CN1692943A - Preparation and Application of CpG DNA Molecular Anti-infection Immune Preparation - Google Patents

Abstract

An anti-infectious immunopotentiator for pig, ox, yak, etc is prepared through artificially synthesizing the CpG oligonucleotide sequce able to excite the reproductive activity of immune cell, preparing the chitosan nanoparticles from deacetyl chitosan, and using said chitosan nanoparticles for molecular packaging of CpG oligonucleotide. It can be used to immunize the experimental animal by intramuscular injection or oral applialtion.

Description

Preparation and application of CpG DNA molecule anti-infection immune preparation

technical field

1. Biotechnology 2. Molecular Immunology

Background technique

In 1984, Tokunaga et al. found that the DNA of Mycobacterium tuberculosis has anti-tumor properties in addition to a genetic material, which opened a new chapter in the study of bacterial DNA with immunostimulatory properties. Subsequently, they concluded through some studies that This immunostimulatory property is caused by hexanucleotides with self-complementary palindromic structures. By 1995, Krieg et al. found that bacterial DNA and some synthetic ODNs (oligodeoxynucleotides) had immunostimulatory effects, but this effect was not necessarily related to whether they had a palindrome structure, but to these The unmethylated CpG structural unit (motif) in DNA is closely related, and it is defined as CpG immunostimulatory sequence (CpG).

CpG sequences mainly exist in the genomes of bacteria, viruses, and invertebrates, while those in vertebrate genomes are rare, and most of them are methylated. CpG usually has certain structural characteristics, that is, CpG is the core, followed by two purine bases at the 5' end and two pyrimidine bases at the 3' end. These unmethylated CpG or artificially synthesized CpG-containing oligonucleotides (CpG-oligideoxynucleotides, CpG-ODN) in bacterial DNA can directly activate B cells and stimulate B cells to secrete immune cells without the presence of T cells. Globulin and IL-6. The antibodies produced by it are of non-antigenic heterosexuality, and the antibodies produced by mouse B cells induced by bacterial DNA can not only react with bacterial DNA, but also cross-react with other DNA. In addition to B cells, CpG can also directly activate monocytes, macrophages and dendritic cells, activate CD8 ⁺ T cells and promote their differentiation, and induce various Th1 cytokines such as IFN, TNE, IL-1 and IL -12, etc., these factors further activate NK cells and secrete IFN-γ, resulting in cytolytic activity.

Although both bacterial DNA and artificially synthesized CpG-ODN have immunostimulatory effects, in some cells such as macrophages, the effect of bacterial DNA is stronger than that of artificially synthesized CpG-ODN. Stcey believes that this may be due to the fact that oligonucleotides are more easily degraded than bacterial DNA, or that the combination of different immunostimulatory sequences in bacterial DNA has a stronger immunostimulatory effect, or that it is more effective for macromolecular DNA.

In the 21st century, human health problems have become increasingly prominent. While antibiotics and other chemical drugs are used to effectively treat infectious diseases, many drug-induced diseases and bacterial resistance to antibiotics have also emerged. At present, due to the excessive reliance on antibiotics to control infectious diseases, the problem of bacterial resistance has resulted. In the past decade or so, the multi-drug resistance and all-drug resistance of bacteria to antibiotics has spread and intensified, making many infectious diseases difficult to control. For example, infectious diseases caused by Mycobacterium tuberculosis and Escherichia coli 0157 have made a comeback, seriously threatening human life and health. At present, there is an urgent need to develop new anti-infection technology and drugs, effectively enhance the body's immune and anti-infection ability, and overcome the harm of antibiotic abuse. In recent years, the emergence of immunostimulatory sequences of nucleic acid molecules has brought a new dawn for this.

At present, it has been determined that the immune activation function of CpG sequences on the animal body is mainly reflected in the following two aspects: one is to stimulate the proliferation of B lymphocytes; the other is to activate monocytes to secrete cytokines, which in turn leads to the activation of natural killer cells and Th1 helper T cells. Elicits humoral and cellular immune responses characteristic of Th1.

