CN110950939B - Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof - Google Patents

Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof Download PDF

Info

Publication number
CN110950939B
CN110950939B CN201911225396.9A CN201911225396A CN110950939B CN 110950939 B CN110950939 B CN 110950939B CN 201911225396 A CN201911225396 A CN 201911225396A CN 110950939 B CN110950939 B CN 110950939B
Authority
CN
China
Prior art keywords
omp22
protein
group
mice
romp22
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911225396.9A
Other languages
Chinese (zh)
Other versions
CN110950939A (en
Inventor
冯旰珠
杜兴冉
姜明子
邓凯丽
陈思遐
林少清
黄榆珍
沈自燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
2nd Affiliated Hospital of Nanjing Medical University
Original Assignee
2nd Affiliated Hospital of Nanjing Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 2nd Affiliated Hospital of Nanjing Medical University filed Critical 2nd Affiliated Hospital of Nanjing Medical University
Priority to CN201911225396.9A priority Critical patent/CN110950939B/en
Publication of CN110950939A publication Critical patent/CN110950939A/en
Application granted granted Critical
Publication of CN110950939B publication Critical patent/CN110950939B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Abstract

The invention belongs to the application of Acinetobacter baumannii Omp 22; in particular to Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof; to be provided withA.bHighly conserved and strong immunogenicity Omp22 protein is taken as a target antigen, dominant B cell epitopes and T cell epitopes of Omp22 protein are screened by bioinformatics and immunology methods, dominant antigen epitope polypeptides are connected in series by 6-aminocaproic acid, and a recombinant multi-antigen epitope polypeptide rOmp22 is chemically synthesized, so that the recombinant multi-antigen epitope polypeptide rOmp22 is reservedA.bThe immunogenicity of Omp22 protein, avoiding the virulence of original structural protein, developing a safe and effective anti-tumor agentA.bNovel vaccine for reducing bacterial infectionsA.bInfection and prevalence in hospitals provide a new tool. Animal experiments show that the recombinant vaccine can induce organisms to generate obvious cellular immunity and humoral immunity reaction, has immune protection effect on acinetobacter baumannii infection, and is high in safety and convenient to use.

