CN111744003A - Application of chemotactic factor CX3CL1 in preparation of vaccine and helicobacter pylori vaccine - Google Patents
Application of chemotactic factor CX3CL1 in preparation of vaccine and helicobacter pylori vaccine Download PDFInfo
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Abstract
The invention provides an application of a chemotactic factor CX3CL1 in preparation of a vaccine and a helicobacter pylori vaccine, and relates to the technical field of vaccines. The application of the chemokine CX3CL1 in preparing the vaccine is beneficial to improving the durability and the effectiveness of the vaccine. The helicobacter pylori vaccine provided by the invention comprises a helicobacter pylori antigen and a chemotactic factor CX3CL1, and solves the problems that the existing helicobacter pylori vaccine is poor in effectiveness and durability and insufficient in recruitment of an excited effective response to a gastric mucosa part.
Description
Technical Field
The invention relates to the technical field of vaccines, in particular to application of a chemotactic factor CX3CL1 in preparation of a vaccine and a helicobacter pylori vaccine.
Background
Helicobacter pylori (h. pylori) is a gram-negative bacterium that colonizes gastric mucosa and causes a range of diseases including gastritis, gastric ulcer, gastric mucosa-associated lymphomas (MALT), and even gastric cancer. The world health organization in 1994 has identified helicobacter pylori as a class I carcinogen for gastric cancer. Epidemiological surveys have shown that H.pylori infects more than half of the world's population, and is particularly serious in developing countries.
The triple therapy of combining bismuth and antibiotics is the main method for treating helicobacter pylori infection at present, but the treatment method has the defects of easy antibiotic tolerance, poor patient compliance, easy relapse and the like. In the face of pathogenic microbial infections, vaccines are probably the most effective means of immunological control. At present, helicobacter pylori vaccine research is widely carried out all over the world, from traditional vaccines in a whole-bacterium form to novel vaccines in a subunit protein or DNA form, the types are various, and the vaccine has a good animal protection effect, but most of the vaccines are still in a preclinical research stage.
CD4T lymphocytes play an important role in combating H.pylori infection. However, protective CD4T cells elicited by H.pylori need to be recruited to the local gastric mucosa in order to effectively exert immunoprotection against H.pylori infection. In the naturally infected state, the immune response elicited by H.pylori is not sufficient to effectively eliminate colonization of the gastric mucosa, leaving many H.pylori infected individuals with bacterial colonization for decades or for life.
The protection rate of the existing helicobacter pylori vaccine needs to be further improved. For example, the oral recombinant helicobacter pylori vaccine developed by third-military medical science has completed all human clinical trial researches and obtained national new drug certificate of class 1.1, becomes the first approved helicobacter pylori vaccine in the world, and greatly promotes the development of the vaccine. The phase III clinical test result of the vaccine shows that: the protection rate in the first year was 71.8% (95% CI 48.2-85.6), but the second year was a sharp decrease to 55.0% (95% CI 0.9-81.0). Thus, the effectiveness and persistence of H.pylori vaccines remains to be further improved. The problem of insufficient local recruitment of the effective response elicited by current H.pylori vaccines to the gastric mucosa is also prevalent.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide the application of the chemokine CX3CL1 in the preparation of vaccines, and the chemokine CX3CL1 is applied to the preparation of vaccines, so that the durability and the effectiveness of the vaccines are improved.
The second purpose of the invention is to provide a helicobacter pylori vaccine, which alleviates the problems of poor effectiveness and durability of the conventional helicobacter pylori vaccine and insufficient recruitment of an excited effective response to the local gastric mucosa.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention, the present invention provides the use of the chemokine CX3CL1 in the preparation of a vaccine.
Preferably, the vaccine comprises a helicobacter pylori vaccine.
According to another aspect of the present invention, there is also provided a helicobacter pylori vaccine comprising a helicobacter pylori antigen and the chemokine CX3CL 1.
Preferably, the helicobacter pylori antigen includes one or more of a natural protein, a recombinant protein, a polypeptide, and a nucleic acid.
Preferably, the helicobacter pylori antigen comprises one or more of adenosine 5-phosphate dehydrogenase, type II citrate synthase, and urease B subunit.
Preferably, the amino acid sequence of the adenosine 5-phosphate dehydrogenase is shown as SEQ ID NO. 1;
and/or the amino acid sequence of the type II citrate synthase is shown as SEQ ID NO. 2;
and/or the amino acid sequence of the urease B subunit is shown as SEQ ID NO. 3.
Preferably, the chemokine CX3CL1 comprises the abcam recombinant chemokine CX3CL1 ab240868.
Preferably, the helicobacter pylori vaccine comprises any one combination of the following (a) to (g):
(a) adenosine 5-phosphate dehydrogenase and chemokine CX3CL 1;
(b) type II citrate synthase and chemokine CX3CL 1;
(c) urease B subunit and chemokine CX3CL 1;
(d) adenosine 5-phosphate dehydrogenase, type II citrate synthase and chemokine CX3CL 1;
(e) 5-adenosine phosphate dehydrogenase, urease B subunit and chemokine CX3CL 1;
(f) type II citrate synthase, urease B subunit, and chemokine CX3CL 1;
(g) 5-phosphoadenosine dehydrogenase, type II citrate synthase, urease B subunit and chemokine CX3CL 1.
Preferably, the vaccine further comprises a vaccine adjuvant.
Preferably, the helicobacter pylori vaccine is a recombinant antigen vaccine, a viral vector vaccine or a gene vaccine.