The immunostimulatory activity of CpG-ODN is not only closely related to the CpG motif and molecular structure, but also has animal species specificity. At present, the research on the application of CpG immunostimulatory sequence mainly focuses on gene therapy, vaccine adjuvant, tumor immunity, etc., while the research on its anti-infection biological effect is less. Recent studies have shown that, in addition to the immunoregulatory function, the CpG motif also plays an important role in controlling the infection of intracellular parasitic bacteria (Listeria, mycobacteria, etc.), which may or may not depend on lymphocytes. Development of immunomodulatory molecular drugs as novel anti-infective agents.

Due to the different immunization pathways and immunization methods, the antigen-presenting cells and antigen presentation methods involved are different, so different types and intensities of immune responses can be produced. Direct intramuscular injection and oral administration are two convenient and effective routes. Intramuscular injection does not require additional equipment and can express foreign genes in muscle cells for a long time, so it is not only an effective means of immunizing animals, but also can be applied to gene therapy. Considering the simplicity of future application, oral administration is also an optional way of immunization. Since intramuscular injection mainly induces immune response through the activation of Th1 cells, the efficiency is low, and a large dose of DNA needs to be injected, and a larger amount of DNA is required for oral administration through the digestive tract. Therefore, choosing an effective encapsulation method to deliver anti-infection DNA preparations to antigen-presenting cells to induce strong humoral immunity and cellular immunity is very important in the application of immune enhancers to prevent and treat infectious diseases. Chitosan is a deacetylated derivative of natural biological polysaccharide chitin, which has good biocompatibility, can be degraded by various enzymes in the body, and the degradation products are safe and non-toxic, and can be completely absorbed by organisms; and because It has unique polycationic properties, and the existing research results show that it is a slow-release material with great potential. In recent years, chitosan-based nanoparticle systems have emerged in the field of gene transfection and expression research. A large number of studies have shown that chitosan can wrap concentrated DNA to form small dispersed particles. When chitosan DNA complex is used for gene delivery and transfection expression, it can effectively carry the target gene into the target cell. The release rate of substances encapsulated in chitosan particles is mainly determined by the biodegradation and erosion of chitosan, so the release of substances is significantly prolonged. Preliminary research results show that: the particle system based on chitosan has broad application prospects in biomedicine. In addition, chitosan also has a variety of biological functions, especially as a drug synergist.

Due to the good biocompatibility of chitosan nanoparticles, it shows broad prospects in the encapsulation of DNA preparations. Therefore, chitosan nanoparticles are used to package oligonucleotides containing CpG motifs to prepare gene preparations, and Applying it to the application technology of improving the body's cellular and humoral immunity and enhancing the anti-infection ability has great potential value in scientific research, medical treatment, animal health care, and industrialization.

Contents of the invention

The technical problem to be solved by the invention is to use chitosan as the encapsulation material to prepare the oligonucleotide gene preparation containing the CpG motif, and use the preparation for anti-infection application technology. The method includes the following steps:

1. Design and synthesis of CpG oligonucleotide sequence and its primers

CpG sequence design and synthesis:

CpG sequence (see the sequence list on page 11 of the instruction manual)

A CpG oligonucleotide sequence containing 11 CpG motifs and 88 bases in length was synthesized, which is characterized by containing 3 5'- AACGTT- 3', 3 5' -GTCGTC- 3', 1 5'- GACGTT- 3', 1 5'- ATCGAT- 3', 1 5'- GTCGTT- 3', 1 5'- GGCGTT- 3', 1 5'- GACGTC- 3' characteristic sequence .

CpG sequence primer design and synthesis:

5' end: CGAGATCTAACGTTGTC

3' end: CGAGATCTAACGTTAAC

2. Preparation of CpG oligonucleotides for immunization

Using the synthesized CpG sequence as a template, it is amplified by PCR (polymerase chain reaction), and the amplified product is dissolved in sterilized saline, and its concentration is measured by an ultraviolet spectrophotometer.

3. CpG oligonucleotides stimulate animal blood immune cell proliferation in vitro

Lymphocytes from pigs, yaks and cattle were cultured in vitro to prepare target cells, and CpG oligonucleotides, CpG oligonucleotides and Escherichia coli vaccine, CpG oligonucleotides and paratyphoid vaccine were added to the target cells respectively, The effect of the CpG oligonucleotide and its co-vaccine on the proliferation of animal immune cells was detected by MTT method, and it was found that the CpG oligonucleotide and its co-vaccine could significantly stimulate the proliferation of lymphocytes in pigs, yaks and cattle active.