Description

Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof
Technical Field
The invention belongs to application of acinetobacter baumannii outer membrane protein Omp22, and particularly relates to acinetobacter baumannii (A), (B), (C) and C)Acinetobacter baumannii，A.b) Infection with immunoprotective effectA.bOmp22 recombinant multi-antigen epitope polypeptide and application thereof.
Background
Acinetobacter baumannii (A), (B), (C), (D)Acinetobacter baumannii，A.b) Is non-fermented gram-negative bacillus, belongs to conditional pathogenic bacteria, can be widely distributed in hospital environment and can survive for a long time, and can cause infection of respiratory system, digestive system, urinary system and central system and even blood stream infection, and morbidityThe rate, mortality and drug resistance are among the leading stages in gram-negative bacilli infection.A.bThe drug resistance mechanism of the biological membrane is complex, and comprises the generation of various inactivated enzymes, the loss of cell outer membrane proteins, the expression of an active efflux system, the variation of drug target positions, the drug resistance shielding of biological membranes and the like, even the drug resistance gene cassette can contain various drug resistance gene mobile elements so that various drug resistance mechanisms coexist, so that the drug resistance gene cassette can contain various drug resistance gene mobile elements to ensure that the various drug resistance mechanisms coexistA.bDrug resistance becomes a clinical problem, and sometimes even faces "no cure". Although it has been relevant for more than twenty yearsA.bMolecular epidemiological and preventive studies of drug resistance are widely conducted worldwide, but no breakthrough progress has been made so far. Therefore, from the immunoprotection perspectiveA.bPrevention and treatment studies of infection have been the direction of much attention of researchers in recent years.
In recent years, many students have a right ofA.bPassive immunizations were beneficially explored by developing small protein A antisera, phospholipase D antisera, 22-kDa outer membrane protein antisera, and outer membrane nuclease antisera, albeit againstA.bInfected mice have a certain protection effect, but non-antibody substances in the infected mice are easy to cause complications such as allergy, diarrhea and the like, and a passive immunization method does not have an active prevention effect and cannot effectively reduce the morbidity. Therefore, an efficient and safe method is developedA.bVaccines have become significant, and immunogenicity and safety are two key issues in vaccine development. McConne II et alA.bThe whole bacterium vaccine has stronger immunogenicity, but the preparation mainly takes formaldehyde inactivation as the main preparationA.bThe approach of whole-bacterium vaccines cannot be guaranteed to beA.bThe bacteria are completely inactivated and these are reactivatedA.bAfter the inoculation, the novel drug resistance can be caused, the blood stream infection can also be caused, and great potential safety hazards exist.A.bThe outer membrane of the bacteria is an important part of the action with a host, although the outer membrane contains rich antigen epitopes, only a few antigens can play a role in protective immune response, and the process for preparing the vaccine by extracting crude outer membrane protein from the whole bacteria is complex, the product purity is extremely low, and the vaccine is not suitable for mass production, so the outer membrane of the bacteria is simply used for preparing the vaccineA.bThe method of vaccination is not advisable. Fattahian et alA.bAntigen egg for bacterial biofilm formationAlthough the recombinant protein vaccine prepared from the white-biofilm-associated protein shows a certain protective effect, the vaccine is not suitable for clinical application because the antigen is single and the immune protective effect is extremely low, an adjuvant is usually added to enhance the immunity, and the coverage of the strain is narrow. Therefore, the antigen with high immunogenicity and avoiding the virulence of structural protein is determined to be preparedA.bThe key to the vaccine is.
Omp22 isA.bA highly conserved protein secreted in outer membrane vesiclesA.bPlays an important role in the pathogenic process, and simultaneously Omp22 has high-efficiency immunogenicity. Weiwei Huang and other researches show that the purified recombinant Omp22 protein can induce mice to generate specific antibodies, has a certain protective effect on mice attacked by Acinetobacter baumannii, and suggests that Omp22 can be used as a vaccineA.bThe bacteria are relatively ideal antigen protein. Many researchers think that it is idealA.bThe vaccine antigen can be generally expressed in the cell outer membranes of various bacterial strains and participates in the adhesion planting of bacteria, the coding sequence of the vaccine antigen is conservative, the protein structure is stable, the antigenicity is not damaged after recombination induction, and the solubility is high.A.bThe Omp22 protein is widely expressed in all clinical cases at presentA.bIsolated strains, having high homology and conservation, are preparedA.bThe vaccine is ideal candidate antigen protein. Omp22 is involvedA.bThe key protein of the metabolic process has certain toxicity, and if the full-length protein is singly used as vaccine antigen to immunize a host, certain harm exists. Therefore, the development of a subunit vaccine (i.e. retaining the epitope of the antigen, deleting the pathogenic fragment) against the epitope of Omp22 protein, which not only retains its corresponding immunogenicity but also avoids its harmfulness to the host, should be made resistantA.bPreferential strategy for infection.
Disclosure of Invention
The invention aims to provide a method for synthesizing Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide, which solves the problem that the prior research is carried outA.bVaccine is insufficient, and a safe and effective vaccine is constructedA.bA subunit vaccine.
The Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide constructed by the invention comprises 3B cell epitopes and 2T cell epitopes, and the specific sequences are as follows:
A.b Omp22 158-172：NIPLSQARAQSVKNY；
A.b Omp22 125-135：YATLDKVAQTL；
A.b Omp22 102-108：SVQLIMP；
A.b Omp22 178-188：VPSSRIDAQGY；
A.b Omp22 112-122：TFDTNKSNIKP；
the nucleotide sequences of the epitope amino acids are respectively as follows:
A.b Omp22 158-172：aacattccgc tgagtcaagc acgcgcacaa agcgttaaaa actat；
A.b Omp22 125-135：tacgcgaccc tggataaagt tgcgcaaacc ctg；
A.b Omp22 102-108：tctgttcagc tgattatgcc g；
A.b Omp22 178-188：gttccgagta gtcgtattga cgcacaaggt tac；
A.b Omp22 112-122：accttcgaca ccaacaaaag caacatcaaa ccg。
the epitope amino acid sequences can be connected in any combination by a linker, and the combination sequence does not influence the immune response. Amino acids commonly used as linkers include: { AHX } fluorenylmethoxycarbonyl-6-aminocaproic acid, { Beta-Ala } Beta-alanine, { Gly } glycine, { GABA } gamma-aminobutyric acid, { Ava } 5-aminopentanoic acid. The linker can be connected by single amino acid or repeated amino acid sequences. Such as- (AHX) n-, - (Beta-Ala) n-, - (Gly) n-, - (GABA) n-, - (Ava) n-where n is 1 or more. In fact, the sequence has no antigen reaction as long as it has a linking function, and does not affectA.bOmp22 recombinant multi-antigen epitope can be used as linker. Among them, { AHX } 6-aminocaproic acid was most used and the effect was also the best, and it was preferably recommended.
Preferably: the epitope polypeptides are connected in series through 6-aminocaproic acid, and the amino acid sequences are as follows:
niplsqaqsvknyxxaytldkvaqtlxsvqlimpxvpssridaqgyxtfdtnksnikp, where X is { AHX }.
The Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide is preparedA.bUse in subunit vaccines.
Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide in preparation of therapeuticA.bUse in antibodies.
The acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and the synthesis of each epitope polypeptide are synthesized by a chemical solid phase method, the purity of the polypeptide is high, the polypeptide is easy to obtain, and the polypeptide does not contain bacterial virulence protein or an amino acid sequence of a virulence structure, so that the potential risk of bacterial infection is avoided.
To be screened outA.bThe dominant antigen epitope polypeptides of Omp22 are connected through 6-aminocaproic acid, so that the linear structure of the recombinant multi-antigen epitope polypeptide rOmp22 can be maintained, and the epitope polypeptides are prevented from being folded and being incapable of exposing antigen sites.
Multiple antigen epitope polypeptide rOmp22 combined with Freund's adjuvant and adjuvant contrast component are used for immunizing female BALB/c mouse respectively for three times, and tracheal perfusion is performed after last immunizationA.bThe survival rate of mice in the standard strain ATCC19606 and rOmp22 groups is higher than that of the adjuvant group, the inflammation degree and bacterial load of lung tissues are lower than those of the adjuvant control group, and the recombinant multi-epitope polypeptide rOmp22 is shown to be directed againstA.bImmunoprotective effects of infection.
The conception of the invention is as follows: to be provided withA.bHighly conserved and strong immunogenicity Omp22 protein is used as a target antigen, dominant B cell epitopes and T cell epitopes of Omp22 protein are screened, dominant antigen epitope polypeptides are connected in series through 6-aminocaproic acid, and a recombinant multi-antigen epitope polypeptide rOmp22 is chemically synthesized, so that the recombinant multi-antigen epitope polypeptide rOmp22 is reservedA.bThe immunogenicity of Omp22 protein, the virulence of structural protein can be avoided, and a safe and effective anti-tumor agent is developedA.bNovel vaccine for reducing bacterial infectionsA.bInfection and prevalence in hospitals provides a new method and tool.
Advantageous effects
Compared with the prior art, the invention has the advantages that: construct aA.bNovel polyepitope polypeptides, both retainingA.bThe antigenicity of Omp22 protein avoids the toxic effect on hostTo construct a safe and effectiveA.bSubunit vaccine, for reductionA.bInfection and prevalence in hospitals provides a new method and tool.
Drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows the electrophoresis results of the recombinant expression vector pET28a-omp22 digested with BamHI + EcoRI, wherein lane M is DNA Marker, lane 1 is the control which is not digested, and lane 2 is the plasmid restriction map;
FIG. 2 isA.bOmp22 protein purification band; the protein Marker in lane M, the Omp22 protein in lane 1 and the Omp22 protein in lane 2, which were purified at 1ug and 2ug, respectively.
FIG. 3 is a drawing showingA.bAnd (3) screening results of dominant B cell epitopes and T cell epitopes of Omp22 protein. FIG. 3A shows that the indirect ELISA result of the B cell epitope peptide fragment of Omp22 protein and Omp22 immune mouse serum shows that the dominant B cell epitope of Omp22 protein is Omp 22B 2: NIPLSQARAQSVKNY, Omp 22B 3: YATLDKVAQTL, Omp 22B 4: SVQLIMP. FIG. 3B shows that the T cell epitope of Omp22 protein stimulates mouse spleen cells and IFN-. gamma.content in the culture supernatant. The dominant T cell epitopes Omp 22T 1: TFDTNKSNIKP and Omp 22T 3: VPSSRIDAQGY are screened out.
FIG. 4 is a mass spectrometry diagram of recombinant multiple antigen epitope polypeptide rOmp 22.
FIG. 5 is a liquid chromatogram of recombinant multiple antigen epitope polypeptide rOmp 22.
Figure 6 is the level of rmop 22 specific IgG expression.
FIG. 7 shows the IFN-r expression level of spleen cell supernatant of mice in rOmp22 immune group and control group.
