CN108486120B

CN108486120B - Novel CpG ODN sequence and application thereof in anti-melanoma

Info

Publication number: CN108486120B
Application number: CN201810402526.0A
Authority: CN
Inventors: 冯志伟; 贾慧婕; 李连涛; 赵铁锁; 郭胜
Original assignee: Xuzhou Medical University; Xinxiang Medical University
Current assignee: Xuzhou Medical University; Xinxiang Medical University
Priority date: 2018-04-28
Filing date: 2018-04-28
Publication date: 2019-12-06
Anticipated expiration: 2038-04-28
Also published as: CN108486120A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a novel CpG ODN sequence and application thereof in resisting melanoma. The novel CpG ODN sequence of the invention has strong immune stimulation function, can effectively stimulate the expression of CD86 on PBMC cells, effectively inhibit the growth of melanoma, increase the expression of CD8 positive T lymphocytes in the spleen of a mouse, and has obvious clinical application prospect.

Description

Novel CpG ODN sequence and application thereof in anti-melanoma

Technical Field

the invention belongs to the technical field of biological medicines, and particularly relates to a novel CpG ODN sequence and application thereof in resisting melanoma.

Background

DNA has been considered to have only weak immunogenicity, and is difficult to induce immune response in the body. However, in recent years, it has been found that synthetic deoxyoligonucleotide single-stranded DNA (CpG ODN) containing CpG motif can activate NK cells of mice and stimulate spleen cells of mice to secrete interferon. CpG is an unmethylated dinucleotide composed of cytosine and guanine linked via a phosphodiester bond (where C represents cytosine, G represents guanine, and p represents a phosphodiester bond). Structural conditions that CpG ODN with immune response stimulating ability must have: (1) the stimulatory sequence must contain an unmethylated CpG motif, (2) sequences with two purine nucleotides at the 5 'end and two pyrimidine nucleotides at the 3' end of the CpG motif have a stronger stimulatory effect, and (3) CpG ODN needs to have a certain length. In a CpG ODN, there may be more than one CpG motif. Due to differences in sequence, particularly sequences flanking CpG, CpG ODN can take a wide variety of forms, exhibit different immunomodulatory activities, and the ability of sequences to be immunostimulatory is affected by purine and pyrimidine nucleotides surrounding CpG motifs, as well as the spacing between CpG motifs, and the like. Therefore, the CpG ODN sequence with stronger immunogenicity is designed.

Disclosure of Invention

the novel CpG ODN sequence of the invention has strong immune stimulation function, can effectively stimulate the expression of CD86 on PBMC cells, effectively inhibit the growth of melanoma, increase the expression of CD8 positive T lymphocytes in the spleen of a mouse, and has obvious clinical application prospect.

The invention provides a novel CpG ODN sequence, and the nucleotide sequence of the novel CpG ODN sequence is as follows:

5’-gacgcgcgtcgacgatcgcgaattcgaacgtacgct-3’。

An application of the novel CpG ODN sequence in resisting melanoma.

Preferably, the novel CpG ODN sequences are applied to the expression of the PBMC cell surface molecule CD86 in melanoma patients.

preferably, the novel CpG ODN sequence is prepared as a drug for treating melanoma mice.

Compared with the prior art, the invention has the beneficial effects that:

the novel CpG ODN sequence of the invention has strong immune stimulation function, can effectively stimulate the expression of CD86 on PBMC cells, causes the immune response of organisms and effectively inhibits the growth of melanoma; increase the expression of CD4 and CD8 positive T lymphocytes in the spleen of the mouse, inhibit the growth of melanoma of the mouse, prolong the life cycle of the mouse and have obvious clinical application prospect.

Drawings

FIG. 1 is a diagram showing the expression of the PBMC cell surface molecule CD 86;

FIG. 2 is a graph of survival of melanoma-bearing mice of the present invention;

FIG. 3 is a tumor entity map of a melanoma-bearing mouse of the present invention (A);

FIG. 4 is a graph of tumor weight for melanoma-bearing mice of the present invention (B);

FIG. 5 is a graph showing the expression of CD4 and CD8 positive T lymphocytes according to the invention;

FIG. 6 is a graph showing the expression of migration and apoptosis-related proteins in tumor tissues according to the present invention;

FIG. 7 is a graph showing the content of anti-tumor cytokine IFN γ in the serum of mice of the present invention.

Detailed Description

several embodiments of the present invention will be described in detail below with reference to fig. 1-7, but it should be understood that the scope of the present invention is not limited to the embodiments, and the reagents involved in the examples can be obtained through common channels.

example 1

a novel CpG ODN sequence, the nucleic acid sequence of the novel CpG ODN sequence is:

5'-gacgcgcgtcgacgatcgcgaattcgaacgtacgct-3', as shown in SEQ ID NO. 1.

The novel CpG ODN sequence has the effect of resisting melanoma and can be applied to the expression of a PBMC cell surface molecule CD86 of a melanoma patient.

in order to verify the application effect of the invention, the following tests were carried out:

(1) Preparing a PBMC cell solution of a melanoma patient, and adjusting the cell concentration to be more than or equal to 1 multiplied by 106 per ml;

(2) inoculating PBMC cells into AIM-V culture medium in a 6-well plate, and incubating at 37 deg.C and 5% CO2 by volume in an incubator;

(3) after the monocytes are adhered to the surface, rhGM-CSF and IL-4 are added, and the test group is incubated for 3 days after CpG ODN (control group is added with equal amount of physiological saline) is added according to the mass concentration of 1 mu g/ml, and the expression of CD86 cell surface molecules is detected by FACS. The results are shown in fig. 1, the left panel is the control group, the expression number of CD86 molecules on the surface of PBMC cells is 5.65%, and the expression number of the right panel is 9.39%, so the CpG ODN sequence of the present application can effectively enhance the expression of the human PBMC surface costimulatory molecule CD 86.