Compared with the prior art, the invention has the following beneficial effects:
the chemokine CX3CL1 provided by the invention can be applied to preparation of vaccines, and can recruit effect memory CD4T cells through CX3CL1-CX3CR1, thereby being beneficial to generating durable immune protection on organisms after the vaccines are immunized. CX3CL1 can also promote Th1, Th2 and Th17 effector cell responses and reduce the response of regulatory T cells, so as to raise the immunogenicity of vaccine and raise the immune response level.
The helicobacter pylori vaccine provided by the invention has the advantages that by using the chemotactic factor CX3CL1 and the helicobacter pylori antigen together, the planting amount of the helicobacter pylori can be obviously reduced, the number and the cell proportion of CD4T cells in gastric mucosa are improved, particularly the proportion of effect memory CD4T cells is improved, the proportion of suppressive Tregs is reduced, the vaccine can effectively play the immune protection role of resisting helicobacter pylori infection, and the durability of the vaccine is improved; meanwhile, the vaccine can promote the response of Th1, Th2 and Th17 effector cells, reduce the response of Treg cells, improve the immune effect of the helicobacter pylori vaccine and has better effectiveness.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the quantitative determination of helicobacter pylori in gastric mucosa of mice after immune challenge experiment;
FIG. 2 shows the number of CD4T cells in gastric mucosa after immune challenge experiment;
FIG. 3 shows the ratio of gastric mucosal CD4T cells after immune challenge experiment;
FIG. 4 shows the gastric mucosal IFN-. gamma.IL-4, IL-17A and Foxp3 mRNA levels after challenge immunization;
FIGS. 5 and 6 show effector memory CD4T (CD 3) chemotactic to gastric mucosa after immune challenge experiments+CD4+CX3CR1+CD25-CD44+CD69-CCR7-T) cells;
FIG. 7 is CD25 of CD4T cells chemotactic to gastric mucosa after immune challenge experiment-The proportion of CD4T cells;
fig. 8 shows the proportion of effector memory CD4T cells among CD4T cells chemotactic to the gastric mucosa after immune challenge experiments.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, the invention provides the use of the chemokine CX3CL1 in the preparation of a vaccine. The chemotactic factor is a cytokine with chemotactic effect on cells, the chemotactic factor CX3CL1 is a CX3C chemokine family member, CX3CR1 is the only specific receptor of CX3CL1, and the CX3CL1-CX3CR1 pathway can recruit effector memory CD4T cells, thereby being beneficial to the long-lasting immune protection of the body after the vaccine is immunized. CX3CL1 can also promote Th1, Th2 and Th17 effector cell responses and reduce the response of regulatory T cells, so as to raise the immunogenicity of vaccine and raise the immune response level. Therefore, the chemokine CX3CL1 is applied to preparing the vaccine, and the effectiveness and the durability of the vaccine can be improved.
Experiments show that after an antigen derived from helicobacter pylori and a chemotactic factor CX3CL1 are used in combination for immunizing a mouse and the helicobacter pylori is used for intragastric lavage, an immune group combined with the chemotactic factor CX3CL1 can obviously reduce the planting amount of the helicobacter pylori, improve the number and the cell proportion of CD4T cells in gastric mucosa, particularly improve the proportion of effector memory CD4T cells and reduce the proportion of suppressive Tregs; meanwhile, after the antigen of the helicobacter pylori and the chemotactic factor CX3CL1 are used in combination for immunizing a mouse, the mRNA levels of IFN-gamma, IL-4, IL-17A and Foxp3 in the gastric mucosa of the mouse are detected, and the mRNA levels of the IFN-gamma, the IL-4 and the IL-17A are all increased, while the mRNA level of the Foxp3 is reduced, which shows that the chemotactic factor CX3CL1 promotes the response of Th1, Th2 and Th17 effector cells and reduces the response of Treg cells. From the foregoing, it can be seen that the chemokine, CX3CL1, may be used in at least some alternative embodiments to prepare a helicobacter pylori vaccine containing a helicobacter pylori antigen.
Based on the synergistic effect of the chemotactic factor CX3CL1 and the helicobacter pylori antigen, the invention also provides a helicobacter pylori vaccine, and the helicobacter pylori vaccine comprises the helicobacter pylori antigen and the chemotactic factor CX3CL 1. Through the synergistic effect of the chemotactic factor CX3CL1 on helicobacter pylori antigens, the helicobacter pylori vaccine provided by the invention can improve the number of CD4T cells in gastric mucosa, particularly can recruit effect memory CD4T cells, and improves the durability of the vaccine; the helicobacter pylori vaccine provided by the invention has better effectiveness by improving the response of Th1, Th2 and Th17 effector cells and reducing the response of Treg cells.
In the present invention, the helicobacter pylori antigen is a substance capable of inducing the immune system of the body to produce an immune response, and these substances include substances derived from natural helicobacter pylori; or a substance artificially synthesized or artificially modified to contain an epitope of helicobacter pylori; or capable of expressing a precursor substance containing an epitope of helicobacter pylori. Examples of the helicobacter pylori antigen include, but are not limited to, proteins or polypeptides having immunogenicity isolated from natural helicobacter pylori; a fusion protein which is transformed by genetic engineering and has helicobacter pylori immunogenicity; artificially synthesized polypeptide containing helicobacter pylori epitope; the helicobacter pylori antigen may also be a nucleic acid, and the term "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of nucleic acids include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acid can be a nucleic acid molecule with self immunogenicity, and can also be a precursor substance for expressing a protein, a fusion protein or a polypeptide containing helicobacter pylori epitope. Therefore, the present invention also does not limit the specific form of the helicobacter pylori vaccine, which may be, but is not limited to, a recombinant antigen vaccine, a viral vector vaccine or a gene vaccine.
