阅读说明：本技术 肺炎衣原体相关抗原短肽及其应用 (Chlamydia pneumoniae-associated antigen short peptide and application thereof ) 是由 黄燕花 赵乙木 罗夫·辛克纳吉 孙晨 于 2019-11-18 设计创作，主要内容包括：本发明公开了肺炎衣原体抗原短肽及其应用。所述肺炎衣原体抗原短肽序列如SEQ ID NO:1-16任一所示。该短肽具有与DC细胞上MHC I类和MHC II分子高度的亲和力,并能有效地使其起到抗原提呈作用,具备良好的多肽疫苗及DC疫苗的潜力。将该短肽中的至少一条体外致敏树突状细胞,制备的DC疫苗能够预防肺炎衣原体相关疾病,尤其是相关慢性疾病,具有良好的临床转化前景。而且,本发明的抗原短肽长度较短,化学合成难度小,可以直接合成得到高纯的产物,应用成本大大降低,同时效果明确,具有很好的应用前景。(The invention discloses a chlamydia pneumoniae antigen short peptide and application thereof. The sequence of the Chlamydia pneumoniae antigen short peptide is shown in any one of SEQ ID NO 1-16. The short peptide has high affinity with MHC class I and MHC class II molecules on DC cells, can effectively play a role in antigen presentation, and has good potential of polypeptide vaccines and DC vaccines. At least one in vitro sensitized dendritic cell in the short peptide can be used for preparing the DC vaccine which can prevent related diseases of the chlamydia pneumoniae, particularly related chronic diseases, and has good clinical transformation prospect. Moreover, the antigen short peptide has short length and small chemical synthesis difficulty, can be directly synthesized to obtain a high-purity product, greatly reduces the application cost, has definite effect and has good application prospect.)

1. The Chlamydia pneumoniae antigen short peptide is characterized in that the sequence of the antigen short peptide is shown in any one of SEQ ID NO 1-16.

2. The use of the short peptide of claim 1 for the preparation of a DC vaccine for the control of chlamydia pneumoniae-associated chronic diseases.

3. The use of the oligopeptide of claim 1 in the preparation of a medicament for the prevention and treatment of chronic diseases associated with chlamydia pneumoniae.

4. A DC vaccine for preventing and treating chronic chlamydia pneumoniae-related diseases, which is loaded with the short peptide and the dendritic cells according to claim 1.

5. The DC vaccine according to claim 4, wherein the DC vaccine is an autologous dendritic cell preparation obtained by sensitizing dendritic cells in vitro with a combination of short peptides represented by at least one of SEQ ID NOS: 1 to 16.

6. The DC vaccine of claim 4, wherein the vaccine is an intravenous infusion vaccine.

7. The DC vaccine of claim 4, wherein the activity of mature DC cells is increased by using stem cell factor or vitrogan factor as adjuvant.

8. The DC vaccine of claim 4, which is prepared by the following steps: the maturation of the DC cells is promoted by taking a maturation promoting factor and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant; then adding the short peptide of claim 1 into a DC cell culture system for inducing maturation, collecting the DC cells loaded with the short peptide fragment, washing with physiological saline, and then resuspending with physiological saline to obtain the DC vaccine.

9. Use of a DC vaccine according to any one of claims 4 to 8 in the manufacture of a medicament for the prevention and treatment of chronic diseases associated with chlamydia pneumoniae.

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to a Chlamydia pneumoniae-associated antigen short peptide and application thereof.

Background

The life expectancy at birth increased by 6.2 years from 1990 to 2013 as reported by "lancet" in 2015. With increasing life expectancy, attention has been directed not only to increased life, but also to improved quality of life. Disability caused by disease is one of the important causes affecting quality of life. At present, the number of disabilities caused by almost all reasons in the world is reduced, and only the number of disabilities caused by cardiovascular and cerebrovascular diseases and tumors is increased. The early stage of chronic diseases such as most cardiovascular and cerebrovascular diseases, tumors and the like lacks of specific clinical symptoms, and irreversible diseases are developed in definite diagnosis. Therefore, the fundamental solution to the chronic diseases such as cardiovascular and cerebrovascular diseases and tumors is important to prevent. The specific pathogenesis of chronic diseases such as cardiovascular and cerebrovascular diseases, diabetes, tumor and the like is not clearly researched, but the pathogenesis of the chronic diseases caused by the pathogenic factor of microbial infection in vivo is proved. Therefore, prevention of microbial infection is also critical to the prevention of the development of chronic diseases.