FIG. 8 shows the perfusion of tracheaA.bThe survival rate, weight change, clinical score and lung tissue bacterial load of mice in an immune group and a control group within one week of the standard strain ATCC 19606. The survival rate of each group of mice is shown in the graph A, the survival rate of the rOmp 2240 ug immune group mice is 100%, and the survival rate of the normal saline control group mice and the Freund's adjuvant control group mice is 50%. FIG. B shows that the weight of the mice changed and decreased to the minimum 48 hours after the tracheal perfusion of each group of mice, and the body weight of the mice in the normal saline control groupThe weight loss is most obvious, the weight loss of the rOmp 2240 ug immune group mice is least, and the body weight is recovered to the body weight before challenge after 4-6 days. The graph C shows that the clinical symptoms are scored, the clinical symptoms of each group of mice are the heaviest within 24 hours after tracheal perfusion, the scores of the normal saline control group are the worst, and the phenomena of death and imminent death occur; the symptoms of the mice in the rOmp 2240 ug immune group are mild, only a part of the mice have slow action and hair fold expression, and the survival mice in each group return to normal after 6 to 7 days. And the figure D shows that the lung tissue homogenized bacterial load of the mice in the rOmp22 immune group is the lowest 24h after tracheal perfusion, and is obviously lower than that of the normal saline group and the adjuvant control group.
FIG. 9 shows tracheal perfusionA.bAnd (3) analyzing the pathological inflammation degree of lung tissue HE staining of mice in an immunized group and a control group after the standard strain ATCC 1960624 h. A picture is lung tissue of a normal saline control group mouse, and large-area solid change, trachea collapse and a large amount of inflammatory cell infiltration of the lung tissue can be seen; the B picture is the lung tissue of a Freund's adjuvant control group mouse, and a large amount of inflammatory cell infiltration, partial consolidation of the lung tissue and structural change of the trachea can be seen; the C picture is rOmp 2210 ug immune group mouse lung tissue, and the trachea structure collapse, cilium lodging and inflammatory cell infiltration can be seen; the D picture is lung tissue of an rOmp 2240 ug immune group mouse, the inflammation degree is relatively low, and most of the lung tissue is of a normal lung tissue structure; e picture is normal mouse lung tissue; and F is the result of semi-quantitative analysis of lung tissue inflammation area of each group of mice.
FIG. 10 shows tracheal perfusionA.bWithin one week of standard strain ATCC19606, the survival rate, body weight change, clinical score and lung tissue bacterial load of mice in a recombinant antigen epitope polypeptide rOmp22 immune group and an Omp22 full-length protein immune group. Panel a shows that there was no statistical difference in survival for each group of mice. One mouse dies in an Omp22 protein nasal drip immunization group, a subcutaneous injection immunization group and a recombinant multi-epitope polypeptide rOmp22 nasal drip immunization group respectively, and no mouse dies in an rOmp22 subcutaneous injection immunization group. Panel B shows that the weight change of the mice, 48 hours after tracheal perfusion challenge, was minimal in each group, and there was no statistical difference in the weight loss in each group. Panel C shows that there were no statistical differences in clinical symptom scores, the mice in each group had the heaviest clinical symptoms within 24 hours after tracheal perfusion, and individual mice had dead and moribund status,only some mice exhibited slow movement, hair fold expression, and the groups of surviving mice recovered to normal activity after 6-7 days. Panel D shows lung homogenate bacterial load, with no statistical difference in lung homogenate bacterial load for each group of mice 24h after tracheal perfusion.
FIG. 11 shows tracheal perfusionA.bAnd (3) analyzing pathological inflammation degree of lung tissue HE staining of mice in a standard strain ATCC 1960624 h post-recombinant antigen epitope polypeptide rOmp22 immune group and an Omp22 full-length protein immune group. A. B, drawing is recombinant multiple antigen epitope polypeptide rOmp22 nasal drip immunization group, subcutaneous injection immunization group mouse lung tissue HE staining pathology respectively; C. d, pictures are respectively the lung tissue HE staining pathology of mice in an Omp22 full-length protein nasal drip immunization group and a subcutaneous injection immunization group; and the E picture is the result of semi-quantitative analysis of the lung tissue inflammation area of each group of mice, and the pathological inflammation degree of each group of mice has no statistical difference.
FIG. 12 is a CCK-8 method for detecting the cytotoxicity of the recombinant multi-epitope polypeptide rOmp22 on human lung adenocarcinoma cells A549. As shown in the figure, rOmp22 and A549 cells with different concentrations are co-cultured for 6h, 24h and 48h, and have no obvious influence on cell proliferation, which indicates that the recombinant multi-antigen epitope polypeptide has no toxic effect in vitro.
FIG. 13 shows the sequencing result of pET28a-omp22, wherein A is the first page of the sequencing report and B is the second page of the sequencing report.
Detailed Description
The present invention will be described in more detail below with reference to the accompanying drawings by way of specific embodiments. The following examples are illustrative only, not limiting, and are not intended to limit the scope of the invention. The chemical reagents and instruments used in the present invention are commercially available without specific reference.
Example 1
A.bThe construction and screening of polypeptide sequences of B cell epitope and T cell epitope of Omp22 protein, and chemical synthesis of recombinant multi-epitope polypeptide rOmp 22. The method comprises the following specific steps:
4B cell epitopes and 4T cell epitopes of Acinetobacter baumannii Omp22 protein, wherein the amino acid sequences are respectively as follows:
A.bOmp22 175-182：GKGVPSSR；
A.bOmp22 158-172：NIPLSQARAQSVKNY；
A.bOmp22 125-135：YATLDKVAQTL；
A.bOmp22 102-108：SVQLIMP；
A.bOmp22 112-122：TFDTNKSNIKP；
A.bOmp22 153-164：GNDSINIPLSQ；
A.bOmp22 178-188：VPSSRIDAQGY；
A.bOmp22 204-215：EQNRRVEISIY。
entrust Nanjing Kingsrei Biotechnology GmbH chemical synthesis, purity is more than 85%, and C end of each epitope short peptide is coupled with BSA molecule;