4. Preparation of nanoparticles

Using the ion cross-linking method, prepare nano-packaging particles with chitosan with a molecular weight of 150,000 and a deacetylation degree of more than 95%, and add CpG oligonucleotides to the chitosan solution in a certain proportion, and magnetically stir to make it fully mixed to prepare CpG. Oligonucleotide chitosan nanoparticles, and the shape, particle size and Zeta potential of the nanoparticles were determined.

The results showed that the prepared CpG oligonucleotide chitosan nanoparticles had a positive charge on the surface, the average particle size was 45nm; the polydispersity was 0.190; the zeta potential was +25.6mV.

5. Application of CpG oligonucleotide chitosan nanoparticle anti-infection preparation

120 Kunming mice were randomly divided into 8 groups, 15 in each group. Among them, 4 groups were non-specific immunization groups, and 4 groups were vaccine immunization groups. Mice were immunized by intramuscular injection and oral administration. The CpG oligonucleotide immunization dose in the oral group was 30 pmol/head mouse, and the chitosan nanoparticle packaging group was 6 pmol/head animal; each mouse in the vaccine immunization group was vaccinated 1/40 portion and 30 pmol of the corresponding CpG oligonucleotide or 6 pmol of the corresponding CpG oligonucleotide packaged in chitosan nanoparticles. Five weeks later, each mouse was orally challenged with 0.4 ml of Escherichia coli K88 and K99 (3×10 ⁹ /ml).

Before and after the mice were immunized, blood was collected from the tail vein every week, the serum was separated, and the humoral immunity (IgG , IgA and IgM content, the determination of specific antibodies) and cellular immunity level (cytokines and the number of peripheral blood immune cells), and the synergistic immune adjuvants of different forms of CpG oligonucleotide molecules in conventional vaccines The changes of cellular immunity and humoral immune response under the influence of the drug were systematically analyzed. Thirty-five days after the mice were immunized, the experimental mice were orally challenged with Escherichia coli K88/K99, and the disease was observed; all mice were dissected on the 49th day, the physiological lesions of various organs were observed, and the immune protection effect was detected.

The results showed that when oligonucleotide chitosan nanoparticles containing CpG motifs were used as immune enhancers, the IgG, IgA, IgM contents and serum IL-2, IL-4, IL-6 and other cytokines in mice At the same time, the number of immune cells such as white blood cells, lymphocytes, monocytes, and neutrophils also increased significantly; combined with conventional vaccines, it can also significantly increase the IgG, IgA, and IgM contents of experimental animals , the levels of cytokines such as IL-2, IL-4, IL-6 and the number of immune cells; the specific immune response to the vaccine was also significantly enhanced. Among them, the content of IgG, IgA, IgM and cytokines IL-2, IL-4, IL-6 in the injection group and oral administration group of oligonucleotides packaged with chitosan nanoparticles packaged with CpG motifs were significantly increased; Compared with the control group, the indicators of humoral immunity and cellular immunity in the intramuscular injection of unpackaged CpG oligonucleotide group were also significantly improved. After chitosan nanoparticles packaged oligonucleotides containing CpG motifs, although the amount of DNA was only 1/5 of that of the unpackaged group, the levels of humoral immunity and cellular immune response in the immunized group were significantly improved, which was significantly different from that in the control group ( P<0.05). Although the immune indexes of the mice in the chitosan nanoparticle-packed CpG oligonucleotide group were significantly higher than those in the control group, the amount of CpG oligonucleotides was 5 times that of the intramuscularly injected chitosan-coated group. .

These results show that oligonucleotides containing CpG motifs can significantly improve the immune response level of mice, and using chitosan nanoparticles as an immune delivery system has the advantages of reducing the amount of plasmids and enhancing humoral and cellular immunity. In the application, it was found that both oral immunization and intramuscular injection can effectively improve the immune response level of immunized mice to conventional vaccines. 5 can achieve a similar enhancement effect. These results suggest that chitosan nanoparticles coated with oligonucleotide sequences containing immunostimulatory sequence CpG can improve the level of cellular immunity and humoral immunity, enhance immune response and immune protection, and it is expected to be an effective new anti-infection immunomodulator , has potential wide application prospects.