Wherein, the chlamydia pneumoniae (chlamydiae) is a prokaryotic microorganism which is parasitic in eukaryotic cells and has a unique development cycle; in the shape of a sphere, an ellipse or a pear. It has been demonstrated that chlamydia pneumoniae can cause the development of various chronic diseases, and the inflammatory reaction induced by chlamydia pneumoniae is involved in the theories of amyloid protein and tau protein and can be involved in the development of alzheimer disease; the chlamydia pneumoniae can cause arteriosclerosis through oxidation stress induced by low-density lipoprotein oxidation, foam cell formation, endothelial dysfunction, platelet adhesion, aggregation and the like to cause cardiovascular diseases, membrane antigens of the chlamydia pneumoniae induce endothelial cells to generate immune response, and the chlamydia pneumoniae can increase smooth muscle cell proliferation through immune regulation to promote atherosclerosis to cause stroke; mycoplasma pneumoniae is also associated with lung cancer onset.

After the chlamydia pneumoniae is infected, the body can be induced to generate specific humoral immunity and cellular immunity, and specific antibodies can block the adsorption of the antibodies to host cells, but the immunity is not durable and strong. There is no effective vaccine for chlamydia pneumoniae at present.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the existing chlamydia pneumoniae vaccine, provide chlamydia pneumoniae antigen polypeptide capable of being combined with MHC class I and MHC class II molecules, provide a chlamydia pneumoniae vaccine based on a DC technology, be applied to preventing and treating chronic diseases related to the chlamydia pneumoniae by adopting the DC technology, increase the activity of mature DC cells by combining stem cell factors/vitrogen, promote the activation of the DC cells by various chlamydia pneumoniae specific antigen peptides, achieve the aim of preventing and treating the chlamydia pneumoniae infection and have good application prospect.

The invention aims to provide a Chlamydia pneumoniae antigen-associated short peptide.

The invention also aims to provide the application of the chlamydia pneumoniae antigen-related short peptide in the preparation of the chlamydia pneumoniae antigen polypeptide DC vaccine.

The invention further aims to provide the chlamydia pneumoniae antigen polypeptide DC vaccine.

The above purpose of the invention is realized by the following technical scheme:

provides a chlamydia pneumoniae antigen short peptide, the sequence of which is shown in any one of SEQ ID NO 1-16.

Provides the application of the chlamydia pneumoniae antigen short peptide in the preparation of the chlamydia pneumoniae antigen polypeptide DC vaccine.

Provides the application of the chlamydia pneumoniae antigen short peptide in preparing the prevention and treatment medicine for chronic diseases related to the chlamydia pneumoniae.

In addition, the DC vaccine for preventing and treating the chlamydia pneumoniae-related chronic diseases is obtained by loading the chlamydia pneumoniae antigen short peptide and dendritic cells. Specifically, the autologous dendritic cell preparation is obtained by sensitizing dendritic cells in vitro by combining short peptides shown by at least one of SEQ ID NO. 1-16.

The Chlamydia pneumoniae antigen short peptide described in SEQ ID NO. 1-SEQ ID NO. 16 can induce DC to play an antigen presenting role.

In particular, the vaccine is an intravenous infusion vaccine.

In the preparation method of the DC vaccine for preventing and treating the chronic disease related to the chlamydia pneumoniae, the stem cell factor or the vitrogen factor is selected as an adjuvant to increase the activity of DC cells, and the specific antigen peptide is used for sensitizing dendritic cells in vitro to obtain an autologous dendritic cell preparation which is used as the vaccine for preventing and treating the chronic disease related to the chlamydia pneumoniae by intravenous infusion. The preparation method comprises the following steps: the maturation of the DC cells is promoted by taking a maturation promoting factor and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant; then adding the short peptide of claim 1 into a DC cell culture system for inducing maturation, collecting the DC cells loaded with the short peptide fragment, washing with physiological saline, and then resuspending with physiological saline to obtain the DC vaccine.