In some preferred embodiments, the helicobacter pylori antigen comprises one or more of adenosine 5' -monophosphate dehydrogenase (IMPDH), type II citrate synthase (CS II), and urease B subunit (urea subunit beta, urea B). The 5-adenosine phosphate dehydrogenase, II type citrate synthase and urease B subunit are taken as helicobacter pylori antigens, and the immunogenicity is better compared with other antigens derived from the helicobacter pylori antigens. The 5-adenosine phosphate dehydrogenase, II-type citrate synthase and urease B subunits are independent protein or polypeptide from natural pyloric spirobacterium, fusion protein after genetic engineering, artificially synthesized polypeptide containing pyloric spirobacterium antigen epitope, nucleic acid capable of expressing the three matters, etc.
In some preferred embodiments, the adenosine 5-phosphate dehydrogenase, type II citrate synthase, and urease B subunits are each independently a protein, the amino acid sequence of the adenosine 5-phosphate dehydrogenase preferably being as set forth in SEQ ID No. 1; the amino acid sequence of the type II citrate synthase is preferably shown as SEQ ID NO. 2; the amino acid sequence of the urease B subunit is preferably shown as SEQ ID NO. 3.
In the present invention, the chemokine CX3CL1 includes a naturally derived chemokine CX3CL1, or a recombinant chemokine CX3CL1 produced by an expression system through genetic engineering, or a precursor substance capable of expressing the chemokine CX3CL1, such as a nucleic acid capable of expressing the chemokine CX3CL 1. The chemokine CX3CL1 preferably comprises the abcam Recombinant chemokine CX3CL1 ab240868(Recombinant mouse CX3CL1 protein (active) (ab 240868)).
When the chemotactic factor CX3CL1 and the helicobacter pylori antigen are used in combination, the chemotactic factor CX3CL1 can play a role in enhancing the immunity of the helicobacter pylori antigen, so the specific dosage of the helicobacter pylori antigen and the chemotactic factor CX3CL1 in the helicobacter pylori vaccine is not limited, and the specific dosage of the helicobacter pylori antigen and the chemotactic factor CX3CL1 can be the immunological effective dosage acceptable in the field. In some embodiments, exemplified by the immunization of mice, the ratio of the helicobacter pylori protein antigen to the recombinant chemokine CX3CL1 is 4:1 by mass, specifically 100. mu.g helicobacter pylori antigen per mouse, and 125. mu.g recombinant chemokine CX3CL per mouse. When the helicobacter pylori antigen contains a plurality of antigens, the amount of each antigen is the same, i.e., each antigen is mixed in an equal ratio and then the mouse is immunized in a dose of 100. mu.g/mouse.
In some preferred embodiments, the immunologically active substance of the helicobacter pylori vaccine is a combination of the chemokine CX3CL1 and at least one of adenosine 5-phosphate dehydrogenase, type II citrate synthase and urease B subunit, and specifically may be any one of the following combinations (a) to (g):
(a) adenosine 5-phosphate dehydrogenase and chemokine CX3CL 1;
(b) type II citrate synthase and chemokine CX3CL 1;
(c) urease B subunit and chemokine CX3CL 1;
(d) adenosine 5-phosphate dehydrogenase, type II citrate synthase and chemokine CX3CL 1;
(e) 5-adenosine phosphate dehydrogenase, urease B subunit and chemokine CX3CL 1;
(f) type II citrate synthase, urease B subunit, and chemokine CX3CL 1;
(g) 5-phosphoadenosine dehydrogenase, type II citrate synthase, urease B subunit and chemokine CX3CL 1.
In some alternative embodiments, the helicobacter pylori vaccine may further comprise conventional adjuvants acceptable in the art to further enhance the immunogenicity, stability, or shelf life of the helicobacter pylori vaccine, and the like. The adjuvant preferably comprises a vaccine adjuvant, and the vaccine adjuvant can nonspecifically enhance the specific immune response of an organism to an antigen, induce the organism to generate long-term and efficient specific immune response, and improve the immunogenicity of the vaccine.
Examples
1. Animal immunization and challenge experimental scheme
Experimental animals: female BALB/c mice, 6-8 weeks old.
Grouping: IMPDH, IMPDH + CX3CL1, CSII + CX3CL1, urea + CX3CL1, IMPDH + CSII + urea + CX3CL 1. Helicobacter pylori antigen 100. mu.g/mouse (if helicobacter pylori antigen contains multiple antigens, the antigens are mixed in equal proportion), CX3CL 125. mu.g/mouse, and PBS control with equal amount. Wherein the amino acid sequence of IMPDH is shown in SEQ ID NO. 1; the amino acid sequence of CSII is shown in SEQ ID NO. 2; the amino acid sequence of UreB is shown in SEQ ID NO. 3; the chemokine CX3CL1 is a commercial product, Recombinant mouse CX3CL1 protein (active) (ab240868), which can be referred to https:// www.abcam.com/Recombinant-mouse-CX3CL 1-protein-active-ab240868.html.
Adjuvant: freund's adjuvant, 100. mu.l/tube.
The immunization mode comprises the following steps: antigen + freund's adjuvant, injected subcutaneously. CX3CL1, i.e. intraperitoneal injection.
Immunization volume: 200 μ l/mouse.