Description of drawings

Table 1 Grouping of non-specifically immunized mice (CNP: chitosan nanoparticles)

Table 2 Grouping of mice immunized with vaccines (CNP: chitosan nanoparticles)

Table 3 CpG in vitro stimulation of animal blood immune cell proliferation response results (OD ₅₇₀ )

Figure 1 Transmission electron micrograph of CpG oligonucleotide chitosan nanoparticles (magnification: 50000)

Figure 2 Size distribution of chitosan nanoparticles

Figure 3 IgG content in the serum of mice in the non-specific immunization group

Figure 4 IgA content in the serum of mice in the non-specific immunization group

Figure 5 IgM content in the serum of mice in the non-specific immunization group

Figure 6 IL-2 content in the serum of mice in the non-specific immunization group

Figure 7 IL-4 content in the serum of mice in the non-specific immunization group

Figure 8 IL-6 content in the serum of mice in the non-specific immunization group

Figure 9 Changes in the number of white blood cells in mice in the non-specific immunization group

Figure 10 Changes in the number of monocytes in mice in the non-specific immunization group

Figure 11 Changes in the number of lymphocytes in mice in the non-specific immunization group

Figure 12 Changes in the number of neutrophils in mice in the non-specific immunization group

Figure 13 IgG content in the serum of mice in the vaccine immunized group

Figure 14 IgA content in the serum of mice in the vaccine immunization group

Figure 15 IgM content in the serum of mice in the vaccine immunization group

Salmonella-specific antibody content in the serum of mice in the vaccine immunization group in Figure 16

Figure 17 Hepatitis B vaccine-specific antibody content in the serum of mice in the vaccine immunization group

Figure 18 Escherichia coli-specific antibody content in the serum of mice in the vaccine immunization group

Figure 19 IL-2 content in the serum of mice in the vaccine immunization group

Figure 20 IL-4 content in the serum of mice in the vaccine immunization group

Figure 21 IL-6 content in the serum of mice in the vaccine immunization group

Figure 22 Changes in the number of white blood cells in mice in the vaccine immunization group

Figure 23 Changes in the number of monocytes in mice in the vaccine immunization group

Figure 24 Changes in the number of lymphocytes in mice in the vaccine immunization group

Figure 25 Changes in the number of neutrophils in mice in the vaccine immunization group

Detailed ways

1. Design and synthesis of CpG oligonucleotide sequence and its primers

CpG sequence design and synthesis:

CpG sequence (see the sequence list on page 11 of the instruction manual)

The sequence is 88 bases in length and contains 11 CpG motifs, which are characterized by 3 5'- AACGTT- 3', 3 5'- GTCGTC- 3', 1 5'- GACGTT- 3', One 5'- ATCGAT- 3', one 5'- GTCGTT- 3', one 5'- GGCGTT- 3', one 5'- GACGTC- 3' characteristic sequences.

CpG sequence primer design and synthesis:

5' end: CGAGATCTAACGTTGTC

3' end: CGAGATCTAACGTTAAC

2. Preparation of CpG oligonucleotides for immunization

Using the synthesized CpG sequence as a template, it is amplified by PCR (polymerase chain reaction), and the amplified product is dissolved in sterilized saline, and its concentration is measured by an ultraviolet spectrophotometer.

3. CpG oligonucleotides stimulate animal blood immune cell proliferation in vitro

Peripheral blood from pigs, yaks and yellow cattle was collected under sterile conditions, anticoagulated with heparin, and blood lymphocytes were separated with lymphocyte separation medium. Then 2ml of 2×10 ⁷ /ml single cell suspension was added to each well of a 6-well culture plate, and cultured for 48h. Target cells were prepared, and the concentration of target cells was adjusted to 8×10 ⁶ /ml with RPMI1640 complete medium containing 20 mg/ml α-MM. Add 50ul of target cells to each well of a 96-well cell culture plate, then add CpG oligonucleotides (0.6μg/well), CpG oligonucleotides (0.6μg/well) and Escherichia coli vaccine (1/500 ), CpG oligonucleotides (0.6 μg/well) and paratyphoid vaccine (1/500 head portion), each sample was set up with 4 replicate wells, and RPMI 1640 complete medium blank control was set up, placed in 5% CO ₂ , 37 ℃ cell culture incubator for 120h. The effect of CpG oligonucleotide and its synergistic vaccine on the proliferation of animal immune cells was detected by MTT method.