A. constructing a recombinant plasmid pET28a-Omp22, transforming the recombinant plasmid into Escherichia coli BL21, obtaining Omp22 protein through steps of induction expression, purification and the like, and detecting the expression of the Omp22 protein in prokaryotic cells through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), wherein the result is shown in figure 1 and figure 2; the sequence of the recombinant plasmid pET28a-omp22 is shown in figure 13.
B. The Omp22 protein is used for immunizing mice, and the specific steps are as follows: SPF-grade female BALB/c mice at 6-8 weeks were divided into Omp22 protein-immunized groups, PBS control groups, and 6 mice per group. Fully emulsifying 100 ug/Omp 22 protein solution with Freund's adjuvant in equal volume, making total volume 200ul, sterilizing mouse back and thigh quadriceps, performing multipoint subcutaneous injection immunization, using Freund's complete adjuvant for the first immunization, using Freund's incomplete adjuvant for the second immunization, and immunizing once every two weeks for 3 times. One week after the last immunization, mouse sera were collected and spleens were isolated.
C. An indirect ELISA method for screening Omp22 protein B cell epitopes comprises the following specific steps: 4 candidate B cell epitope peptide fragments of Omp22 are coated on an ELISA (enzyme-linked immunosorbent assay) plate, the dosage of the ELISA plate is 20ug +100ul of coating buffer solution, each hole of a negative control group uses an isovolumetric PBS (phosphate buffer solution), each hole of a BSA (bovine serum albumin) control group uses an isovolumetric 1% BSA solution, each group is provided with 3 multiple holes, and the ELISA plate is coated overnight at 4 ℃. The liquid in the hole was reversed and the plate was washed 5 times. 100ul Omp22 protein immune mouse serum (1: 500 diluted with PBS) was added, incubated at 37 ℃ for 2h, and the plate was washed 5 times. Fresh diluted enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG 1:10000 diluted) was added at 100 ul/well, incubated at room temperature for 1h, and the plate was washed 5 times. TMB substrate was added, 100 ul/well, and developed in the dark at room temperature for 20 mins. A stop solution of 10% H2SO4 was added at 50 ul/well and the value read at OD 450. 3 dominant B cell epitopes are screened out according to the OD value, the screened B cell epitopes have strong hydrophilicity, strong surface accessibility and high antigen index, and are shown in figure 3A;
the amino acid sequences of the three B cell epitope polypeptides are respectively as follows:
A.bOmp22 158-172：NIPLSQARAQSVKNY；
A.bOmp22 125-135：YATLDKVAQTL；
A.bOmp22 102-108：SVQLIMP；
D. the method for screening the Omp22 protein T cell epitope by the double sandwich ELISA method comprises the following specific steps: spleen cells were collected from mice, isolated and cultured in 24-well plates at 1X 10 per well6Cells, total volume 1 ml. Stimulating spleen cells by using 20ug/ml of candidate T cell epitope, culturing for 72h, collecting cell supernatant, and detecting IFN-gamma level by using an ELISA method, wherein the steps are as follows: according to the set standard substance and the collected cell supernatant, 100 ul/hole are added in turn, each group is provided with three multiple holes, the incubation is carried out for 2h at room temperature, the plate is washed for 5 times, and the plate is patted dry. Adding 100ul of pre-diluted biotin-labeled antibody working solution per hole, incubating at room temperature for 1h, washing the plate for 5 times, and patting dry. Adding 100 ul/hole of HRP enzyme-labeled conjugate working solution, incubating for 20min at room temperature in the dark, washing the plate for 5 times, and drying by patting. Adding 100ul of TMB color developing agent per hole, and incubating for 20min at room temperature in the dark. Stop solution was added at 50 ul/well and data was read at OD450 nm. 2 dominant T cell epitopes are screened according to the level of the T cell epitope stimulating splenocytes to generate IFN-gamma, and the T cell epitope is shown in figure 3B;
the amino acid sequences of the two T cell epitopes are respectively:
A.bOmp22 178-188：VPSSRIDAQGY；
A.bOmp22 112-122：TFDTNKSNIKP。
E. the screened dominant B cell epitope and T cell epitope are connected in series through 6-aminocaproic acid to prepare Omp22 recombinant multi-antigen epitope polypeptide rOmp22, as shown in figure 4 and figure 5, and the amino acid sequences are as follows:
NIPLSQARAQSVKNY{AHX}YATLDKVAQTL{AHX}SVQLIMP{AHX}VPSSRIDAQGY{AHX}TFDTNKSNIKP
example 2
The detection of the immune effect of the recombinant multi-antigen epitope polypeptide rOmp22 comprises the following specific implementation steps:
female BALB/c mice are randomly divided into 4 groups in 6-8 weeks, each group comprises 6 mice, namely a recombinant multi-epitope polypeptide rOmp22 high dose group (40 ug/mouse) and an rOmp22 low dose group (10 ug/mouse), the groups are fully mixed and emulsified with Freund's adjuvant 1:1, a normal saline + adjuvant group and a normal saline control group are additionally arranged, the total volume is 200ul, subcutaneous injection is carried out for immunization, Freund's complete adjuvant is used for primary immunization, Freund's incomplete adjuvant is used for secondary immunization, and the immunization is carried out once in two weeks and three times in total. One week after the last immunization, mouse sera were collected and spleens were isolated.
The ELISA method for detecting the rOmp22 specific IgG expression level in the mouse serum comprises the following steps: the recombinant multi-epitope polypeptide rOmp 2220 ug +100ul coating buffer solution coats an ELISA plate and is coated overnight at 4 ℃. The liquid in the hole was reversed and the plate was washed 5 times. 100ul of serum (diluted 1:500 and diluted by PBS) freshly diluted by mice immunized in a high dose group of rOmp22 and a low dose group of rOmp22 are added respectively, 100ul of serum freshly diluted by mice immunized in a normal saline immunization group and a normal saline plus adjuvant immunization group are added in a control group, 5 multiple wells are arranged on the serum of each mouse, the mice are incubated for 2h at 37 ℃, and the plates are washed for 5 times. Fresh diluted enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG 1:10000 dilution) was added at 100 ul/well, incubated at room temperature for 1h, and the plate was washed 5 times. TMB substrate was added, 100 ul/well, and developed in the dark at room temperature for 20 mins. Adding 50 ul/hole 10% H2SO4 stop solution, reading the value at OD450, the result is shown in figure 6, the recombinant multi-antigen epitope polypeptide rOmp22 is combined with the antibody in the serum, and then the secondary antibody (HRP marked goat anti-mouse IgG) is used for color development, which indicates that the recombinant multi-antigen epitope polypeptide rOmp22 has antigen-antibody reaction with the immunized mouse serum, and the rOmp22 can induce the organism to generate humoral immune reaction after immunizing the mouse, SO as to generate rOmp22 specific antibody IgG.