Immunization protocol: subcutaneous immunization was performed 3 times ( weeks 0, 2, 4). The first time, the second time and the third time, complete Freund's adjuvant is used, incomplete Freund's adjuvant is used. Ip injection of CX3CL1 was performed 6 times (once per week from week 3).
One week after the last immunization (i.e., week 5), 1.0 × 109CFU H.pylori was gavaged once a day for 4 consecutive days. Mice were sacrificed at 4 weeks after challenge, and the quantitative amount of helicobacter pylori, pathological lesions, CD4T cell responses in the gastric tissues of the mice were examined to analyze the immunoprotection effect.
2. Quantitative detection of helicobacter pylori in gastric mucosa of mouse
The fixed planting amount of helicobacter pylori on gastric mucosa of the mouse is detected by a Real-time quantitative Real-time RT-PCR method.
(1) After the mice were euthanized, the stomach was dissected and removed.
(2) The stomach tissue was cut into pieces with surgical scissors, and 1ml of sterile physiological saline was added.
(3) Further homogenizing the stomach tissue by a homogenizer at low temperature. Then extracting the helicobacter pylori DNA by using a bacterial genome DNA extraction kit. The homogenate was centrifuged at high speed and the supernatant was discarded.
(4) The pellet was resuspended in tissue debris by adding 200. mu.l of GA buffer. Then 20. mu.l of protease K was added and mixed well.
(5) Adding GB buffer solution, shaking and mixing uniformly.
(6) Incubation at 70 ℃ for 10 min, the solution becomes clear and centrifuged instantaneously.
(7) Adding 220 μ l of absolute ethyl alcohol, shaking for mixing, separating out flocculent precipitate, and centrifuging instantly.
(8) The whole mixture was transferred to a centrifugal column CB3 and centrifuged at high speed (13200g) for 1 minute.
(9) After washing with buffer solution GD and PW in sequence, transferring the centrifugal column into a new centrifugal tube and airing.
(10) Then, 200. mu.l of deionized water was added thereto, and the mixture was allowed to stand at room temperature for 2 minutes. Centrifuging at high speed for 1 minute, and collecting filtrate which is the water solution of the target DNA.
(11) And taking 2 mul of the DNA solution and carrying out quantitative detection on the helicobacter pylori 16S rDNA by an RT-PCR method. A helicobacter pylori 16S rDNA standard substance with different concentration gradients is used as a standard curve, and a positive control and a negative control are simultaneously set. 3 duplicate wells were set for each condition, and the average was taken as the DNA concentration of the test sample. The test results were multiplied by the dilution factor to obtain the amount of helicobacter pylori colonized in the stomach of each mouse. The final results are expressed logarithmically (log 10).
The primers and probes used in Real-time RT-PCR are shown in the following table.
The Real-time PCR reaction system is as follows.
| Reagent | Dosage of |
| Premix Ex Taq(2×) | 12.5μl |
| sense primer(10μM) | 0.5μl |
| anti-sense primer(10μM) | 0.5μl |
| H.pylori 16s probe(5μM) | 1μl |
| DNA template | 2μl |
| Deionized water | 8.5μl |
| Total volume | 25μl |
The Real-time PCR reaction conditions were as follows.
3. Mouse gastric mucosal lymphocyte isolation
Mouse stomach tissue was dissected along the greater and lesser curvature of the stomach and gently washed 2 times with sterile PBS to remove food debris. Then, the cells were incubated in 10ml of Hank's balanced salt solution (HBSS, Ca-free, My-free) containing 1mM Dithiothreitol (DTT), 1mM ethylenediaminetetraacetic acid (EDTA), and 2% Fetal Calf Serum (FCS) at 37 ℃ for 45 min. The resulting mixture was then passed through a sterile steel mesh to remove undigested tissue to obtain a single cell suspension. The digested single cell suspension was washed twice with sterile PBS and used.
4. Mouse gastric mucosa CD4T cell phenotype detection
The lymphocyte suspension separated above was stained with a fluorescently labeled monoclonal antibody. The antibodies used were APClabeled anti-mouse CD3, APC/Cy7 labbeled anti-mouse CD4, PerCP/Cy5.5 labeledanti-mouse CX3CR1, FITC labeled anti-mouse CD25, PE/Cy7 labeled anti-mouse CD44, BV421 labeled anti-mouse CD69 and PE labeled anti-mouse CCR7 antibodies. After staining, the cells were washed with PBS, resuspended in PBS and examined by flow cytometry.
5. Detection of mouse gastric mucosa CD4T cell response level
Mouse gastric mucosa IFN-gamma, IL-4, IL-17A and Foxp3 mRNA levels were detected using real-time fluorescent quantitative PCR methods.
RNA was extracted from the isolated gastric mucosal lymphocytes using a total RNA extraction kit (steps as described in the specification).
(1) The cell pellet was dissolved in the lysate RZ and allowed to stand at room temperature for 5 minutes to completely separate the nucleic acid-protein complex.
(2) Then, 200. mu.l of chloroform was added thereto, followed by shaking vigorously and mixing them for 15 seconds, followed by standing at room temperature for 3 minutes.
(3) Then, at 4 ℃, 13200g is centrifuged for 10 minutes to obtain three layers: a colorless aqueous phase, an intermediate layer and a yellow organic phase. The colorless aqueous phase was transferred to a new tube for further operation.
(4) Adding 0.5 volume of absolute ethyl alcohol into the colorless aqueous phase, mixing uniformly, and transferring into an adsorption column CR 3.