The results are shown in Table 3 of the attached description. Table 3 shows: CpG, CpG and Escherichia coli vaccine, CpG and paratyphoid vaccine three treatment groups can significantly stimulate the proliferation of lymphocytes, significantly different from the control group (P<0.05). Among them, the stimulating effects of CpG and E. coli vaccines were the most obvious.

4. Preparation of nanoparticles

Chitosan with a molecular weight of 150,000 and a deacetylation degree of over 95% is dispersed in 1% acetic acid solution (liquid A); CpG oligonucleotides are dissolved in tripolyphosphate solution (liquid B). Put liquid A and liquid B in a water bath at 50°C for 20 minutes, add the DNA solution into the chitosan solution in a certain proportion (ratio of the molar number of amino groups to the molar number of phosphate groups of DNA), stir it with magnetic force to make it fully mixed, and let it stand Composite particles with a diameter of 40-60 nm were obtained by filtration. That is, the CpG oligonucleotide chitosan nanoparticle sample solution.

5. Determination of morphology, particle size and Zeta potential of nanoparticles

Take a small amount of CpG oligonucleotide chitosan nano sample solution to observe the shape of nanoparticles under a transmission electron microscope and take pictures. Another appropriate amount of nanoparticle sample solution was taken, diluted with double distilled water, and then measured with a Zetasizer3000HS/IHPL nanoparticle size analyzer for particle size, dispersion and Zeta potential.

The transmission electron microscope observation results show that the chitosan nanoparticles are mostly spherical (see accompanying drawing 1 of the description). The measurement results of the nanoparticle size analyzer are: the average particle diameter is 45nm; the polydispersity is 0.190 (see accompanying drawing 2 of the specification); the zeta potential is +25.6mV, indicating that the surface of the nanoparticle has a positive charge.

6. Immunization of experimental animals

120 Kunming mice were randomly divided into 8 groups, 15 in each group. Among them, 4 groups were non-specific immunization groups, and 4 groups were vaccine immunization groups. By intramuscular injection, the mice were immunized by injecting the quadriceps muscles on both sides of the mice.

The CpG oligonucleotide immunization dose in the oral group was 30 pmol/head mouse, and the chitosan nanoparticle packaging group was 6 pmol/head animal; each mouse in the vaccine immunization group was inoculated with 1/40 head portion and the corresponding CpG oligonucleotide 30 pmol of acid or 6 pmol of corresponding CpG oligonucleotide packaged in chitosan nanoparticles. Five weeks later, each mouse was orally challenged with 0.4 ml of Escherichia coli K88 and K99 (3×10 ⁹ /ml). Animal immunization groups are shown in Table 1 and Table 2 of the accompanying instructions. Table 1 shows the grouping of non-specific immune components, and Table 2 shows the grouping of vaccine immune components.

7. The effect of chitosan nanoparticles packaged CpG oligonucleotides on the non-specific immune response of mice

Blood was collected from the tail vein of the mice before and after immunization every week, and the serum was separated to detect various immune indicators.

(1) Determination of IgG, IgA and IgM content

Experimental mouse serum was diluted to 10 ⁵ (IgG) and 10 ⁴ (IgM, IgA) respectively and coated on Costar microtiter plates, and specific rabbit anti-mouse IgG, IgM and IgA heavy chain antibodies (produced by Bethyl Company, USA) were used as primary antibodies. , Enzyme-labeled goat anti-rabbit IgG was used as the secondary antibody (Huamei Bioengineering Company), TMB (tetramethylbenzidine) was used as the substrate, and the contents of IgG, IgM and IgA in mouse serum were determined.

The results are shown in Figure 3, Figure 4, and Figure 5 of the description. It can be seen from the figure that after the mice were injected with CpG oligonucleotides, the levels of IgG, IgA, and IgM in the serum of the mice were significantly higher than those of the control group. After packaging CpG oligonucleotides with chitosan nanoparticles, although the amount of DNA was only 1/5 of that of the unpackaged group, the IgG, IgA, and IgM contents in the serum of the immunized group were still significantly increased, which was significantly different from that of the control group (P <0.05). Although the levels of IgG, IgA, and IgM in the serum of mice in the oral administration of chitosan nanoparticles packaged CpG oligonucleotides group were significantly higher than those in the control group, the amount of CpG oligonucleotides was significantly higher than that in the intramuscular injection of chitosan packaged group. The dosage is 5 times higher. After Escherichia coli challenge in the 6th week, the serum IgG, IgA and IgM contents of mice in the immunized group increased significantly compared with those in the control group.

