床症状， 本部分实验用
Ure+、 CagA+、 VacA+的 I型幽门螺杆菌 (Mel icobacter pylori, Hp) SSI 菌 株对三种 SPF级小鼠（BALB/c、 NIH、 KM)采用循环滴喂攻击等方法处理， 从 菌体定值、 抗体水平、 病理变化三个方面分 14、 39、 69、 105天进行检测。 结果证明 可持续定植于各供试小鼠胃粘膜上， 并刺激 Φ抗体（IgG)维持 一较高水平， 其中 BALB/c感染后的细菌定植早， 定植率高， 引起的胃病变最 明显。 φ在小鼠身上主要引起以胃粘膜出血、 变性、 坏死为主， 伴有淋巴细 胞浸润的炎症反应， 这与 Φ致人胃病的症状相似。 故本实验选取了 BALB/c 小鼠做核酸疫苗评价用 HP感染动物模型。
4. 2 核酸疫苗预防效果验证
设实验组和对照组，每组小鼠 30只，实验组小鼠肌肉注射去内毒素核酸 疫苗质粒， 对照组同样注射空载体质粒。 初次免疫后， 第 7， 14天再次注射， 共注射三次。
末次免疫后一周进行 Hp感染实验。取布氏琼脂培养的 Hp,用 0. 02mol/l Ph 7. 4 PBS无菌迅速洗脱至无菌试管，调整菌浓度至 109CFU/ml作感染菌液。 采用循环滴喂感染两组小鼠。
末次滴喂后第四周宰杀小鼠， 分离血清及胃粘膜。 胃粘膜组织进行快速 尿素酶实验和直接涂片， 革兰氏染色镜检。
结果判读方法为： 胃粘膜组织直接涂片革兰氏染色镜检， 出现革兰氏阴 性螺杆状细菌且尿素酶实验阳性则判定为 Hp感染。
保护率 = (未感染数量 /存活数量） X 100%
感染率 = (感染数量 /存活数量） X 100%
Figure imgf000012_0001
组别 心 感染数量 未感染数量 感染率 保护率 实验组 30 6 24 80% 对照组 30 27 3 90%
4. 3 核酸疫苗治疗效果验证
设实验组和对照组， 每组小鼠 30只。 首先进行 Hp染实验， 方法同上。 感染 14天后采血清测定抗 Hp抗体效价确认感染效果。 各组取确认感染的小 鼠， 实验组肌肉多点注射去内毒素核酸疫苗质粒， 对照组同样注射空载体质 粒。 初次注射后， 第 7， 14天再次注射， 共注射三次。
治疗效果评价：每日观察小鼠，在肌肉注射重组核酸疫苗质粒和空质粒后， 在 14、 39、 69天通过观察治疗小鼠临床症状 （被毛粗乱等得 0分， 症状消失 -1分）、 ELISA检测模型小鼠外周血的 Hp抗体 IgG (下降 -1分， 上升或不变 0 分）、 尿素酶实验（阴性 -1分， 阳性 0分）、 细菌培养菌落计数测定 Hp的定植 量（数目下降 -1分， 上升或不变 0分）； 病理组织检测胃组织的腺体炎症（减 轻 -1分， 加重或无变化 0分）、 出血 （减轻 -1分， 加重或无变化 0分）、 水肿 (减轻 -1分， 加重或无变化 0分）、 萎缩 （减轻 -1分， 加重或无变化 0分）、 坏死（减轻 -1分， 加重或无变化 0分）， 综合评价肌肉注射重组核酸疫苗对各 感染小鼠的治疗效果。
统计学分析：根据细菌 定植量、抗体 IgG水平、 胃组织病历损害、 IFN- Y变化综合分值判定治疗的效果。 其计算方法为： 在发病期间， 一组内动物 最高分值的总和除以动物数， 即为该组动物的平均临床分值， 各组之间的临 床评分士 SD值及外周血抗体 IgG、 细胞因子水平的士 SD采用 Kruskal-Wallis 检验进行比较， 当？<0. 05时， 组之间比较采用 Mann-Whitney U检验。
假设临床综合积分总共为 8分， 相对临床评分为 100%。 两两比较发现， 重组核酸疫苗免疫 14天后， 相对临床评分不变， 均为 100%。 核酸疫苗免疫 39天后， 相对对照组， 实验组相对临床评分为 78%。 69天后， 相对对照组， 实验组相对临床评分为 27. 9%。对照组和实验组在免疫 14天差异无统计学意 义（P〉0. 05), 免疫 39天和 69天后， 对照组和实验组差异有统计学意义（P <0. 01 )， 具体如表二。