(5) Then, the mixture is centrifuged for a short time to adsorb RNA, and the waste liquid is discarded.
(6) The protein was washed out with buffer RD and the impurities were washed out with rinsing solution RW.
(7) The column was transferred to a new centrifuge tube and air dried.
(8) Then, 50. mu.l of deionized water was added thereto, and the mixture was allowed to stand at room temperature for 2 minutes.
(9) And centrifuging at high speed for 1 minute, and collecting filtrate which is the aqueous solution of the target RNA.
The RNA was reverse transcribed into cDNA using a reverse transcription kit under the following reaction conditions.
| Temperature of | Time of day | |
| 42℃ | 45min | |
| 95 | 5min | |
| 4℃ | keep |
Based on the cDNA, the expression levels of IFN-gamma and IL-17A are detected by a SYBR green incorporation method Real-time RT-PCR. Setting positive control blank and negative control blank, and taking beta-actin as reference gene. Three duplicate wells were set for each condition and the average was taken as the final result.
The primers used in RT-PCR are as follows.
The Real-time PCR reaction system is as follows.
| Reagent | Dosage of |
| SYBR Green Realtime PCR Master Mix | 12.5μl |
| sense primer(10μM) | 0.5μl |
| anti-sense primer(10μM) | 0.5μl |
| cDNA template | 2μl |
| Deionized water | 9.5μl |
| Total volume | 25μl |
The Real-time PCR reaction conditions were as follows.
The experimental results are shown in fig. 1 to 8:
FIG. 1 shows the determination of the colonization of helicobacter pylori of the gastric mucosa after immunological challenge, in order to evaluate whether the combined use of CX3XL1 enhances the immunoprotective effect. As can be seen from FIG. 1, the test group immunized with CX3CL1 in combination with H.pylori antigen showed a lower colonization amount of H.pylori in gastric mucosa than the test group immunized with H.pylori antigen alone, such as the IMPDH group and the IMPDH + CX3CL1 group, which showed a lower colonization amount of H.pylori, indicating a stronger immune protection function, and similarly, the results of several other pairs of comparison also indicate that CX3CL1 enhances the immune protection function of H.pylori antigen.
FIGS. 2 and 3 show the number and ratio of mouse gastric mucosal CD4T cells evaluated in the immune challenge experiment. Numerous studies have demonstrated that CD4T cells play an important protective role against H.pylori infection. In contrast, it was found that the combined use of the H.pylori antigen CX3CL1 recruited more CD4T cells to the gastric mucosa.
FIG. 4 shows the RT-PCR method to measure the levels of IFN-r, IL-17A, IL-4 and Foxp3 elicited by mouse gastric mucosa after immune challenge protection experiments from mRNA levels, which are representative molecules of th1, th17, th2 and Treg cell responses, respectively. It is shown at the molecular level that, when CX3CL1 is used in combination, stronger th1(IFN-r), th2(IL-4), th17(IL-17A) effector cell responses are stimulated, and the response of regulatory T cell Treg (Foxp3) is reduced.
FIGS. 5 and 6 show CX3CR1 recruited to mouse gastric mucosa following challenge-immune protection+The CD4T cells of (1) were immunophenotyped. FIG. 6 is a phenotypic analysis of cells within the boxed region of section B of FIG. 5, and the finding that the predominant effector memory CD4T cells (CD 3) chemotactic to the gastric mucosa via the CX3CL1-CX3CR1 pathway following the combination of CX3CL1 (CD 3)+CD4+CX3CR1+CD25-CD44+CD69-CCR7-T cells) that are capable of increasing long-lasting protection; in addition, CX3CR1 is the only receptor currently known for CX3CL 1. It can be seen that the combination of the H.pylori antigen and CX3CL1 enhances the persistence of H.pylori vaccine protection.
Further analysis of CD25 in CD4T cells recruited via CX3CL1-CX3CR1 pathway following immune challenge protection experiments-Proportion of cells, the CD25 molecule is an important surface marker for regulatory T cell tregs with suppressive function. The results are shown in figure 7, and the data demonstrate that recruitment of tregs is reduced when the combination of CX3CL1 is used to recruit CD4T cells, primarily effector T cells, but not suppressor T cells. It is suggested that CX3CL1 promotes the recruitment of effector CD4T cells and inhibits the recruitment of tregs, thereby exerting a protective effect.