More specifically, as an alternative, the DC vaccine for preventing and treating the chlamydia pneumoniae-related chronic disease is prepared by the following steps:

s1, extracting and inducing DC cells:

s11, obtaining immature DC cells

Collecting peripheral blood of healthy donor, separating mononuclear cells by lymphocyte separation, culturing at 37 deg.C in culture medium with 5% CO₂After culturing for 3 hours under the conventional condition, the adherent cells are immature DC cells;

s12, amplification culture of immature DC cells

37℃、5％CO₂Culturing for 5 days under the condition, and changing the culture solution every other day to complete the amplification culture of immature DC cells (imDC cells);

S13.Induction of DC cells

Adding a maturation promoting factor, and simultaneously taking a stem cell factor or a Vitrogen factor as an adjuvant to promote the maturation of the DC cells;

s2, loading of polypeptide:

adding the short peptide of claim 1 to the culture system 5 days after inducing DC cell maturation;

s3, preparing a DC vaccine:

and (3) centrifuging to collect the DC cells loaded with the short peptide fragments, washing the cells for 3 times by using physiological saline, and finally, resuspending the DC cells loaded with the short peptide fragments by using the physiological saline to obtain the DC vaccine.

In addition, the application of the DC vaccine in preparing the prevention and treatment medicine for chronic diseases related to the chlamydia pneumoniae also belongs to the protection scope of the invention.

Immunology has shown that Dendritic Cells (DC) are the most powerful antigen-presenting cells known at present, and are considered to be the initiator of the immune response of the body, and are centrally located in the immune response. Mature DCs express abundant MHC class I and MHC class II molecules associated with antigen presentation. It can be ingested, processed and presented with antigen; participate in the maintenance of immunological memory; secretion of cytokines modulates immune responses.

The invention predicts that the chlamydia pneumoniae can be used as a sequence cluster of the antigen by combining with a bioinformatics technology, and through a large number of researches and searches, the obtained 16 chlamydia pneumoniae antigen peptides have high affinity with MHC I and MHC II molecules on DC cells, can effectively play a role in antigen presentation, have good potential of polypeptide vaccines and DC vaccines, and prompt that the chlamydia pneumoniae antigen peptides have good clinical transformation and disease prevention and treatment prospects.

The invention selects specific epitope polypeptide, sensitizes autologous DC cells in vitro, prepares DC cell preparation, and carries out venous return transfusion on patients, reconstructs the whole body immune balance of organisms, starts immune system, and prevents the specificity of infected microorganisms.

The DC technology adopted by the invention jointly activates dendritic cells by using a plurality of specific antigen peptides, the antigen peptides have extremely strong specificity, induce the dendritic cells with higher activity to carry a plurality of antigen information, can stimulate the immunity of an organism after being back-infused into the human body, can achieve the aim of inducing the human body to generate specific antibodies aiming at the chlamydia pneumoniae and CTL cells aiming at the chlamydia pneumoniae specificity, and thus can effectively prevent and treat the occurrence and development of chronic diseases related to the chlamydia pneumoniae.

The invention has the following beneficial effects:

the invention provides chlamydia pneumoniae antigen polypeptides with sequences shown as SEQ ID NO. 1-16, and the antigen peptides have high affinity with MHC class I and MHC class II molecules on DC cells, can effectively play a role in antigen presentation, and have good potential of polypeptide vaccines and DC vaccines.

The short peptide has short length and small chemical synthesis difficulty, can be directly synthesized to obtain a high-purity product, greatly reduces the application cost, has good potential of a polypeptide vaccine and a DC vaccine, can prevent related diseases of the chlamydia pneumoniae, particularly related chronic diseases, and has good clinical transformation and practical application prospects.

The technology for preparing the DC vaccine for preventing and treating the chlamydia pneumoniae based on the chlamydia pneumoniae antigen peptide has the following advantages: (1) after DC is activated in vitro, the stem cell factor/Vitrogen factor can increase the activity of DC cells, increase the antigen-recognizing ability, and promote the generation of immune response after being returned into the body. (2) Long-term immunological memory: because the specific antigen peptide is fully contacted with the immune cells with memory function, 2 the immune cells have accurate and long-term immune memory after being infused back into the body, thereby enhancing the immune control effect and providing long-term protection for preventing reinfection or prevention. (3) After the DC is back-transfused in vivo, the immunity of the organism can be reestablished, thereby achieving the purpose of preventing and treating the in vivo infection virus.

Drawings

FIG. 1 is a comparison of killing activity against target cells in the DC vaccine treated group and the polypeptide-DC vaccine control group.

FIG. 2 shows the activity change of T cells in a blank control group, a DC vaccine group, a polypeptide vaccine group and a polypeptide-DC vaccine group detected by a CCK-8 method in vitro DC sensitized T cells.

FIG. 3 shows the levels of antibodies in sera of the placebo, DC, polypeptide, and polypeptide-DC vaccine groups measured by ELISA 6 weeks after immunization of mice.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise specified; the reagents and materials used in the following examples are all commercially available.