Example 2

5'-gacgcgcgtcgacgatcgcgaattcgaacgtacgct-3', as shown in SEQ ID NO. 1.

the novel CpG ODN sequence is prepared into a medicament for treating melanoma mice.

1. establishing a mouse melanoma model:

(1) Digesting and resuspending melanoma cells B16, counting, calculating the ratio of viable cells, and selecting a sample with the ratio of the viable cells being more than or equal to 98% for later use;

(2) Preparing a melanoma cell sample with the ratio of live cells being more than or equal to 98% into a cell suspension by using a serum-free culture medium, adjusting the concentration to be more than or equal to 1 multiplied by 107 cells/ml, refrigerating, and injecting 0.1ml subcutaneously at a position which is close to the root of the tail and is about 0.7cm below the lower part of the right side of the mouse;

(3) After injection of melanoma cells, mice with skin cumulus of 6-8mm in diameter in the injection area were selected as animal test mice.

2. Mouse animal test:

(1) the animal test mice 7 days after the inoculation were randomly divided into three groups: PBS group, CpG ODN 1 group and CpG ODN 2 group, wherein the PBS group is controlled by normal saline 20 mug/piece each time, the CpG ODN 1 group dosage is 10 mug/piece each time, the CpG ODN 2 group dosage is 20 mug/piece each time, the injection mode is as follows: the inguinal lymph node drainage area of the test mouse is injected once every other day for four times;

(2) the differences in survival time of the mice were observed, and the results are shown in FIG. 2;

(3) after 14 days of the last treatment, the melanoma tissues of the test mice are separated, and the growth conditions of the melanoma tissues of each group are observed, and the results are shown in figures 3-4; FACS is used for detecting CD4+ and CD8+ T in the spleen of the tumor-bearing mouse, and the result is shown in FIG. 5; the results of detecting the expression of proteins related to melanoma migration and apoptosis in tissues are shown in FIG. 6; the results of measuring IFN γ levels in serum using Elisa are shown in FIG. 7.

As can be seen from fig. 2, the survival rate of the melanoma-bearing mice injected with the novel CpG ODN sequence group is higher than that of the mice injected with the physiological saline, and the effect of the CpG ODN 2 group is better than that of the CpG ODN 1 group, so that the novel CpG ODN sequence can effectively prolong the survival time of the melanoma-bearing mice.

from fig. 3 to fig. 4, it can be seen that the size and weight of the tumor of the melanoma-bearing mice injected with the novel CpG ODN sequence group are both significantly smaller than those of the normal saline group, so that the novel CpG ODN sequence can effectively inhibit the tumor growth of the melanoma-bearing mice.

FIG. 5 shows the ratio of CD4 and CD8 positive T lymphocytes in splenocytes detected by flow cytometry after incubation with antibodies labeled with different color fluorescence CD4 and CD 8. The CD4T cell ratio was 23.5. + -. 2.6 for the CpG ODN 20. mu.g group and 20.3. + -. 2.1 for the CD8T cell ratio, both of which were greater than the CD4T cell ratio and the CD8T cell ratio for the PBS group and CpG ODN 10. mu.g, respectively. It can be seen that the novel CpG ODN sequence significantly increased the ratio of CD4T cells and CD8T cells in splenocytes from melanoma-bearing mice. Therefore, the novel CpG ODN sequence improves the anti-tumor immune response of mice

FIG. 6 shows that the expression level of apoptosis-related protein Cleaved-Caspase3 in the CpG ODN 2 group is the most, and the expression of migration-related protein MMP2 is obviously inhibited, which indicates that the novel CpG ODN sequence can effectively inhibit the tumor growth of melanoma mice.

Fig. 7 shows that the content of IFN γ, which is an antitumor cytokine, in serum was the highest in the CpG ODN 2 group, and that the novel CpG ODN sequence had an antitumor effect.

In conclusion, the novel CpG ODN sequence can effectively improve the expression of PBMC (peripheral blood mononuclear cell) co-stimulatory molecules, inhibit the growth of melanoma of mice, improve the anti-tumor immune response of the mice and have certain application value.

it should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

it will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<120> novel CpG ODN sequence and application thereof in anti-melanoma

<141> 2018-04-23

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> DNA

<213> Artificial sequence

<400> 1

gacgcgcgtc gacgatcgcg aattcgaacg tacgct 36

Claims

1. A novel CpG ODN sequence, wherein the nucleic acid sequence of the novel CpG ODN sequence is:

5’-gacgcgcgtcgacgatcgcgaattcgaacgtacgct-3’。

2. Use of a novel CpG ODN sequence according to claim 1 for the preparation of an anti-melanoma drug.

3. The use of the novel CpG ODN sequence of claim 2 in the preparation of an anti-melanoma drug, wherein the novel CpG ODN sequence is used for expression of the PBMC cell surface molecule CD86 in melanoma patients.

4. The use of the novel CpG ODN sequence of claim 2 for the preparation of an anti-melanoma drug, wherein the novel CpG ODN sequence is prepared as a drug for the treatment of melanoma mice.