Memory T cells are of great significance in exerting durable anti-infective immunity. FIG. 8 is the effector memory CD4T (CD 3) in CD4T cells chemotactic to the gastric mucosa via the CX3CL1-CX3CR1 pathway following immune challenge experiments+CD4+CX3CR1+CD25-CD44+CD69-CCR7-T) ratio of cells, as can be seen from FIG. 8, the combination of CX3CL1, promotes the recruitment of more effector memory CD4T cells to the gastric mucosa, contributing to long-lasting immune protection.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> second medical center of general hospital of people liberation force of China
Application of <120> chemokine CX3CL1 in preparation of vaccine and helicobacter pylori vaccine
<160>16
<170>PatentIn version 3.5
<210>1
<211>481
<212>PRT
<213> helicobacter pylori (helicobacter pylori)
<400>1
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Pro ArgLys Ser Ser Val Leu Pro Lys Asp Val Ser Leu Lys Ser Arg
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Leu Thr Lys Asn Ile Ser Leu Asn Ile Pro Phe Ile Ser Ala Ala Met
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Asp Thr Val Thr Glu His Lys Thr Ala Ile Ala Met Ala Arg Leu Gly
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Gly Ile Gly Ile Val His Lys Asn Met Asp Ile Gln Thr Gln Val Lys
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Ile Phe Ile His Ala His Arg Thr Leu Ala Asp Ala Lys Val Ile Thr
100 105 110
Asp Asn Tyr Lys Ile Ser Gly Val Pro Val Val Asp Asp Lys Gly Leu
115 120 125
Leu Ile Gly Ile Leu Thr Asn Arg Asp Val Arg Phe Glu Thr Asp Leu
130 135 140
Ser Lys Lys Val Gly Asp Val Met Thr Lys Met Pro Leu Val Thr Ala
145 150 155 160
Arg Val Gly Ile Ser Leu Glu Glu Ala Arg Asp Leu Met His Lys His
165 170 175
Lys Ile Glu Lys LeuPro Ile Val Asp Lys Asp Asn Val Leu Lys Gly
180 185 190
Leu Ile Thr Ile Lys Asp Ile Gln Lys Arg Ile Glu Tyr Pro Glu Ala
195 200 205
Asn Lys Asp Asp Phe Gly Arg Leu Arg Val Gly Ala Ala Ile Gly Val
210 215 220
Gly Gln Leu Asp Arg Ala Glu Met Leu Val Lys Ala Gly Val Asp Ala
225 230 235 240
Leu Val Leu Asp Ser Ala His Gly His Ser Ala Asn Ile Leu His Thr
245 250 255
Leu Glu Glu Ile Lys Lys Ser Leu Val Val Asp Val Ile Val Gly Asn
260 265 270
Val Val Thr Lys Glu Ala Thr Ser Asp Leu Ile Ser Ala Gly Ala Asp
275 280 285
Ala Ile Lys Val Gly Ile Gly Pro Gly Ser Ile Cys Thr Thr Arg Ile
290 295 300
Val Ala Gly Val Gly Met Pro Gln Val Ser Ala Ile Asp Asn Cys Val
305 310 315 320
Glu Val Ala Ser Lys Phe Asp Ile Pro Val Ile Ala Asp Gly Gly Ile
325 330 335
Arg Tyr Ser Gly Asp Val AlaLys Ala Leu Ala Leu Gly Ala Ser Ser
340 345 350
Val Met Ile Gly Ser Leu Leu Ala Gly Thr Glu Glu Ser Pro Gly Asp
355 360 365
Phe Met Ile Tyr Gln Gly Arg Gln Tyr Lys Ser Tyr Arg Gly Met Gly
370 375 380
Ser Ile Gly Ala Met Thr Lys Gly Ser Ser Asp Arg Tyr Phe Gln Glu
385 390 395 400
Gly Val Ala Ser Glu Lys Leu Val Pro Glu Gly Ile Glu Gly Arg Val
405 410 415
Pro Tyr Arg Gly Lys Val Ser Asp Met Ile Phe Gln Leu Val Gly Gly
420 425 430
Val Arg Ser Ser Met Gly Tyr Gln Gly Ala Lys Asn Ile Leu Glu Leu
435 440 445
Tyr Gln Asn Ala Glu Phe Val Glu Ile Thr Ser Ala Gly Leu Lys Glu
450 455 460
Ser His Val His Gly Val Asp Ile Thr Lys Glu Ala Pro Asn Tyr Tyr
465 470 475 480
Gly
<210>2
<211>426
<212>PRT
<213> helicobacter pylori (helicobacter pylori)
<400>2
Met Ser Val Thr Leu Val Asn Asn Glu Asn Asn Glu Arg Tyr Glu Phe
1 5 10 15
Glu Thr Ile Glu Ser Thr Arg Gly Pro Lys Ala Val Asp Phe Ser Lys
20 25 30
Leu Phe Glu Thr Thr Gly Phe Phe Ser Tyr Asp Pro Gly Tyr Ser Ser
35 40 45
Thr Ala Gly Cys Gln Ser Lys Ile Ser Tyr Val Asn Gly Lys Lys Gly
50 55 60
Glu Leu Tyr Tyr Arg Gly His Arg Ile Glu Asp Leu Val Ala Lys Tyr
65 70 75 80
Lys Tyr Val Asp Val Cys Lys Leu Leu Leu Thr Gly Glu Leu Pro Lys
85 90 95
Asn Gln Asp Glu Ser Leu Glu Phe Glu Leu Glu Leu Arg His Arg Ser
100 105 110
Phe Val His Glu Ser Leu Leu Asn Met Phe Ser Ala Phe Pro Ser Asn
115 120 125
Ala His Pro Met Ala Lys Leu Ser Ser Gly Val Ser Ile Leu Ser Thr
130 135 140
Leu Tyr Ser Thr His Gln Asn Met His Thr Glu Glu Asp Tyr Gln Thr
145 150 155 160
Met Ala Arg Arg Ile Val Ala Lys Ile Pro Thr Leu Ala Ala Ile Cys
165 170 175
Tyr Arg Asn Glu Val Gly Ala Pro Ile Ile Tyr Pro Asp Ile Ala Arg
180 185 190
Ser Tyr Val Glu Asn Ile Leu Phe Met Leu Arg Gly Tyr Pro Tyr Ser
195 200 205
Arg Leu Lys His Thr Thr Gln Gly Glu Val Glu Ile Thr Pro Leu Glu
210 215 220
Val Glu Ala Phe Asp Lys Ile Leu Thr Leu His Ala Asp His Ser Gln
225 230 235 240
Asn Ala Ser Ser Thr Thr Val Arg Asn Val Ala Ser Thr Gly Val His
245 250 255
Pro Tyr Ala Ala Ile Ser Ala Gly Ile Ser Ala Leu Trp Gly His Leu
260 265 270
His Gly Gly Ala Asn Glu Lys Val Leu Leu Gln Leu Glu Glu Ile Gly
275 280 285
Asp Val Lys Asn Val Asp Lys Tyr Ile Ala Arg Val Lys Asp Lys Asn
290 295 300
Asp Asn Phe Lys Leu Met Gly Phe Gly His Arg Val Tyr Lys Ser Tyr
305310 315 320
Asp Pro Arg Ala Lys Ile Leu Lys Gly Leu Lys Asp Glu Leu His Gln
325 330 335
Lys Gly Val Lys Met Asp Glu Arg Leu Ser Glu Ile Ala Ala Lys Val
340 345 350
Glu Glu Ile Ala Leu Lys Asp Glu Tyr Phe Ile Glu Arg Asn Leu Tyr
355 360 365
Pro Asn Val Asp Phe Tyr Ser Gly Thr Ile Leu Arg Ala Leu Lys Ile
370 375 380
Pro Val Arg Phe Phe Thr Pro Val Phe Val Ile Gly Arg Thr Val Gly
385 390 395 400
Trp Cys Ala Gln Leu Leu Glu His Val Lys Ser Pro Gln Ala Arg Ile
405 410 415
Thr Arg Pro Arg Gln Val Tyr Val Gly Asp
420 425
<210>3
<211>569
<212>PRT
<213> helicobacter pylori (helicobacter pylori)
<400>3
Met Lys Lys Ile Ser Arg Lys Glu Tyr Ala Ser Met Tyr Gly Pro Thr
1 5 10 15
Thr Gly Asp Lys Val Arg LeuGly Asp Thr Asp Leu Ile Ala Glu Val
20 25 30
Glu His Asp Tyr Thr Ile Tyr Gly Glu Glu Leu Lys Phe Gly Gly Gly
35 40 45
Lys Thr Leu Arg Glu Gly Met Ser Gln Ser Asn Asn Pro Ser Lys Glu
50 55 60
Glu Leu Asp Leu Ile Ile Thr Asn Ala Leu Ile Val Asp Tyr Thr Gly
65 70 75 80
Ile Tyr Lys Ala Asp Ile Gly Ile Lys Asp Gly Lys Ile Ala Gly Ile
85 90 95
Gly Lys Gly Gly Asn Lys Asp Met Gln Asp Gly Val Lys Asn Asn Leu
100 105 110
Ser Val Gly Pro Ala Thr Glu Ala Leu Ala Gly Glu Gly Leu Ile Val
115 120 125
Thr Ala Gly Gly Ile Asp Thr His Ile His Phe Ile Ser Pro Gln Gln
130 135 140
Ile Pro Thr Ala Phe Ala Ser Gly Val Thr Thr Met Ile Gly Gly Gly
145 150 155 160
Thr Gly Pro Ala Asp Gly Thr Asn Ala Thr Thr Ile Thr Pro Gly Arg
165 170 175
Arg Asn Leu Lys Phe Met Leu Arg Ala Ala Glu Glu Tyr Ser Met Asn
180 185 190
Ile Gly Phe Leu Ala Lys Gly Asn Ala Ser Asn Asp Ala Ser Leu Ala
195 200 205
Asp Gln Ile Glu Ala Gly Ala Ile Gly Leu Lys Ile His Glu Asp Trp
210 215 220
Gly Thr Thr Pro Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp Lys
225 230 235 240
Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Leu Asn Glu Ala Gly
245 250 255
Cys Val Glu Asp Thr Met Ala Ala Ile Ala Gly Arg Thr Met His Thr
260 265 270
Tyr His Thr Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys
275 280 285
Val Ala Gly Glu His Asn Ile Leu Pro Ala Ser Thr Asn Pro Thr Ile
290 295 300
Pro Phe Thr Val Asn Thr Glu Ala Glu His Met Asp Met Leu Met Val
305 310 315 320
Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp
325 330 335
Ser Arg Ile Arg Pro Gln Thr Ile Ala Ala Glu Asp Thr Leu His Asp
340 345 350
Met Gly Ile Phe Ser Ile Thr Ser Ser Asp Ser Gln Ala Met Gly Arg
355 360 365
Val Gly Glu Val Ile Thr Arg Thr Trp Gln Thr Ala Asp Lys Asn Lys
370 375 380
Lys Glu Phe Gly Arg Leu Lys Glu Glu Lys Gly Asp Asn Asp Asn Phe
385 390 395 400
Arg Ile Lys Arg Tyr Leu Ser Lys Tyr Thr Ile Asn Pro Ala Ile Ala
405 410 415
His Gly Ile Ser Glu Tyr Val Gly Ser Val Glu Val Gly Lys Val Ala
420 425 430
Asp Leu Val Leu Trp Ser Pro Ala Phe Phe Gly Val Lys Pro Asn Met
435 440 445
Ile Ile Lys Gly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn
450 455 460
Ala Ser Ile Pro Thr Pro Gln Pro Val Tyr Tyr Arg Glu Met Phe Ala
465 470 475 480
His His Gly Lys Ala Lys Tyr Asp Ala Asn Ile Thr Phe Val Ser Gln
485 490 495
Ala Ala Tyr Asp Lys Gly Ile Lys Glu Glu Leu Gly Leu Glu Arg Gln
500 505 510
Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gln
515 520 525
Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Ser Glu Thr Tyr His
530 535 540
Val Phe Val Asp Gly Lys Glu Val Thr Ser Lys Pro Ala Asn Lys Val
545 550 555 560
Ser Leu Ala Gln Leu Phe Ser Ile Phe
565
<210>4
<211>31
<212>DNA
<213> Artificial sequence
<400>4
tttgttagag aagataatga cggtatctaa c 31
<210>5
<211>28
<212>DNA
<213> Artificial sequence
<400>5
cataggattt cacacctgac tgactatc 28
<210>6
<211>18
<212>DNA
<213> Artificial sequence
<400>6
cgtgccagca gccgcggt 18
<210>7
<211>22
<212>DNA
<213> Artificial sequence
<400>7
gatcctttgg accctctgac tt 22
<210>8
<211>20
<212>DNA
<213> Artificial sequence
<400>8
<210>9
<211>20
<212>DNA
<213> Artificial sequence
<400>9
<210>10
<211>21
<212>DNA
<213> Artificial sequence
<400>10
actcattcat ggtgcagctt a 21
<210>11
<211>22
<212>DNA
<213> Artificial sequence
<400>11
ctccagaagg ccctcagact ac 22
<210>12
<211>18
<212>DNA
<213> Artificial sequence
<400>12
gggtcttcat tgcggtgg 18
<210>13
<211>20
<212>DNA
<213> Artificial sequence
<400>13
<210>14
<211>23
<212>DNA
<213> Artificial sequence
<400>14
agctgatgca gcatgaagtg tgg 23
<210>15
<211>22
<212>DNA
<213> Artificial sequence
<400>15
cctgcagagt taagcatgcc ag 22
<210>16
<211>23
<212>DNA
<213> Artificial sequence
<400>16
tgcttgatca catgtctcga tcc 23
Claims (10)
1. The use of the chemokine CX3CL1 in the preparation of a vaccine.
2. The use of claim 1, wherein the vaccine comprises a helicobacter pylori vaccine.
3. A helicobacter pylori vaccine, comprising a helicobacter pylori antigen and a chemokine CX3CL 1.
4. The helicobacter pylori vaccine of claim 3, wherein the helicobacter pylori antigen comprises one or more of a native protein, a recombinant protein, a polypeptide, and a nucleic acid.
5. The helicobacter pylori vaccine of claim 3, wherein the helicobacter pylori antigen comprises one or more of adenosine 5-phosphate dehydrogenase, type II citrate synthase, and urease B subunits.
6. The helicobacter pylori vaccine according to claim 5, wherein the amino acid sequence of the adenosine 5-phosphate dehydrogenase is shown in SEQ ID No. 1;
and/or the amino acid sequence of the type II citrate synthase is shown as SEQ ID NO. 2;
and/or the amino acid sequence of the urease B subunit is shown as SEQ ID NO. 3.
7. The helicobacter pylori vaccine according to claim 3, wherein the chemokine CX3CL1 comprises abcam recombinant chemokine CX3CL1 ab240868.
8. The helicobacter pylori vaccine according to any one of claims 3 to 7, wherein the helicobacter pylori vaccine comprises any one of the following combinations (a) to (g):
(a) adenosine 5-phosphate dehydrogenase and chemokine CX3CL 1;
(b) type II citrate synthase and chemokine CX3CL 1;
(c) urease B subunit and chemokine CX3CL 1;
(d) adenosine 5-phosphate dehydrogenase, type II citrate synthase and chemokine CX3CL 1;
(e) 5-adenosine phosphate dehydrogenase, urease B subunit and chemokine CX3CL 1;
(f) type II citrate synthase, urease B subunit, and chemokine CX3CL 1;
(g) 5-phosphoadenosine dehydrogenase, type II citrate synthase, urease B subunit and chemokine CX3CL 1.
9. The helicobacter pylori vaccine of any one of claims 3 to 7, wherein the vaccine further comprises a vaccine adjuvant.
10. The helicobacter pylori vaccine of any one of claims 3 to 7, wherein the helicobacter pylori vaccine is a recombinant antigen vaccine, a viral vector vaccine, or a gene vaccine.
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| CN113209285A (en) * | 2021-04-22 | 2021-08-06 | 成都亿妙生物科技有限公司 | Application of helicobacter pylori chemotactic factor chemotaxis gene |
| CN115975053A (en) * | 2022-12-07 | 2023-04-18 | 南开大学 | Vaccine targeting novel coronaviruses |
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| CN106421768A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第三军医大学 | Helicobacter pylori multi-subunit vaccine based on CD4+T cellular immunity and preparing method |
| CN106480003A (en) * | 2016-10-31 | 2017-03-08 | 中国人民解放军第三军医大学 | Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique |
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| CN106421768A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第三军医大学 | Helicobacter pylori multi-subunit vaccine based on CD4+T cellular immunity and preparing method |
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Cited By (3)
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| CN113209285A (en) * | 2021-04-22 | 2021-08-06 | 成都亿妙生物科技有限公司 | Application of helicobacter pylori chemotactic factor chemotaxis gene |
| CN113209285B (en) * | 2021-04-22 | 2023-03-10 | 成都欧林生物科技股份有限公司 | Application of helicobacter pylori chemotactic factor chemotaxis gene |
| CN115975053A (en) * | 2022-12-07 | 2023-04-18 | 南开大学 | Vaccine targeting novel coronaviruses |
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| CN111744003B (en) | 2022-11-01 |
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