ELISA method for detecting rOmp22 immune group and control group mouse spleen cell supernatantIFN- γ expression level: spleen cells were collected from mice, isolated and cultured in 24-well plates at 1X 10 per well6Cells, total volume 1 ml. 20ug/ml of recombinant multi-antigen epitope polypeptide rOmp22 stimulates spleen cells, the spleen cells are cultured for 72h, cell supernatant is collected, and IFN-gamma level is detected by ELISA method, the steps are as follows: adding 100 ul/hole in turn according to the set standard substance and the collected cell supernatant, setting five multiple holes in each group, incubating for 2h at room temperature, washing the plate for 5 times, and patting to dry. Adding 100 ul/hole of pre-diluted biotin-labeled antibody working solution, incubating for 1h at room temperature, washing the plate for 5 times, and patting dry. Adding 100 ul/hole of HRP enzyme-labeled conjugate working solution, incubating for 20min at room temperature in the dark, washing the plate for 5 times, and drying by patting. Adding 100ul of TMB color developing agent per hole, and incubating for 20min at room temperature in the dark. Stop solution was added at 50 ul/well and data was read at OD450 nm. Drawing a standard curve according to the standard substance and the OD450 value, calculating the IFN-r expression level of the spleen cell supernatant of each group of mice, wherein the result is shown in figure 7, the IFN-r level secreted in the spleen cell supernatant of the mice in the recombinant multi-antigen epitope polypeptide rOmp22 immune group is obviously higher than that of the control group, the IFN-r level in the spleen cell supernatant of the mice in the rOmp22 high-dose immune group is higher, and the result shows that the rOmp22 immune mice can induce the organism to generate cellular immune response and promote the secretion of Th1 type cell factor IFN-r.
Example 3
Recombinant multiple antigen epitope polypeptide rOmp22 pairA.bThe mouse pneumonia caused by the traditional Chinese medicine has an immune protection effect, and the specific implementation steps are as follows:
female BALB/c mice are randomly divided into 4 groups (10 mice in each group) in 6-8 weeks, namely a recombinant multi-antigen epitope polypeptide rOmp22 high-dose group (40 ug/mouse) and an rOmp22 low-dose group (10 ug/mouse), are mixed with Freund's adjuvant 1:1 to be fully emulsified, a normal saline + adjuvant group and a normal saline control group are additionally arranged, the total volume is 200ul, subcutaneous injection is carried out for immunization, Freund's complete adjuvant is used for primary immunization, Freund's incomplete adjuvant is used for secondary immunization, and immunization is carried out for three times in two weeks.
2 weeks after the last immunization, 50ul of half of the lethal dose was infused into the tracheaA.bStandard strain ATCC19606 (1X 10)8CFU containing 5% porcine mucin), and establishing a mouse acute pneumonia model.
24h after trachea perfusion, 4 mice are killed in each group, right lung tissues of the mice are weighed and prepared into homogenate, the homogenate is diluted according to a 1:10 gradient, 100ul of the homogenate is taken to a blood plate and is evenly coated, the blood plate is placed in a bacterial incubator at 37 ℃, and bacterial colony counting is carried out after 48 hours. The results are shown in fig. 8, and the colony number of the rOmp22 high-dose immunization group is obviously reduced compared with the adjuvant group and the normal saline control group.
After the left lung tissue section is sliced, 4% paraformaldehyde is used for fixing, HE staining is carried out, histopathological changes are observed, the result is shown in figure 9, and the lung tissue inflammation degree of a multi-epitope polypeptide rOmp22 immune group is obviously reduced compared with that of a control group.
Observing clinical symptoms, weight change and survival rate of each group of mice every day within 1 week after trachea perfusion; the results are shown in FIG. 10, and the rOmp22 high dose group mice have only mild clinical symptoms, no death and minimal weight loss, indicating that the high dose of recombinant multiple epitope polypeptide rOmp22 pairsA.bThe mouse pneumonia caused by the standard strain ATCC19606 has an immunoprotective effect.
Example 4
Recombinant multi-antigen polypeptide rOmp22 and Omp22 full-length protein pairA.bAnd comparing the immune protection effect of the induced pneumonia of the mice. The specific implementation steps are as follows:
female BALB/c mice at 6-8 weeks were randomly divided into 4 groups (10 mice per group), namely a recombinant multiple antigen epitope polypeptide rOmp22 nasal mucosa immune group, a rOmp22 subcutaneous injection immune group, a recombinant Omp22 full-length protein nasal mucosa immune group and an Omp22 full-length protein subcutaneous injection immune group. The immune dose is that each mouse is dripped or injected with rOmp22 polypeptide or Omp22 full-length protein 20ug through nasal mucosa, dissolved by normal saline, mixed with Freund's adjuvant 1:1 and fully emulsified, the total volume of the nasal mucosa administration group is 20 ul/mouse, and the total volume of the subcutaneous injection immune group is 200 ul/mouse. The primary immunization was performed with Freund's complete adjuvant, and the latter two immunizations were performed with Freund's incomplete adjuvant once in two weeks, three times in total.