(2) Detection of cytokines

Blood was collected from the tail vein of the mice before and after immunization every week, and the serum of the experimental mice was diluted 100 times to coat the Costar microtiter plate, and the rabbit anti-mouse IL2, IL4, IL6 IgG antibody (provided by Wuhan Boster Bioengineering Company) was used as the primary antibody. TMB (tetramethylbenzidine) was used as the substrate, and the contents of IL2, IL4 and IL6 in serum were detected by SABC method, and the cellular immune response of mice was observed.

The results are shown in accompanying drawings 6, 7 and 8 of the description. It can be seen from the figure that the contents of IL-2, IL-4 and IL-6 in the serum of mice in the oligonucleotide immunization group containing CpG motifs were significantly higher than those in the control group (P<0.05). Compared with the non-encapsulated group, the particle-packed CpG oligonucleotide group had no significant difference in the increase of IL-2, IL-4 and IL-6 levels in serum (P>0.05). Oral administration of chitosan nanoparticles packaged CpG oligonucleotides can also effectively increase the levels of IL-2, IL-4 and IL-6 in serum, but the amount of CpG oligonucleotides in the oral group is lower than that in the intramuscular injection group. 5 times.

(3) Dynamic changes in the number of immune cells in the peripheral blood of immunized mice

Blood was collected from the tail vein of the mice before and after immunization every week, and the total number of white blood cells was counted by conventional methods; blood smears were stained with Giemsa, and neutrophils, mononuclear nuclei, and lymphocytes were classified and counted under a microscope.

The results are shown in Figures 9, 10, 11 and 12 of the description. The dynamic change of the number of immune cells in the peripheral blood of immunized mice is an important index to investigate the cellular immune response of mice. The results showed that after the mice were immunized with CpG oligonucleotides, the number of peripheral blood leukocytes, monocytes, and lymphocytes in the immunized mice increased significantly compared with the control group, and the number of neutrophils also increased significantly compared with the control group before challenge. High, but the number of neutrophils after challenge was lower than that of the control group; after immunizing mice with chitosan nanoparticles packaged with CpG oligonucleotides, it was found that the amount of DNA used was reduced by 5 times, and the same To achieve the effect of increasing the number of immune cells.

8. Regulation of vaccine-specific immune response by packaging CpG oligonucleotides in chitosan nanoparticles

(1) Determination of IgG, IgA and IgM content

Experimental mouse serum was diluted to 10 ⁵ (IgG) and 10 ⁴ (IgM, IgA) respectively and coated on Costar microtiter plates, and specific rabbit anti-mouse IgG, IgM and IgA heavy chain antibodies (produced by Bethyl Company, USA) were used as primary antibodies. , Enzyme-labeled goat anti-rabbit IgG was used as the secondary antibody (Huamei Bioengineering Company), TMB (tetramethylbenzidine) was used as the substrate, and the contents of IgG, IgM and IgA in mouse serum were determined.

The results are shown in Figures 13, 14, and 15 of the description. Figures 13, 14, and 15 show the changes in serum immunoglobulins in mice after mice were immunized with CpG oligonucleotides, hepatitis B vaccine, typhoid fever vaccine and Escherichia coli vaccine. The results showed that: after being immunized with CpG oligonucleotides at the same time, the IgG, IgA, and IgM contents of the experimental animals were significantly increased compared with those of the control group. Among them, chitosan nanoparticles packaged CpG oligonucleotides, and then combined with hepatitis B vaccine, typhoid vaccine and Escherichia coli vaccine to inject mice, the results showed that although the amount of DNA used by this immunization method was only 1% of that of the unpackaged group /5, but can cooperate with vaccine immunization, its IgG, IgA, IgM content is significantly increased compared with the control group (P<0.05), and there is no significant difference with the packaging immunization group (p>0.05). Oral administration and intramuscular injection of chitosan nanoparticles packaged CpG oligonucleotide group had similar increases in serum IgG, IgA, and IgM levels, but the oral administration group required a larger dose of DNA.

(2) Determination of specific antibodies

Prepare hepatitis B antigen, Salmonella antigen, Escherichia coli antigen coated Costar microtiter plate, experimental mouse serum 100 times diluted as primary antibody, TMB (tetramethylbenzidine) as substrate, SABC method for determination of hepatitis B specific antibody in mouse serum , typhoid-specific antibody, Escherichia coli specific antibody content.

The results are shown in Figures 16, 17, and 18 of the description. Figures 16, 17 and 18 show the changes in the specific antibodies of the experimental animals after the hepatitis B vaccine, typhoid vaccine and Escherichia coli vaccine were co-immunized with the corresponding CpG oligonucleotides. The results showed that: one week after the animals were immunized, the production of specific antibodies was detected; after simultaneous immunization with CpG oligonucleotides, the above-mentioned specific antibodies of the experimental mice were significantly higher than those of the control group (P<0.05). After packaging CpG oligonucleotides with chitosan nanoparticles and co-injecting immunized mice with vaccines, the CpG oligonucleotides can improve the specificity of experimental animals to vaccines when the amount of nucleic acid is only 1/5 of the unpackaged group. The effect of sexual immune response is also significant. Among them, the way of intramuscular injection is better for raising the specific antibody of hepatitis B vaccine and typhoid vaccine, while the way of oral immunization is slightly better for the increase of specific antibody of Escherichia coli.

(3) Detection of cytokines

Blood was collected from the tail vein of the mice before and after immunization every week, and the serum of the experimental mice was diluted 100 times and coated with a Costar microtiter plate. Rabbit anti-mouse IL2, IL4, and IL6 IgG antibodies (provided by Wuhan Boster Bioengineering Co., Ltd.) were used as primary antibodies. TMB (tetramethylbenzidine) was used as the substrate, and the contents of IL2, IL4 and IL6 in serum were detected by SABC method, and the cellular immune response of mice was observed.

The results are shown in Figures 19, 20, and 21 of the description. The results showed that after CpG oligonucleotide combined with hepatitis B vaccine, typhoid fever vaccine and Escherichia coli vaccine to immunize mice, the levels of IL-2, IL-4 and IL-6 in serum of mice were higher than those of the control group. Compared with the unpackaged group, the packaged DNA was only 1/5 of the amount of the unpacked group, but it also played a role in improving the serum IL- 2. The role of IL-4 and IL-6 content. There was no significant difference between the oral group and the intramuscular injection group (P>0.05).

(4) Dynamic changes in the number of immune cells in peripheral blood of immunized mice

Blood was collected from the tail vein of the mice before and after immunization every week, and the total number of white blood cells was counted by conventional methods; blood smears were stained with Giemsa, and neutrophils, mononuclear nuclei, and lymphocytes were classified and counted under a microscope.

The results are shown in Figures 22, 23, 24, and 25 of the description. With hepatitis B vaccine, typhoid vaccine and Escherichia coli vaccine combined with CpG oligonucleotides to immunize mice, the changes in the number of immune cells in the peripheral blood of the mice were also found that oral administration and intramuscular injection of CpG oligonucleotides all affected the peripheral blood of experimental mice. Blood immune cells have a better ability to improve; but the number of neutrophils in mice after challenge is slightly lower than that in the control group; white blood cells, lymphocytes, and neutrophils in mice in the immune group reached the highest value 14 days after immunization. Monocytes reached the highest value at 49 days after immunization. Packaging CpG oligonucleotides with chitosan nanoparticles can not only reduce the amount of DNA to 1/5 of the unpackaged amount, but also effectively increase the number of peripheral blood immune cells in mice. Both oral and intramuscular injection immunization methods can effectively increase the number of immune cells, but the amount of DNA in the intramuscular injection method is 4/5 less than that in the oral method.

9. Detection of organ lesions in challenged mice

Thirty-five days after the mice were immunized, the experimental mice were orally challenged with Escherichia coli K88 and K99, and the growth of the mice was observed; on the 49th day, all mice were dissected by conventional methods, and the physiological lesions of various organs were observed.

After 3 weeks of inoculation, the mice were dissected and found that the mice inoculated with CpG oligonucleotides were healthy and survived, and there were no obvious lesions in the liver, spleen, duodenum, and other digestive tract and respiratory tract tissues and organs, while the control mice had no obvious lesions. Onset, lethargy, erect coat, thin and soft stools with brown color; autopsy observation found that the mice in the control group had bleeding, hepatosplenomegaly and other lesions mainly in the stomach, duodenum, jejunum mucosa .

10. Data Analysis

All the above-mentioned data were compared by variance analysis, and P<0.05 was taken as the standard of significant difference.

CpG sequence list

<110>Sichuan University

<120> Preparation and application of oligonucleotide anti-infection gene preparation containing CpG motif

<160>1

<210>1

<211>88

<212>DNA

<213> Artificial sequence

<220>

<400>1

cgagatctaa cgttgtcgtc gacgtcgtcg tcaggcctga cgttatcgat ggcgttgtcg 60

tcaacgttgt cgttaacgtt agatctcg 88

Table 1 Grouping of non-specifically immunized mice group Immunization program A1 CpG intramuscular injection inoculated mice A2 CNP-encapsulated CpG intramuscularly injected mice B CNP-encapsulated CpG orally vaccinated mice C normal saline

Note: CNP: chitosan nanoparticles

Table 2 Grouping of vaccine immunized mice group Immunization program YA1 CpG intramuscular injection group mice+vaccine YA2 GNP-encapsulated CpG intramuscular injection of mice + vaccine YB GNP-encapsulated CpG orally vaccinated mice + vaccine YC Normal saline + vaccine

Note: CNP: chitosan nanoparticles

Table 3 CpG in vitro stimulation of animal blood immune cell proliferation response results (OD ₅₇₀ ) deal with yak cattle pig CpG 0.293± ^0.012c 0.280± ^0.011c 0.288± ^0.059c control 0.041±0.019 ^d 0.046±0.009 ^d 0.055± ^0.015d CpG+ E. coli vaccine 0.645±0.024 ^a 0.798±0.093 ^a 1.041±0.089 ^a coli vaccine 0.368± ^0.036b 0.545± ^0.021b 0.643± ^0.107b CpG+ paratyphoid vaccine 0.428± ^0.029b 0.495± ^0.028b 0.448± ^0.061b Paratyphoid vaccine 0.235± ^0.019c 0.248± ^0.022c 0.275± ^0.031c

Claims (4)

1, contain the preparation and the application of the oligonucleotide anti-infectious immunity preparation of CpG motif, the preparation, chitosan nano particle that its characteristics comprise synthetic, the chitosan nano particle of the oligonucleotide that contains the CpG motif is applied to the technology that animal infection enhancing human body immunity such as pig, cattle, yak are replied to the branch subpack of the oligonucleotide that contains the CpG motif and with this chitosan CpG nano-particle immune formulation.

2, the preparation of CpG oligonucleotide anti-infectious immunity preparation according to claim 1, the substrate that it is characterized in that described immune formulation be synthetic contain 11 CpG characteristic sequences, length is the oligonucleotide of 88 bases, its characteristics contain 35 '- AACGTT-3 ', 35 '- GTCGTC-3 ', 15 '- GACGTT-3 ', 15 '- ATCGAT-3 ', 15 ' -GTCGTT-3 ', 15 '- GGCGTT-3 ', 15 '- GACGTC-3 ' characteristic sequence.

3, the preparation that contains the oligonucleotide anti-infectious immunity preparation of CpG motif according to claim 1, the lapping that it is characterized in that described immune formulation is that molecular weight is 150000, the chitosan of deacetylation more than 95% prepares chitosan CpG nano-particle molecule with ionic cross-linking.

4, the chitosan packing of using claim 1 to require contains the application technology of the oligonucleotide nano-particle of CpG motif as animal novel anti-infection molecular immune regulators such as pig, cattle, yaks, it is characterized in that following several respects:

(1) with the mice be laboratory animal, with the preparation chitosan CpG oligonucleotide nano-particle with two kinds of different delivery modes (oral and intramuscular injection) immunity, compared of the influence of different immunization routes to immune effect;

(2) with the chitosan CpG oligonucleotide nano-particle nonspecific immunity laboratory animal of preparation, the result shows that chitosan CpG nano-particle can effectively improve laboratory animal cell and humoral immunity level, strengthens its non-specific immunity;

(3) the chitosan CpG nano-particle combined vaccine (Hepatitis B virus vaccine, antityphoid vaccine and bacillus coli vaccine) with preparation is total to the immunization experiment animal, and the result shows that chitosan CpG oligonucleotide nano-particle can significantly strengthen cellular immunization and the humoral immunoresponse(HI) reaction of laboratory animal to vaccine.

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