表二 时间 （天） 对照组 实验组
14 7.91±0.16 8.02±0· 17
39 8.36±0.34 6.50±0.26
69 8.13±0· 35 2.20±0.21 注： η=5 实施例 5: 多靶点融合多肽原核表达载体设计、 构建、 鉴定 选择 PET28a(+)原核表达载体，将合成的融合多肽编码 DNA序列与该载 体用 EcoRI及 Xhol双酶切后连接转化 DH5a感受态细胞。 通过对 PCR和双酶 切鉴定的阳性克隆提质粒转化表达宿主菌 Rosseta garni II， 通过 PCR鉴定 为阳性的克隆送出测序。 准备诱导表达目的表位肽。 实施例 6: 多靶点融合多肽的诱导表达、 纯化及免疫印记鉴定 按照 PET原核表达系统诱导操作流程，使用诱导剂 IPTG诱导，诱导前后取样
SDS-PAGE鉴定分析， SDS-PAGE鉴定分析结果如图 2所示， 图中： 泳道 1: 表达菌诱导前；
泳道 2: 表达菌诱导后；
泳道 3： Protein MARKER; 结果显示诱导后在 30KD处出现明显的蛋白条带， 与预测的蛋白分子量 相当。 通过 PET28a(+)载体上的 His标签纯化该多靶点融合多肽， 纯化结果如 图 3所示， 图中： 泳道 1: Protein MARKER;
泳道 2:菌体超声破碎后沉淀；
泳道 3:菌体超声破碎后上清；
泳道 4: Ni柱洗脱； 结果表明， 该目的蛋白以可溶形式存在于细胞质中， 纯化后电泳纯度达 到 90%以上。 将纯化后的多靶点融合多肽电转至 NC膜上， 分别用抗 Hp 人血清及抗 UreB单抗做一抗验证多靶点融合多肽的免疫特异性，结果如图 4所示，图中： 泳道 1： Prote in MARKER ;
泳道 2 : 多靶点融合多肽， 一抗为抗人 Hp阳性血清； 泳道 3 : 多靶点融合多肽， 一抗为抗 UreB单抗； 泳道 4 : 多靶点融合多肽， 一抗为正常人血清； 泳道 5 : 空表达菌裂解液， 一抗为抗 UreB单抗；
结果显示该多靶点融合多肽与抗 Hp人血清和 UreB单抗的反应性良好。 实施例 7 : 多靶点融合多肽免疫动物效果验证 取多靶点融合多肽免疫兔子， 500ug/只， 共免疫 5只。 首次免疫后每 14 天免疫一次。 每次免疫后一周采血， 分离血清。 用制备的多靶点融合多肽包
Figure imgf000015_0001
免疫天数 0 7 21 35 49 抗 HP效价 0 102 103 104 105 效价变化图如附图 5所示。 数据显示多靶点融合多肽免疫原性良好， 在免疫
7天后产生抗体且效价上升较快。 实施例 8 : 抗多靶点融合多肽的特异抗体制备 当兔子免疫效价达到 105 时心脏采血处死， 收集血清， 准备亲和层析。 将多靶点融合多肽偶联至 CNBr 活化的 sepharose 4FF填料上， 偶联方 法按照填料说明实施。 装柱平衡后， 将上步收集的兔血清用 Loading Buffer 稀释 5 倍上样， 循环上样 4-5 次。 Elution Buffer 洗脱， 收集洗脱峰。 0D260/280分析抗体浓度， SDS-PAGE鉴定抗体纯度。 结果表明该抗体纯度均 为 95% 以上。 将抗体透析至 10 mM PBS中浓缩至 2 mg/ml以上， 备用。
实施例 9:
抗多靶点融合多肽的特异抗体治疗 Hp感染效果验证
设实验组和对照组， 每组小鼠 30只。首先进行 Hp感染实验， 方法同上。 感染 14天后采血清测定抗 Hp抗体，确认感染效果。 各组取确认感染的小鼠， 实验组口服抗多靶点融合多肽特异抗体，对照组口服 PBS缓冲液。每日口服， 疗程 14天。
治疗效果评价： 每日观察小鼠， 口服抗体后，在 14、 39、 69天通过观察治 疗小鼠临床症状 （被毛粗乱等得 0分， 症状消失 -1分）、 ELISA检测模型小鼠 外周血的^抗体 IgG (下降 -1分， 上升或不变 0分）、 尿素酶实验 （阴性 -1 分， 阳性 0分）、 细菌培养菌落计数测定 的定植量 （数目下降 -1分， 上升 或不变 0分）； 病理组织检测胃组织的腺体炎症 （减轻 -1分， 加重或无变化 0 分）、 出血 （减轻 -1分， 加重或无变化 0分）、 水肿 （减轻 -1分， 加重或无变 化 0分）、 萎缩 （减轻 -1分， 加重或无变化 0分）、 坏死 （减轻 -1分， 加重或 无变化 0分）， 综合评价治疗效果。
统计学分析：根据细菌 定植量、抗体 IgG水平、 胃组织病历损害、 IFN- Y变化综合分值判定治疗的效果。 其计算方法为： 在发病期间， 一组内动物 最高分值的总和除以动物数， 即为该组动物的平均临床分值， 各组之间的临 床评分士 SD值及外周血抗体 IgG、 细胞因子水平的士 SD采用 Kruskal-Wallis 检验进行比较， 当？<0.05时， 组之间比较采用 Mann-Whitney U检验。 假设临床综合积分总共为 8分， 相对临床评分为 100%。 两两比较发现， 口服特异抗体 14天后， 相对对照组， 实验组相对临床评分为 56%。 口服特异 抗体 39天后， 相对对照组， 实验组相对临床评分为 23%。 69天后， 相对对照 组， 实验组相对临床评分为 10%。 对照组和实验组在口服抗体 14天、 39天 和 69天后， 差异均有统计学意义 （P<0.01)， 具体如表四。
表四
时间 （天） 对照组 实验组
14 7.61±0.16 4.42±0.17
39 8.56±0.34 1.92±0.26
69 8.83±0· 35 0.82±0· 21 注： η=5 ί果显示了该特异抗体在口服治疗幽门螺旋杆菌感染方面具有极好的疗 % 如上所述， 便可较好的实现本发明。

Claims

权 利 要 求
1、 一种具有 SEQ ID NO : 2所示氨基酸序列的多靶点融合多肽或其衍生
2、 根据权利要求 1所述的具有 SEQ ID NO ： 2所示氨基酸序列的多靶点 融合多肽， 其特征在于， 所述多靶点融合多肽由幽门螺旋杆菌尿素膜通道蛋 白及幽门螺旋杆菌尿素酶 B亚单位的 B细胞及 T细胞优势表位肽融合而成， 该多靶点融合多肽采用原核或真核表达或化学合成方法制备。
3、 编码 SEQ ID NO : 2所示氨基酸序列的核苷酸序列， 其特征在于， 为 SEQ ID NO : 1所示核苷酸序列的多靶点重组基因。
4、 根据权利要求 3所述的编码 SEQ ID NO ： 2所示氨基酸序列的核苷酸 序列， 其特征在于， SEQ ID NO : 1所示核苷酸序列由幽门螺旋杆菌尿素膜通 道蛋白及幽门螺旋杆菌尿素酶 B亚单位的 B细胞及 T细胞优势表位肽所对应 的核苷酸序列重组而成， 该重组序列采用 PCR或人工合成的方法制备。
5、 包含权利要求 3所述核苷酸序列的原核表达载体或真核表达载体。
6、一种多靶点核酸疫苗， 其特征在于， 包含权利要求 3所述核苷酸序列 的真核表达载体， 该真核表达载体在预防或治疗幽门螺旋杆菌感染的生物制 品中的应用。
7、 一种多靶点融合多肽疫苗， 其特征在于， 包含 SEQ ID NO : 2所示氨 基酸序列的多靶点融合多肽在预防或治疗幽门螺旋杆菌感染的生物制品中的 应用。
8、 一种抗体制品， 其特征在于， 为包含抗 SEQ ID NO : 2所示氨基酸序 列的多靶点融合多肽表位的特异性抗体， 该特异性抗体在预防或治疗幽门螺 旋杆菌感染的生物制品中的应用
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