2 weeks after the last immunization, 50ul of half of the lethal dose was infused into the tracheaA.bStandard strain ATCC19606 (1X 10)8CFU containing 5% porcine mucin), and establishing a mouse acute pneumonia model.
24h after trachea perfusion, 4 mice are killed in each group, right lung tissues of the mice are weighed and prepared into homogenate, the homogenate is diluted according to a 1:10 gradient, 100ul of the homogenate is taken to a blood plate and is evenly coated, the blood plate is placed in a bacterial incubator at 37 ℃, and bacterial colony counting is carried out after 48 hours. As a result, no statistical difference was found in the colony counts of the mice in each group.
After the left lung tissue is sliced, 4% paraformaldehyde is fixed, HE staining is carried out, histopathological changes are observed, the result shows that lung tissues of all groups of mice have slight solid change and inflammatory cell infiltration, and semi-quantitative analysis shows that the inflammatory degree of the lung tissues of all groups of mice has no statistical difference.
Within 1 week after tracheal perfusion, each group of mice was observed daily for clinical symptoms, weight change and survival rate. The results are shown in figure 11, and it is found that one mouse dies in the Omp22 protein nasal drip immunization group, the subcutaneous injection immunization group and the recombinant multi-epitope polypeptide rOmp22 nasal drip immunization group within one week after virus challenge, no mouse dies in the rOmp22 subcutaneous injection immunization group, and the survival rate of the mice in each group is not statistically different. The weight of the mice is reduced to the minimum 48 hours after the tracheal perfusion of the mice for attacking, and the weight reduction of the mice of each group has no statistical difference. The clinical symptom scores have no statistical difference, the clinical symptoms of each group of mice are the heaviest within 24 hours after tracheal perfusion, individual mice are in death and dying states, only part of mice have slow action and hair fold expression, and the activity of each group of surviving mice returns to normal after 6-7 days.
The results show that the recombinant multi-epitope polypeptide rOmp22 and the full-length protein Omp22 have immune protection effects on acinetobacter baumannii tracheal perfusion mice, and the immune protection effects have no statistical difference. The recombinant multi-epitope polypeptide rOmp22 nasal drop immunity and subcutaneous injection immunity are found, and the survival rate, the weight change, the clinical symptom score and the pathological inflammation degree of the mouse have no statistical difference with the full-length protein Omp22 nasal drop immunity and subcutaneous injection immunity. The immune protection effect of the recombinant multi-epitope polypeptide rOmp22 on the mouse acute pneumonia caused by acinetobacter baumannii ATCC19606 is not inferior to that of Omp22 full-length proteome.
Example 5
The CCK-8 method detects that the recombinant multi-antigen polypeptide rOmp22 has no toxic effect on human lung adenocarcinoma cells A549. The specific implementation steps are as follows:
when the A549 cells are cultured to the logarithmic phase, pancreatin digestive cells without EDTA are added, the cells are centrifuged at 1000rpm for 5min, the supernatant is discarded, 1ml of complete culture medium is added, and the cells are counted. 1640 complete Medium cells were diluted to 1.0X 106cells/ml, cells were plated in 96-well plates at 100ul per well (1.0X 10 contained)4cells), set up 1640 complete medium control wells without cells, and incubate overnight at 37 ℃ in a carbon dioxide-containing incubator.
Weighing 1mg of recombinant multi-antigen epitope polypeptide rOmp22, diluting 1640 complete culture medium to 800ug/ml, adding 1640 complete culture medium to be diluted to 400ug/ml, 200ug/ml, 100ug/ml and 50ug/ml in a multiple ratio manner, respectively taking 10ul of rOmp22 solution with corresponding concentration to stimulate A549 cells, and setting 5 multiple wells for each concentration. Incubation in a carbon dioxide-containing incubator at 37 ℃.
After the recombinant multi-epitope polypeptide rOmp22 and A549 cells are co-cultured for 6 hours, 24 hours or 48 hours, 10ul of CCK-8 reagent is added into each well, the wells are incubated for 1 hour in a dark place, and data are read at OD450 nm. The ratio of cells per well to control wells was calculated from OD450 nm. The results are shown in FIG. 12, which shows that the recombinant multiple epitope polypeptide rOmp22 has no toxicity to A549 cells.
The above description is only for the preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Sequence listing
<110> second subsidiary hospital of Nanjing medical university
<120> Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
Asn Ile Pro Leu Ser Gln Ala Arg Ala Gln Ser Val Lys Asn Tyr
1 5 10 15
<210> 2
<211> 11
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
Tyr Ala Thr Leu Asp Lys Val Ala Gln Thr Leu
1 5 10
<210> 3
<211> 7
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
Ser Val Gln Leu Ile Met Pro
1 5
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
Val Pro Ser Ser Arg Ile Asp Ala Gln Gly Tyr
1 5 10
<210> 5
<211> 11
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 5
Thr Phe Asp Thr Asn Lys Ser Asn Ile Lys Pro
1 5 10
<210> 6
<211> 45
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 6
aacattccgc tgagtcaagc acgcgcacaa agcgttaaaa actat 45
<210> 7
<211> 33
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 7
tacgcgaccc tggataaagt tgcgcaaacc ctg 33
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 8
tctgttcagc tgattatgcc g 21
<210> 9
<211> 33
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 9
gttccgagta gtcgtattga cgcacaaggt tac 33
<210> 10
<211> 33
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 10
accttcgaca ccaacaaaag caacatcaaa ccg 33
<210> 11
<211> 59
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 11
Asn Ile Pro Leu Ser Gln Ala Arg Ala Gln Ser Val Lys Asn Tyr Xaa
1 5 10 15
Tyr Ala Thr Leu Asp Lys Val Ala Gln Thr Leu Xaa Ser Val Gln Leu
20 25 30
Ile Met Pro Xaa Val Pro Ser Ser Arg Ile Asp Ala Gln Gly Tyr Xaa
35 40 45
Thr Phe Asp Thr Asn Lys Ser Asn Ile Lys Pro
50 55

Claims (2)

1. Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide, which is characterized in that: the amino acid sequence is as follows:
NIPLSQARAQSVKNY { AHX } YATLDKVAQTL { AHX } SVQLIMP { AHX } VPSSRIDAQGY { AHX } TFDTNKSNIKP, wherein AHX is fluorenylmethoxycarbonyl-6-aminocaproic acid.
2. The use of the polypeptide of claim 1 in the preparation of an acinetobacter baumannii subunit vaccine.
CN201911225396.9A 2019-12-04 2019-12-04 Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof Active CN110950939B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911225396.9A CN110950939B (en) 2019-12-04 2019-12-04 Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911225396.9A CN110950939B (en) 2019-12-04 2019-12-04 Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof

Publications (2)

Publication Number Publication Date
CN110950939A CN110950939A (en) 2020-04-03
CN110950939B true CN110950939B (en) 2022-05-17

Family

ID=69979729

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911225396.9A Active CN110950939B (en) 2019-12-04 2019-12-04 Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof

Country Status (1)

Country Link
CN (1) CN110950939B (en)

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104725492B (en) * 2015-04-18 2017-08-25 吉林大学 A kind of Acinetobacter bauamnnii surface antigen S urA1 with immune protective
CN104877019B (en) * 2015-04-24 2018-06-08 中国人民解放军第三军医大学 Acinetobacter bauamnnii assumes the albumen and preparation method and application of albumin A 1S_1523
CN104861050B (en) * 2015-04-24 2018-06-08 中国人民解放军第三军医大学 Acinetobacter bauamnnii zinc relies on Gly-Lys-Ala-Phe-Val-Lys-Lys 1S_1610 albumen and its preparation method and application
CN105861402B (en) * 2015-07-20 2020-02-14 中国医学科学院医学生物学研究所 Novel vaccine form for presenting heterologous proteins on the surface of outer membrane vesicles of E.coli
CN105111288A (en) * 2015-07-24 2015-12-02 中国医学科学院医学生物学研究所 Acinetobacter baumannii subunit vaccine antigen protein, and applications thereof
CN105497884B (en) * 2015-12-23 2019-11-15 中国医学科学院医学生物学研究所 Application of the outer membrane protein Omp22 as Acinetobacter bauamnnii vaccine target spot
CN105582531B (en) * 2016-01-27 2020-06-12 中南大学湘雅医院 Acinetobacter baumannii combined subunit protein vaccine and preparation method thereof
CN108503698B (en) * 2018-05-24 2021-03-05 中国人民解放军军事科学院军事医学研究院 Immunogenicity of Acinetobacter baumannii Ata protein

Also Published As

Publication number Publication date
CN110950939A (en) 2020-04-03

Similar Documents

Publication Publication Date Title
US11020468B2 (en) Compositions, methods and therapies for administering antigen peptide
CN105934441A (en) A novel sars immunogenic composition
CN101955545B (en) Multi-target recombination gene and application of protein thereof in preventing and treating infection of helicobacter pylori
CN105106945B (en) A kind of helicobacter pylori tetravalence virulence factor polyepitope vaccines and preparation method thereof
Wang et al. Oral immunization with recombinant Lactococcus lactis expressing the hemagglutinin of the avian influenza virus induces mucosal and systemic immune responses
Yang et al. Subunit vaccines for Acinetobacter baumannii
CN104861050B (en) Acinetobacter bauamnnii zinc relies on Gly-Lys-Ala-Phe-Val-Lys-Lys 1S_1610 albumen and its preparation method and application
Zhang et al. Immune responses to orally administered recombinant lactococcus lactis expressing multi-epitope proteins targeting M cells of foot-and-mouth disease virus
Wang et al. Evaluation of the immune response afforded by combined immunization with orf virus DNA and subunit vaccine in mice
Chen et al. Recombinant PAL/PilE/FlaA DNA vaccine provides protective immunity against Legionella pneumophila in BALB/c mice
CN110950939B (en) Acinetobacter baumannii Omp22 recombinant multi-antigen epitope polypeptide and application thereof
CN106967174B (en) Application of the polypeptide amalgamation protein GWP in the immunoprotection of anti-rickettsia rickettsii
CN102058881A (en) Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof
CN105037561B (en) A kind of fusion protein CMFO and its application
CN103450353B (en) Application of protein AIG_01780 in Rickettsia rickettsii resistant immune protection
CN109369809A (en) Multi-epitope antigen and preparation method thereof and the application in the drug of preparation prevention and treatment chlamydia psitacci infection
CN113248578B (en) Novel coronavirus (2019-nCoV) recombinant antigen and polyclonal antibody
CN103483430B (en) Application of protein A1G_07050 to Rickettsia rickettsii-resisting immunoprotection
CN106075416A (en) The design of a kind of novel Echinococcus moltilocularis subunit vaccine, preparation method and application
He et al. Recombinant Mip-PilE-FlaA dominant epitopes vaccine candidate against Legionella pneumophila
CN105753992A (en) Pseudomonas aeruginosa recombinant protein Vac11 as well as preparation method and application
CN105732817A (en) PA (pseudomonas aeruginosa) recombinant protein Vac33, as well as preparation method and application thereof
CN105732778A (en) Pseudomonas aeruginosa recombinant protein OprL as well as preparation method and application thereof
CN101961487A (en) Echinococcosis granulosa gene engineering vaccine candidate P-29
CN105198982B (en) IL-6-based epitope and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant