WO2010081257A1

WO2010081257A1 - Composition comprising bacillus calmette guerin polysaccharides and bacillus calmette guerin nucleic acids and use of preparing medicament thereof

Info

Publication number: WO2010081257A1
Application number: PCT/CN2009/000069
Authority: WO
Inventors: 宁云山; 关继峰
Original assignee: 九芝堂股份有限公司
Priority date: 2009-01-16
Filing date: 2009-01-16
Publication date: 2010-07-22
Also published as: CN102238959A; CN102238959B

Abstract

A composition comprising Bacillus Calmette Guerin polysaccharides and Bacillus Calmette Guerin nucleic acids is provided, wherein said polysaccharides are comprising 10%-69% by mass of the composition and said nucleic acids are comprising 30%-89% by mass of the composition. The composition is made by mixing said polysaccharides and said nucleic acids, wherein said polysaccharides and said nucleic acids are obtained from the Bacillus Calmette Guerin cultures by the following process: extracting the mixture of said polysaccharides and said nucleic acids from said Bacillus Calmette Guerin cultures, and separating said polysaccharides and said nucleic acids from the mixture by ion exchange chromatography. The use of the composition comprising said polysaccharides and said nucleic acids for preparation of a biological medicament for treatment or prevention of microorganism infections, tumors, allergic reactions and the like is also provided.

Description

TECHNICAL FIELD The present invention relates to a composition comprising a BCG polysaccharide and a BCG nucleic acid and the use of the composition. BACKGROUND OF THE INVENTION Bacillus Calmette-Guerin is a bovine attenuated tuberculosis, non-pathogenic and immunogenic. It is the most widely used strain in the world by using Bacillus vaccination instead of the first infection of tuberculosis to obtain immunity against tuberculosis. One of the seedlings.

In 1882, Robert successfully isolated tubercle bacilli and identified the pathogens of tuberculosis. In 1908, Calnette and Guerin of the Pasteur Institute in France successfully developed an attenuated Bovine Mycobacterium tuberculosis, named Bacillus Calmette-Guerin (BCG). In 1921, Weill-Halle successfully applied the bacteria to the human body to prevent tuberculosis. In 1971, the Changsha prevention and treatment group of bronchitis first used the BCG scratch method to prevent chronic bronchitis, asthma, rheumatoid arthritis and achieved good results. In 1987, Hunan Jiuzhitang Siqi Bio-Pharmaceutical Co., Ltd. (formerly Changsha Shenjian Pharmaceutical Factory) cooperated with Changsha Medical Laboratories to conduct BCG extraction based on the research of Professor Tan Lizhi, a famous respiratory disease expert at Hunan Medical University. The study of the substance, using the hot phenol method to remove the bacterial protein, and then using ethanol precipitation to extract the bacterial polysaccharide and nucleic acid and a small amount of protein mixture, made a significant effect in clinical application. In 1994, Hunan Jiuzhitang Siqi Bio-Pharmaceutical Co., Ltd. cooperated with China National Institute of Biological Products to improve the process, improve the quality standards, further reduce the side reaction of this product, develop a new generation of Bacillus extract, and make the product enter The 95th edition, 2000 edition of the "Chinese Biological Products Regulations", and named as BCG polysaccharide, nucleic acid injection, the preparation is a mixture of polysaccharides, nucleic acids and proteins, wherein the polysaccharide is about 70% ~ 80%, the nucleic acid is 10% ~ 20%, the protein content is less than 1%. Since the introduction of BCG polysaccharide and nucleic acid injection, it has become a hot spot in clinical application research. Domestic clinical studies have shown that BCG polysaccharides and nucleic acid injections have Extensive immunomodulatory effects, in addition to enhancing cellular immune function, also have two-way immunomodulatory effects. It has a good effect in preventing and treating respiratory diseases such as colds, asthma, allergic rhinitis and skin diseases such as eczema, urticaria, flat warts, warts, and condyloma acuminata.

The results of modern biochemistry, immunology, genomics and proteomics have revealed the material basis and mechanism of action of M. tuberculosis (Bacillus) in the prevention and treatment of diseases such as respiratory tract and skin diseases. Studies have shown that the main components of BCG include lipids, polysaccharides, proteins and nucleic acids, in which polysaccharides and nucleic acid components play important physiological functions. In Bacillus, polysaccharides (mainly including peptidoglycan, arabinogalactan and mycolic acid) and lipids often bind together to form a major component of the cell wall. Current studies indicate that there are ligands that bind to human and animal Toll receptors (TLRs) on BCG cell walls and other organelles. These ligands bind to at least five Toll receptors (TLR1, TLR2, TLR4, TLR6, TLR9). When they bind to TLR to promote the maturation of dendritic cells (DC) and induce the secretion of IL-12, IL-12 promotes the transformation of ThH cells into a subset of TH _H cells and IFN-γ secretion, thereby producing T _H 1 Predominant non-specific immune response (J Exp Med 2003; 197: 403-411) ₀

CpG motifs (CpG motifs) are a class of oligodeoxynucleotides (ODN) with unmethylated cytosine and guanine nucleotides (CpG) as the core. The base arrangements mostly follow the following rules: 5T _Ur P _Ur CGPyPyr3', that is, two 嘌呤 at the 5' end and two pyrimidines at the 3' end. This sequence activates a variety of immune effector cells and is therefore referred to as the immuno-stimulatory DNA sequence (ISS). Yamamoto et al. (Yamamoto ST et al. J Immunol. 1991; 148: 4072) first reported that a 30-base oligodeoxynucleotide (ODN) isolated from BCG (BCG) activates the killing activity of mouse NK cells. Stimulation of its Y-interferon secretion is thought to be due to a palindrome sequence containing six bases in the ODN. In 1995, Krieg et al. found that synthetic and bacterial-derived DNA fragments containing unmethylated CpG activated B cell proliferation, differentiation, and production of antibodies. Comparison of bacterial, viral, and vertebrate (including human) genome sequences revealed that bacterial, viral genomic DNA is rich in CpG motifs, whereas vertebrate DNA is relatively lacking in CpG motifs. Among the analyzed bacterial genomes, M. tuberculosis contains the highest proportion of CpG motifs (analytical analysis of M. tuberculosis H37Rv genome data by bioinformatics techniques: GC% content in the M. tuberculosis H37Rv genome It is about 66.1%, and the percentage of CpG is The amount is about 22%).

Numerous studies have confirmed that the bacterial genome DNA and synthetic CpG ODNs rely mainly on the CpG motif in their sequence to activate the cellular and immune responses of specific and non-specific systems. Bacterial CpG DNA activates a variety of immunoreactive cells, including monocytes/macrophages, natural killer cells (NK), dendritic cells (DC), B cells, and T cells, in vitro and in vivo, stimulating the secretion of immune-active cells. cytokines and to increase certain surface molecules, chemokines, adhesion molecules, and costimulatory molecules, induction of innate immune system activation and antigen-specific adaptive immune response, Similarly, CpG DNA can induce T _H type 1 immune response, inhibition of T _H 2 type immune response, regulating T _H 1 / T _H 2 balance, because the correction T _H 1 / T _H 2 disease caused by imbalance plays an important role. Currently, CpG DNA is mainly used in the following research areas:

1. The role of prevention and treatment of infectious diseases

The ability of CpG DNA as an immunoadjuvant to enhance the immunity of vaccines to the body has been demonstrated in a variety of animal models. The co-injection of Davis and other CPG ODNs with the HBV vaccine to orangutans has a serum yang conversion rate of 100%, which is significantly higher than the 15% seroconversion rate of the single injection of HBV vaccine. Since orangutans are genetically very similar to humans, this positive result indicates that CpG-ODN can be developed into a successful human adjuvant (1⁄2zcd« 2000, 18 (18): 1920-1924. Currently, CpG ODN has been adjuvanted. In human clinical trials, preliminary results indicate that seroconversion can be greatly accelerated and antibody titers can be increased without significant adverse reactions. In addition, the effects of CpG-ODN and recombinant HBsAg on transgenic mice with chronic asymptomatic carriers of HBV were observed and CpG was found. After DNA and HBsAg interacted with the experimental animals, the antigen-specific antibodies in the animals increased significantly. Transplanting CD8 ⁺ T cells into another transgenic mouse can eliminate HBsAg in the recipient mice, indicating that CpG DNA is used in the treatment of chronic HBV infection. It has potential application value (J 'ra/, 2001, 75 (14); 6482-6491:). CpG DNA as an immunoadjuvant has also shown great potential in HIV vaccine research. It will contain CpG motifs. The plasmid stimulates DC cells in vitro, and then co-injects it with the HIV coat protein into Balb/c mice, which can significantly enhance the ability of HIV-specific cellular and humoral immunity in mice (1⁄2zcd). « 2S57-2Sd5. ). In addition, the gp-120/CpGODN linker can significantly stimulate mouse-specific immune responses, including the induction of large amounts of a chemokine, specific antibody production, and specific CTL responses ( / Immunol, 2001, 167 (3 ): 1584-1591. ) 2. Research on CpG-DNA in tumor immunotherapy

Experiments have shown that CpG DNA is an effective adjuvant for tumor vaccines, which can induce protective immunity in the body and thus resist the attack of lethal tumors. Auf et al. reported that CpG DNA can effectively fight tumor development by activating macrophages and plays an important role in early tumor treatment (Clin Cancer Res, 2001, 7 (11) · 3540-3543). In addition, Sipos et al reported that CpG ODN was co-injected with antigen-stimulated DC cells into colon cancer-bearing mice, which significantly increased the anti-tumor activity of DC; and systemic administration inhibited tumor growth compared to 5-FU and hyperthyroidism. 7th Intenational Symposinum on dendritic cell [C], Germany, 2002, Austria: Robidruck, 2002.29. ) Similarly, CPG DNA inhibits apoptosis of WHEI231 B lymphocytes and increases immunocompetent cells (eg spleen cells, macrophages) The ability of cells and hematopoietic cells tolerate λ-rays, thereby enhancing the tolerance of tumor patients to radiotherapy and improving the efficacy of tumor radiotherapy. (Mol Immunol, 2006, 43c8: 1163-71.

3. Research on CpG-ODN in allergic diseases

Allergic diseases (allergic diseases) are caused by environmental allergens inducing a T _H 2 type immune response in the body. Since CpG DNA strong T _H 1 immune response enhancer effect, not only to prevent the occurrence of type T _H 2 immune response, and can make T _H 2 type immune response has occurred steering T _H 1-type immune responses. Sur S et al demonstrated 48h after CpG DNA vaccination in mice sensitive to ragweed in mice by synthetic T _H 2-type cytokine suppressing allergen specific IgE production by reducing the number of cells, significantly reduced allergic response, and there It has a specific immune memory effect, suggesting that CpG DNA can be used to immunize the body with specific antigens of allergic diseases, in order to prevent and treat the corresponding allergic diseases. Immunol, 1999, 162 (10): 6284-6293) CpG DNA is not only in the mouse model of asthma Prevents the occurrence of allergic diseases and prevents allergic reactions in sensitized mice when allergens are encountered; through studies on various animal models of asthma, it is found that whether administered intraperitoneally or intranasally, whether it is Before or after induction, CpG DNA can completely prevent allergen-induced asthma eosinophilia, reduce bronchial hyperresponsiveness, inhibit lung and systemic T _H 2 immune response, reduce allergies The original induced IL-4 level and IgE synthesis, especially in the case of simultaneous use of CpG DNA and antigen, is particularly effective, and more meaningful is provided by CpG DNA. Protection can last for a considerable period of time (J Clin Immunol, 2001, 21: 175-182; Wild JS Allergy 2001, 6: 365-376; J Allergy Clin Immunol 2002; 111: 706- 7l.

In summary, bacterial CPG DNA or synthetic CPG DNA is used in the prevention and treatment of microbial infections (alone or as an immunoadjuvant), immunotherapy or adjuvant therapy of tumors, and correction of allergic reactions to T _H 1/T _H 2 cell imbalance. The disease will have a very broad application prospect.

Currently, the Bacillus polysaccharide and nucleic acid injections which have been marketed are polysaccharide nucleic acid preparations extracted by Bacillus subtilis by hot phenol method, and the preparation is a mixture of polysaccharides, nucleic acids and proteins, wherein the polysaccharide is about 70% to 80%, nucleic acid. It is 10%~20%, and the protein content is less than about 1%. According to the data of the current research, the preparation has the advantages of slow effect and short effective duration in clinical application. The ratio of polysaccharide to nucleic acid in the preparation is determined by the level of BCG polysaccharide and nucleic acid extraction process at that time. Whether the ratio is appropriate and optimal is not optimized by scientific methods. Therefore, research on the ratio of polysaccharides to nucleic acids in BCG polysaccharides and nucleic acid mixtures will be widely used in the treatment or prevention of improving the efficacy of existing BCG polysaccharides and nucleic acid injections or for the development of therapeutic effects, long-lasting and safe. A new generation of BCG-derived preparations such as infectious diseases, allergic diseases and tumors provides a material basis. SUMMARY OF THE INVENTION One object of the present invention is to provide a composition comprising a BCG polysaccharide and a BCG nucleic acid which can exert different effects in different diseases, the Bacillus polysaccharide and the BCG nucleic acid in the composition. The ratio can be ratio adjusted as needed; another object of the present invention is to provide the use of the above composition comprising a BCG polysaccharide and a BCG nucleic acid.

The present invention provides a composition comprising a BCG polysaccharide and a BCG nucleic acid, wherein the mass percentage of the polysaccharide in the composition is 10% to 69%, and the mass percentage of the nucleic acid in the composition is 30%~ 89%.

The above composition is prepared by mixing a BCG polysaccharide and a BCG nucleic acid; wherein the BCG polysaccharide and the BCG nucleic acid are extracted from the BCG culture by the following method:

(1) extracting a BCG polysaccharide and a nucleic acid mixture from a BCG culture;

(2) separating Bacillus polysaccharides from BCG polysaccharide and nucleic acid mixture by ion exchange chromatography; (3) mixing the BCG polysaccharide obtained in the step (2) with the BCG nucleic acid in proportion.

Wherein, the above step (1) may include the following steps:

(11) inoculating the Bacillus species in a medium suitable for the growth of mycobacteria to collect the cells in the log phase;

(12) the above-mentioned cells are crushed, and the supernatant is collected by centrifugation;

(13) After the supernatant is extracted with an organic solvent, it is removed by dialysis or gel chromatography. The ion exchange chromatography described in the above step (2) may be column chromatography using an anion exchange medium. the anion exchange medium is preferably _Q Seph arase ^TM XL.

The above step (2) may include the following steps:

(21) loading the BCG polysaccharide and the nucleic acid mixture obtained in the step (1), and simultaneously collecting the flow-through liquid;

(22) After the sample is finished, the column is washed with physiological saline, the column washing liquid is collected, and the flow-through liquid and the column washing liquid are combined;

(23) using the desalting column for desalting to obtain a BCG polysaccharide;

(24) Step (22) After the completion of the washing column, the elution is carried out by using a sodium chloride solution having a concentration of 0.1 to 2 mol/L to collect the elution peak;

(24) The collected eluate is desalted using a desalting column to obtain a BCG nucleic acid. The present invention also provides the use of the above composition comprising a BCG polysaccharide and a BCG nucleic acid for the preparation of a medicament for treating or preventing a microbial infection, a tumor or an allergic disease.

The composition comprising the BCG polysaccharide and the BCG nucleic acid provided by the invention can regulate the cellular immune function of the body, activate the mononuclear-macrophage system and induce cytokines such as interferon, thereby regulating humoral immunity, and having antiviral activity. , anti-inflammatory, anti-allergic and other extensive immunomodulatory effects, more importantly, its immunomodulatory function is significantly better than the listed BCG nucleic acid nucleic acid injection, so the present invention provides Bacillus polysaccharides and cardinal The composition of the bacterial nucleic acid can be formulated into a novel biological preparation for treating or preventing diseases such as microbial infections, tumors, allergies and the like. The invention provides a composition comprising a BCG polysaccharide and a BCG nucleic acid, and an important feature is a natural product derived from Bacillus Calmette-Guerin (Bacillus Calmette-Guerin), Bacillus Calmette-Guerin or Bacillus polysaccharide, and a nucleic acid preparation. The human clinical application for decades has shown good safety and effectiveness in the prevention and prevention of diseases such as microbial infections, tumors, allergies, etc., thus providing the possibility of developing new biological agents. BEST MODE FOR CARRYING OUT THE INVENTION The innovation and application of the present invention will be described in detail below by way of specific examples and experimental results, in order to help the reader to better understand the spirit and substance of the invention, but not to limit the scope of the invention.

Example 1 Cultivation and harvesting of BCG

(1) Bacterial culture: The strains cryopreserved in liquid (Chinese BCG strain D2PB302, supplied by the Chinese Medicine and Biological Products Laboratory) were dissolved at room temperature and inoculated into potato Sutong medium at 37 °C. Continuous culture for 14-20 days; or after 15 days of continuous culture at 37 ° C, transfer to modified liquid Sutong medium, continuous culture at 37 ° C for 14-20 days.

Among them, the preparation method of the potato Sutong medium can be:

Wash fresh potatoes (1), pierce them into a cylinder with a puncture, and cut into 4 cm long bevels with a knife;

Rinse the potato bevel with running drinking water for 1 hour;

Rinse the potato bevel block with purified water;

Flush the bevel block with Sutong medium;

Take 20 ml of Sutong medium and add it to a 100 ml sterilized large tube; place the washed potato slant into a sterilized large tube containing Sutong medium;

O.llMPa atmospheric pressure 121 ° C, sterilization for 20 minutes. Allow to cool to room temperature to be inoculated Sutong medium preparation ratio:

Every 1000ml: : Large 0.5g dipotassium hydrogen phosphate 1.04g

8.0g citric acid 2.0g glycerin 60ml 10% ferric ammonium citrate 0.5ml Add purified water to ammonia water to ρΗ8.0

Improved liquid sutong based formulation and preparation:

Aspartate (AR): 4g

Limonic acid (AR) : 2g

K2HP043H20 (AR) : 0.5g

MGS047H20 (AR) : 0.5g

Ammonium ferric citrate (CP): 0.05g

Glycerin (medicinal): 60ml

Distilled water: 940ml

Then add 1% (w/v) zinc sulphate lml, 10 lbs for 20 minutes.

(2) Collection of bacteria: When the cells are grown to the logarithmic phase, after the bottles are inspected, the membrane is collected, washed with deionized distilled water, dried, and weighed.

Example 2 Preparation of Bacillus polysaccharides and nucleic acid mixture

(1) Preparation method 1: Dialysis method

1. Cell disruption and hot phenol treatment: The cells collected in Example 1 were added to purified water in a ratio of 10:1, and the cells were disrupted by tissue mashing machine (12000 rpm/min) for 3 min×3 times. The body is mashed, and then the same amount of hot phenol (60 to 65 ° C) as the suspension of the broken bacteria is added, and the mixture is kept for 30 minutes to 1 hour under low-speed stirring.

2. Extraction of BCG polysaccharide nucleic acid mixture: The mixture of hot phenolic mixture is naturally precipitated for 1 to 10 days, the supernatant is aspirated, and the supernatant is loaded into a dialysis bag after centrifugation in a tube centrifuge (molecular weight cutoff > 5000 dols) The phenol was removed by dialysis for 1 to 10 days in 100 volumes of water for injection. Adding an appropriate amount of ethanol to the dialysis liquid to make the alcohol content 60~85%, natural precipitation for 1~10 days, precipitation The mixture was thoroughly stirred with absolute ethanol and washed by centrifugation 3 times. After centrifugation with diethyl ether for 3 times, it is dried in a desiccator and dried for 2 to 5 days to form a BCG polysaccharide nucleic acid mixture.

(2) Preparation method 2: gel chromatography

1. Cell disruption and hot phenol treatment: The cells collected in Example 1 were added to purified water in a ratio of 10:1, and the cells were disrupted by tissue mashing machine (12000 rpm/min) for 3 min×3 times. The body is mashed, and then the same amount of hot phenol (60~65 ° C) as the suspension of the broken bacteria is added, and the mixture is kept in a low speed stirring for 30 minutes to 1 hour.

2. Extraction of BCG-polysaccharide nucleic acid mixture: The mixture of hot phenolic mixture is naturally precipitated for 1 to 10 days, the supernatant is aspirated, and after centrifugation in a tube centrifuge, the supernatant is applied to the supernatant through a purification system at a volume of 30%. Pre-equilibrated gel column (GH-25 or G-25 for chromatography, 0.9% saline for balance), effluent was detected by UV spectrometer (wavelength at 260 nm or 280 nm), and the peak of interest was collected. The collected peaks of interest are filtered through a 0.45 μηι sterile filter, which is a BCG polysaccharide, a nucleic acid mixture, and stored at 4 ° C or -20 ° C until use.

Example 3 Preparation of Bacillus polysaccharides and Bacillus Calmette from a BCG-polysaccharide nucleic acid mixture

1. Preparation of ion exchange chromatography column: 1L~100L ion exchange packing material (Q SepharoseTM XL) is loaded into the corresponding chromatography column according to the method provided in the manual, and is sprayed with 2~10 times column volume of water for injection. Wash the column and equilibrate the column with 2 to 10 column volumes of saline for injection;

2. Isolation of polysaccharides and nucleic acids from the BCG polysaccharide nucleic acid mixture: 10 column volumes of the BCG polysaccharide and nucleic acid mixture prepared by the second embodiment were loaded while collecting the flow through. After the loading is completed, the column is washed with 2 column volumes of physiological saline, the column washing liquid is collected, the flow-through liquid and the washing column are combined, and named as component I (Bacillus polysaccharide), and sterilized by 0.45 μηι The filter is filtered and stored at 4 ° C or -20 ° C until use. After washing the column, use lmol/L sodium chloride solution

(Water for injection) Continuous elution was carried out and detected by an ultraviolet spectrometer (wavelength at 260 nm or 280 nm), and the eluted peak was collected and designated as component II (Bacillus acid). The desalting column is used for desalting (the mobile phase is physiological saline for injection), and the desalted component II is filtered through a 0.45 μηι sterile filter and stored at 4 ° C or -20 ° C until use. Example 4 Detecting and identifying the prepared BCG polysaccharide and BCG nucleic acid components According to the method provided by the present invention, three batches of BCG polysaccharide and BCG nucleic acid components are continuously prepared from Bacillus culture. Bacillus polysaccharides and Bacillus Calmette-Guerin were prepared from the components I and II obtained in the third example by the standard methods provided in the Chinese Biological Products Regulations (2000) and Molecular Cloning, Third Edition. Identification and content determination of nucleic acids, the results are shown in Table 1. Table 1 Bacillus polysaccharides and BCG nucleic acid composition determination results Polysaccharide nucleic acid CpG Volume polysaccharide content Nucleic acid content Nucleic acid purity

Project proportion proportion content

(ml) (ug/ml) (ug/ml) A260/A280

( ) ( ) BCG

Glucose and nucleic acid mixing 600ml 3400 1280 — 72.65 27.35

Fluid

Bacillus

979.19 1739.45 55 96.94 3.06 —— Bacillus sclerotium

486.29 63.275 792.5 1.86 7.98 92.6 17.6 acid

Bacillus

Glucose and nucleic acid mixing 600ml 3400 1280 — 72.65 27.35 —

Bacillus

913.67 1807.75 23.75 98.7 1.03 - Bacillus nucleus

353.66 85.27 812.6 1.89 9.62 90.38 18.3 Acid

Bacillus

Glucose and nucleic acid mixing 600ml 3400 1280 — 72.65 27.35 —

Bacillus

955.18 1955.6 75 96.28 3.72 —— Bacillus nucleus

393.4 81.3 935 1.83 7.98 92.2 19.6 Acid Note:

1. Determination of polysaccharide content: Refer to the Enketone method of the Chinese Biological Products Regulations (2000).

2. Determination of nucleic acid content: Reference "Molecular Cloning 3rd Edition".

3. Determination of nucleic acid purity: Reference "Molecular Cloning 3rd Edition".

4. Determination of protein content: Refer to the lowry method of the Chinese Biological Products Regulations (2000).

5. Determination of CpG content in nucleic acids:

(1) Methylation modification reaction

The methylation modification reaction was carried out in strict accordance with the method provided by the supplier, that is, the DNA was treated with Sssl methylase in a suitable NEBuffer, S-adenosylmethionine (SAM) was added at 37 ° C, and SAM was supplemented every 4 hours for 24 hours. After phenol: chloroform: isoamyl alcohol (25:24:1) extraction 2 times, chloroform: isoamyl alcohol (24:1) extraction 2 times, respectively, diethyl ether, ethanol precipitation, 70% ethanol washing, air drying , dissolved in TE buffer.

(2) Detection of the degree of methylation modification

DNA stock and methylase-treated DNA were treated with restriction endonuclease Hpa II for 1 hour, 1% agarose gel electrophoresis for 60 minutes, and EB staining.

(3) Enzymatic hydrolysis

DNA was hydrolyzed using nuclease PI (Nuclease Pl) and bacterial alkaline phosphatase (BAP) (Sigma). Take a 50μl solution (500ug/ml dissolved in TE buffer, pH 8.0) into a 1.5ml centrifuge tube, denaturing in boiling water for 10 minutes, and then inactivated by ice water for 5 minutes. Add 100μ1 30ηιΜ sodium acetate, ρΗ5.3.

5μ1 20mM zinc sulfate and lOul nuclease PI (lmg/ml 200units per mg in 30mM sodium acetate pH5.3) in a water bath at 37 °C for 2 hours, add 20μ10.5Μ Tris-cl to adjust pH 8.5, then add lOul bacterial alkaline phosphatase (BAP) (10. 8mg / ml 43.7units / mg.) 37 ° C water bath for 2 hours, stored at -20 ° C for use.

(4) RP-HPLC HPLC system: waters 2695 high performance liquid chromatography, 2998 dual channel UV detector. HPLC conditions:

Column: C-18 reverse phase column;

Mobile phase: Buffer A (2.5% v/v methanol, 0.05 M KH ₂ PO ₄ , pH 4.0) 22.5 min;

Buffer B (8.0% v/v methanol, 0.05 M KH ₂ PO ₄ , pH 4.0) for 30 minutes; eluent: 70% methanol-water for 10 minutes;

Flow rate: lml/min;

Column temperature: 35 ° C;

Detection wavelength: 254nm, 280nm dual wavelength simultaneous detection.

(5) Data processing

Due to the principle of special base complementation of double-stranded DNA, theoretically dC/dG=ldT/dA=l, the detection of the molar ratio of dC/dG and dT/dA can reflect the accuracy of detection. Calculate dC/dG, dT/dA by quantitatively calculating various deoxynucleosides in the hydrolyzed BCG-CpG-DNA with a known amount of various deoxynucleosides as standard and 8Br-Guo as an internal standard. Molar ratio, and the molar percentage of GC, calculate the mass of the nucleic acid by adding the molar amount of various deoxynucleosides and the molecular weight of its corresponding deoxymononucleotide; according to the 5th power of d to the molar of dC The product of the amount of CpG and the molecular weight of CpG are calculated, and the mass of CpG is calculated, and the mass percentage of CpG is obtained.

Effects of different components of Bacillus subtilis on T lymphocyte subsets and cytokines in immunocompromised mice

1. Establishment of immunocompromised mouse model of immunocompromised model is based on the "Guidelines for Pharmacodynamics of Anti-inflammatory Immunopharmaceuticals. New Drugs (Western Medicine) Compilation of Preclinical Research Guidelines (Pharmacology and Pharmacology Toxicology)", ie daily Hydrocortisone (100 mg/kg, ic, 0.1 ml / 10 g body weight) was injected once daily for 7 days. 2. The experimental grouping test was divided into 8 groups: normal animal control group, immunocompromised model group, BCG polysaccharide injection group (concentration = 1.0 mg/ml, polysaccharide content 96%), BCG nucleic acid injection group (concentration = 1.0mg/ml, nucleic acid content 92%, purity>1.8), New BCG polysaccharide nucleic acid injection group I (concentration=1.0mg/ml, polysaccharide content=69%, nucleic acid content=30%), New Bacillus Polysaccharide nucleic acid injection group II (concentration = 1.0 mg / ml, polysaccharide content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group III (concentration = 1.0 mg / ml, polysaccharide content = 10%) , nucleic acid content = 89%), has been listed in the BCG polysaccharide nucleic acid injection group (concentration = 1.0mg / ml, polysaccharide content of 75%, nucleic acid content of 15%). In the BCG-polysaccharide injection group, the BCG-nucleic acid injection group and the BCG-nucleic acid polysaccharide nucleic acid injection group, the corresponding samples (0.1 ml/10 g) were injected intramuscularly in the hind limbs from 2 weeks before the modeling to the modeling period. Body weight), once every other day, 24 hours after the last administration. In the normal animal control group, normal saline (ic, 0.1 ml / 10 g body weight) was injected intramuscularly every day for 21 days.

3. Detection of T lymphocyte subsets and cytokines in mice

1 Total cell percentage: The lymphocyte suspension was routinely prepared, and the cell concentration was adjusted to 4×10 ⁹ /L with RPMI1640 complete medium. The SRBC was washed 3 times with physiological saline and adjusted to a concentration of 1% with the culture solution. In a 1 ml plastic centrifuge tube, add 50 μL of the lymphocyte suspension and the culture solution to each tube, and incubate for 1 hour in a 37 ° C water bath. Add 100 μ SR of SRBC suspension and mix for 10 minutes in a 37 ° C water bath. Centrifuge (800 rpm, 5 minutes) water bath for 2 hours. The cells were gently suspended and mixed with 100 μΐ 4% glutaraldehyde fixative (formulated with zinc acid buffer). The supernatant was completely removed before counting, and 100 μL of physiological saline solution and 20 μl diluted 1% methylene blue staining solution were added for staining, and counted after 20 minutes to obtain a total of 1 cell%.

2 Theophylline-resistant cell garland%: Mix 50 μl of the culture medium containing 100 mmol/L theophylline in a 50 μL lymphocyte suspension, and the rest of the steps are the same as above.

3 Th, 1 ₈ cells%: Calculated as follows. Number of positive rings

Total butyl cells % χ 100%

Total number of lymphocytes

Th cell% ₌ tea resistant wreath% 00 _%

73⁄4 cells% = 100% - 73⁄4 cells%

4 Determination of serum IFN-γ: by ELISA method, according to the kit instructions. 4. Results: Continuous subcutaneous injection of hydrocortisone (100 mg/kg, 1 time/day) for 7 days can significantly reduce the total T cells, Th cells, Ts cells and blood IFN-γ levels. Bacterial polysaccharide injection group, BCG nucleic acid injection group, New Bacillus nucleic acid polysaccharide nucleic acid injection group I, II, III, the listed BCG nucleic acid polysaccharide nucleic acid injection group can increase total T cells, Th cells, Ts The number of cells and the level of ovarian IFN-γ were significantly different between the new BCG polysaccharide nucleic acid injections I and II and the BCG-polysaccharide nucleic acid injection (Ρ<0.05, Table 2).

Table 2 Effect of BCG-polysaccharide nucleic acid injection on cellular immune function in mice ( _± s, n =9-10) Total τ ( ) Th ( ) Ts ( ) IFN-γ (pg/ml) Normal animal control group 44.6 ±4.5 33.4 ±4.1 11.1 ±4.1 209.1 ±113.0

**

Immunocompromised group 38.0 ± 3.6 29.2 ± 2.8 * 8.8 ± 2. 137.0 ± 88 * BCG polysaccharide injection group 49.1 ± 4.2 ⁺⁺ 35.5 ± 2.6 ⁺⁺ 16.7 ± 4.4 ⁺⁺ 291.6 ± 70.2 ⁺⁺ BCG nucleic acid injection Group 50.0±3.9 ⁺⁺ 38.6± 1.7++ 17.9±4.9 ⁺⁺ 293.6 ± 126.2 ⁺⁺ New BCG polysaccharide nucleic acid

Injection group I 53.0±3.3 ^++# 39.6± 1.5 ^++# 19.4±5.0 ^++# 323.6士131.2 ^++# New BCG polysaccharide nucleic acid

Injection group Π 55.0±3.9 ^++# 43.6± 1.6 ^++# 22.4±5.2 ^++# 353.6+ 109.3 ⁺⁺ New BCG polysaccharide nucleic acid

Injection group m 51.0±3.9 ⁺⁺ 39.3± 1.4++ 18.6±4.7 ⁺⁺ 293.6±125.8 ⁺⁺ Bacillus polysaccharides

43.0±3.9 ⁺⁺ 30.6± 1.4++ 15.4±3.9 ⁺⁺ 263.6 ± 76.2 ⁺⁺ nucleic acid injection group

<0.05, /XOl normal animal control group; ⁺ P<0.05, ⁺⁺ Ρ<0.01 immunocompromised group;

^# <0.05 The BCG-polysaccharide nucleic acid injection group has been listed.

Effects of different components of BCG on genital herpes simplex virus (HSV-2) infection in guinea pigs

1. Establishment of animal models

For modeling methods, see (Be atew/)/, et al. J Infect Bis, 2001; 183 (6): 844), that is, first clean the vulva with saline, dry cotton swab rubs the vagina several times, causing vaginal mucosa Injury, then use HSV-2 (potential 10_ ⁴ ~_ ⁶ TCID ₅ . provided by Wuhan Institute of Virology, Chinese Academy of Sciences) with a syringe with a gavage needle to 0.1 ml, insert the needle into the vaginal vagina about 3 ~ 4 After cm injecting the virus into the vagina, slowly withdraw it. Use a gelatin sponge to insert the vagina to maintain the venom. A little toxic droplet on the needle is in the vulva. Gently spread it with a glass rod to make the virus penetrate into the skin.

2. Animal grouping

After 14 days of virus infusion, they were randomly divided into seven groups according to the degree of skin lesions: model group, BCG-injected group (concentration = 1.0 mg/ml, polysaccharide content 96%), BCG-injected group (concentration = 1.0 mg) /ml, nucleic acid content 92%, purity >1.8), New BCG polysaccharide nucleic acid injection group I (concentration = 1.0 mg / ml, polysaccharide content = 69%, nucleic acid content = 30%), new BCG polysaccharide nucleic acid Injection group II (concentration = 1.0 mg / ml, polysaccharide content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group III (concentration = 1.0 mg / ml, polysaccharide content = 10%, nucleic acid Content = 89%), BCG-polysaccharide nucleic acid injection group (concentration = 1.0 mg / ml, polysaccharide content 75%, nucleic acid content ^ 15%). On the 15th day, the model group, the BCG-injected group, the BCG-nucleic acid injection group, the New Bacillus nucleic acid polysaccharide nucleic acid injection group, and the BCG-nucleic acid polysaccharide nucleic acid injection group were respectively provided in the hind limb femoral muscle. Injection of normal saline and different components of BCG (dosing volume is 0.1 ml/100 g body weight), once every other day for 3 weeks; observe the recurrence rate during the administration (the number and days of skin lesions) and skin In the case of loss, serum IFN-γ levels were measured 24 hours after the last administration.

Skin lesions were scored according to the literature (Be ate « et al. J Infect Dis, 2001; 183 (6) · 844). Score: 0 points, asymptomatic; 0.5 points, reddish; 1.0 points, red and swollen nopes; 1.5 Points, single small blisters (≤2 mm); 2.0 points, single large blisters (>2 mm); 2.5 points, multiple small blisters and/or vaginal ulcers (bleeding); 3.0 points, multiple large blisters; 3.5 points , severe vulvar swelling; 4.0 points, multiple small (large) blister fusion; 4.5 points, hind limb paralysis; 5.0 points, vulvar ulcers.

3. Experimental results

After inoculation with HSV-2, guinea pigs showed symptoms similar to human genital genital herpes, mainly manifested by vulvar redness, blisters, ulcers, some animals and even hind limb paralysis. The symptoms of skin lesions began to appear 2 to 3 days after seeding, first manifested as scattered redness and small blisters, and developed in days 4-10. For blister-ulcerative lesions, scarring begins on days 8-12, and ecdysis falls off on days 13-15. On days 7-12, 8 animals developed hind limb paralysis, and the symptoms disappeared on days 12-14.

Fourteen days after HSV-2 inoculation, the guinea pigs were randomly divided into seven groups according to the degree of skin lesions, and different components of saline and Bacillus were separately administered. The results showed that BCG-injected group, Bacillus acid injection group, New Bacillus nucleic acid polysaccharide nucleic acid injection group I, New Bacillus nucleic acid polysaccharide nucleic acid injection group II, New Bacillus nucleic acid polysaccharide nucleic acid injection Group III, the listed BCG nucleic acid polysaccharide nucleic acid injection group can significantly reduce the number of recurrence days of skin lesions, and can significantly increase the level of blood IFN-γ, but the new BCG polysaccharide nucleic acid injection II, III and Compared with the listed BCG polysaccharide nucleic acid injection, there was a significant difference (PO.05, Table 3).

Table 3 Effects of different components of BCG on genital herpes recurrence days and blood IFN-γ levels in guinea pigs (n = 15-16) Recurrence days IFN-γ (pg/ml) Model group 4.2 ± 2.3 100.3 ± 37.6

Bacillus polysaccharide nucleic acid has been marketed 2.9 ± 1.8* 161.5 ± 113.7*

Injection group

*

BCG polysaccharide injection group 2.5 ± 1.6 170.2 ± 107.9* BCG nucleic acid injection group 2.3 ± 1.5 191.5 Shi 112.3* New BCG polysaccharide nucleic acid injection group I 1.9 ± 1.6"# 208.0士123.6"# New card Mediated polysaccharide nucleic acid injection group 1.7 1.7 ± 1.6"# 228.0 ± 121.3"#

**

New BCG Polysaccharide Nucleic Acid Injection Group ΠΙ 2.0 ± 1.7 190.0 ± 111.6"

<0.05, "PO.Ol ra model group; Ρ<0.05 Μ has been listed in the BCG polysaccharide nucleic acid injection group.

Example 7 Effect of dinitrofluorobenzene on delayed (IV) allergic reaction in mice

Test group

The test was divided into six groups: saline control group, hydrocortisone positive control group (25 mg/kg), BCG polysaccharide injection group (concentration = 1.0 mg/ml, polysaccharide content 96%), BCG nucleic acid injection. group (concentration = 1.0 mg / ml, nucleic acid content 92%, purity > 1.8), New BCG polysaccharide nucleic acid injection group I (concentration = 1.0 mg / ml, polysaccharide content = 69%, nucleic acid content = 30%), new BCG polysaccharide nucleic acid injection group II (concentration = 1.0 mg / ml, polysaccharide content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group 浓度 (concentration = 1.0 mg / ml, polysaccharide content =10%, nucleic acid content=89%), BCG-polysaccharide nucleic acid injection group (concentration=1.0mg/ml, polysaccharide content^75%, nucleic acid content 15%), (n=8-12

2. Sensitized contact

24 hours before sensitization, the abdomen of the mice was defibrated with 8% Na ₂ S, and the range was about 3 cm x 3 cm. On the first and second days of the test, 1% 2,4-dinitrofluorobenzene solution was applied to the hair removal. (DNFB, 1:1 with acetone and sesame oil as solvent) 50 μ1 sensitization.

3. Antigen attack

On the 6th day, 1% DNFB solution 10 μΐ was evenly applied to the left ear (both sides) of the mouse to induce dermatitis, and the right ear was applied with an equal volume of vehicle for self-control. 24 hours after the induction of dermatitis, the mice were sacrificed by cervical dislocation. After weighing, the left and right auricles were cut. The ear piece with a diameter of 8 mm was removed with a puncher in the middle of the left and right ears. Weighing was performed to calculate the difference in weight between the left and right ears (ie, the degree of swelling). At the same time, the mouse thymus and spleen were weighed, and the thymus index and spleen index (mg/10 g mouse body weight) were calculated.

Bacillus subtilis injection group mice were injected intramuscularly (0.1 ml/10 g body weight) 3 weeks before the antigen challenge, once every day, and 24 hours after the last administration. Physiological saline and hydrocortisone control group were injected intramuscularly with normal saline and hydrocortisone on the 1st day before sensitization. The dosage volume was 0.1 ml/10 g body weight, once daily, for 7 days. The antigen challenge was performed 1 h after the last administration.

4. Experimental results

There was no significant difference in mouse body weight between groups. Compared with the saline group, the BCG polysaccharide injection group (concentration = 1.0 mg / ml, polysaccharide content 96%), BCG nucleic acid injection group (concentration = 1.0 mg / ml, nucleic acid content 92%, purity > 1.8), New BCG polysaccharide nucleic acid injection group 1 (concentration = 1.0 mg / ml, polysaccharide content = 69%, nucleic acid content = 30%), New BCG polysaccharide nucleic acid injection group II (concentration = 1.0 mg / ml, polysaccharide Content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group III (concentration = 1.0 mg / ml, polysaccharide content = 10%, nucleic acid content = 89%), has been listed The polysaccharide polysaccharide nucleic acid injection group (concentration = 1.0 mg / ml, polysaccharide content ^ 75%, nucleic acid content ^ 15%) can significantly reduce the mouse thymus index and spleen index, while the new BCG nucleic acid polysaccharide injection I and has Compared with the listed BCG nucleic acid polysaccharide nucleic acid injection, there was a significant difference (Ρ<0.05, see Table 4). Table 4: Effect of BCG-polysaccharide nucleic acid injection on delayed allergic reaction induced by dinitrofluorobenzene (n=8-12)

Body weight thymus index spleen index (g) (mg/10g body weight) (mg/10g body weight) saline control group 37.8 ± 1.5 34.45 ± 4.72 48.72 ± 7.66 hydrocortisone control group 36.9 ± 5.4 11.91 ± 3.02 37.07 ± 8.92** Bacillus polysaccharide nucleic acid

Injection group 34.7 ± 3.6 31.33 士 10.64** 43.58 ± 11.68 ¹ " BCG polysaccharide injection group 35.9 + 3.8 30.93 士 8.64 41.23士 11.56 BCG nucleic acid injection group 35.9 + 3.6 30.22 士 7.23** 40.38士 10.62 ** New Bacillus Polysaccharide Nucleic Acid

Injection group I 34.8 ±3.9 26.32 + 6.73 35.88 ±8.86 New BCG polysaccharide nucleic acid

Injection group II 34.9 ±3.7 23.61 ±7.03 33.27 ±6.76 New BCG polysaccharide nucleic acid

Injection group III 34.8 ± 3.9 30.61 ±9.03 41.22±8.29 <0.05, PO.Olra saline control group; ^# P<0.05 Listed BCG polysaccharide nucleic acid injection group Example 8 Effect on immediate (I type) allergy - Rat allergic passive skin allergic reaction

Test group

The test was divided into eight groups: saline control group, hydrocortisone positive control group (25 mg/kg), BCG polysaccharide injection group (concentration = 1.0 mg/ml, polysaccharide content 96%), BCG nucleic acid injection. Group (concentration = 1.0 mg / ml, nucleic acid content 92%, purity > 1.8), New BCG polysaccharide nucleic acid injection group I (concentration = 1.0 mg / ml, polysaccharide content = 69%, nucleic acid content = 30%), New BCG polysaccharide nucleic acid injection group II (concentration = 1.0 mg / ml, polysaccharide content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group (concentration = 1.0 mg / ml, polysaccharide Content = 10%, nucleic acid content = 89%), BCG-polysaccharide nucleic acid injection group (concentration = 1.0 mg / ml, polysaccharide content ^ 75%, nucleic acid content 15%), (n = 10) o

2. Antiserum preparation

Six rats weighing 90-100 g were taken, and the trichosanthin protein was mixed with 5 mg/ml suspension of 4% aluminum hydroxide gel, and 0.1 ml of each foot of the rats was injected into a total of 0.4 ml. Fourteen days after sensitization, the common carotid artery was cannulated for blood collection, and the serum was separated by centrifugation at GOOO rpm, 15 min, 4 ° C), and stored at -20 ° C until use.

3. Passive skin sensitization and antigen attack

After ether anesthesia in rats, the hair on both sides of the back spine was cut (about 3 cm X 3 cm on each side), and 1:20, 1:40 diluted antiserum was injected intradermally into the back of the rat, and two injections were injected for each dilution. Antigen challenge was performed after 0.1 ml of each point for 0.1 h, ie intravenous injection of 0.5 ml of Evans blue solution (containing 1 mg of trichosanthin). After 20 minutes, the animals were killed by decapitation, the back skin was turned over, and the blue spot skin was cut. Each point was soaked in 5 ml of acetone physiological saline solution (3:7 V/V) for 48 h, centrifuged at GOOO rpm, 15 min, 4 ° C) The supernatant was taken, and the optical density value was measured at a wavelength of 590 nm to calculate the percent inhibition of blue spot.

Blue spot optical density of the control group - Blue spot optical density of the drug group

Blue spot inhibition percentage : 100%

Blue spot optical density

- Intramuscular injection of hind limbs 3 weeks before antigen challenge in rats with intracellular polysaccharide injection Each component of Bacillus Calmette-Guerin (0.1 ml/100 g body weight) was administered once a day, and antigen challenge was performed 24 hours after the last administration. Physiological saline and chlorpheniramine maleate control antigens were intramuscularly injected with normal saline and chlorpheniramine maleate 1 h before challenge, and the dose was 0.1 ml/100 go.

4. Test results

Compared with the saline group, the BCG polysaccharide injection group (concentration = 1.0 mg / ml, polysaccharide content 96%), BCG nucleic acid injection group (concentration = 1.0 mg / ml, nucleic acid content 92%, purity > 1.8), New BCG polysaccharide nucleic acid injection group I (concentration = 1.0 mg / ml, polysaccharide content = 69%, nucleic acid content = 30%), New BCG polysaccharide nucleic acid injection group 11 (concentration = 1.0 mg / ml, polysaccharide Content = 54%, nucleic acid content = 45%), New BCG polysaccharide nucleic acid injection group III (concentration = 1.0 mg / ml, polysaccharide content = 10%, nucleic acid content = 89%), Bacillus polysaccharide nucleic acid The injection group (concentration = 1.0 mg / ml, polysaccharide content 75%, nucleic acid content 15%), (n = 8-12) can inhibit the formation of blue spot on the skin of rats, showing a significant decrease in absorbance; The nucleic acid polysaccharide nucleic acid injections I and II were significantly different from the commercially available BCG polysaccharide nucleic acid injections (Ρ<0.05, Table 5).

Antiserum dilution

1:20 1:40

Inhibition rate inhibition rate absorbance value absorbance value

(%) (%) Physiological saline water control group 0.085 ± 0.033 - 0.038 ± 0.016 - chlorpheniramine maleate positive 0.033 ± 0.018" 61 0.019 ± 0.009" 50 control group

BCG-polysaccharide nucleic acid has been marketed 0.069 ±0.038 19 0.032 ±0.012 38 Injection group

BCG-polysaccharide injection group 0.042 ±0.016" 51 0.028 ±0.011" 39 BCG-nucleic acid injection group 0.043 ±0.017" 52 0.029士 0.006 41 New BCG polysaccharide nucleic acid

Injection group I 0.035 ±0.012 ^# 59 0.022士0.009"# 51 New BCG polysaccharide nucleic acid

Injection group Π 0.0359±0.016"# 62 0.020士 0.007"# 52 New BCG polysaccharide nucleic acid

Injection group m 0.035 ±0.019" 51 0.028+ 0.003" 42 <0.05, PO.Olra saline control group; #Ρ<0.05 Μ has been listed in the BCG polysaccharide nucleic acid injection group.

From the fifth, sixth, seventh and eighth examples, the following conclusions can be drawn: BCG-polysaccharide injection prepared by the present invention (polysaccharide content>95%), BCG nucleic acid injection (nucleic acid content>90%, purity) >1.8) and the new BCG nucleic acid polysaccharide nucleic acid injection prepared by the above-mentioned BCG polysaccharide and BCG nucleic acid can enhance the total T cell, Th cell, Ts cell number and blood IFN-γ level; In the model of guinea pig genital herpes, the above injections can also significantly reduce the number of recurrence days of skin lesions and increase the level of IFN-γ in blood. Similarly, the above injections significantly inhibited the delayed allergic reaction caused by xylene and the same passive skin allergic reaction in rats. The above results suggest that: the BCG polysaccharide, the BCG nucleic acid prepared by the present invention, and the BCG- and BCG-nucleic acid The new Bacillus nucleic acid polysaccharide nucleic acid preparation prepared according to the new ratio can regulate the cellular immune function, activate the mononuclear-macrophage system and induce cytokines such as interferon, thereby regulating humoral immunity, and has antiviral, anti-inflammatory and anti-inflammatory effects. A wide range of immunomodulatory effects such as allergies, and more importantly, the immunomodulatory function of preparing a new Bacillus nucleic acid polysaccharide nucleic acid preparation in a new ratio is significantly superior to that of a commercially available BCG nucleic acid nucleic acid injection, and thus is prepared by the present invention. BCG polysaccharides and BCG nucleic acids can be combined in a single mode or in a certain ratio to form new biological agents for treating or preventing diseases such as microbial infections, tumors, allergies and the like.

Finally, it should be noted that the above embodiments are merely illustrative and not limiting of the technical solutions of the present invention. Although the present invention has been described in detail with reference to the embodiments of the present invention, it should be understood that the invention may be modified or equivalently modified without departing from the spirit and scope of the invention. It is intended to be included within the scope of the appended claims.

Claims

A composition comprising a BCG polysaccharide and a Bacillus nucleic acid, wherein the mass percentage of the polysaccharide in the composition is 10% to 69%, and the mass percentage of the nucleic acid in the composition is 30%~89%.

The composition comprising a BCG polysaccharide and a BCG nucleic acid according to claim 1, wherein the composition is prepared by mixing a BCG polysaccharide and a BCG nucleic acid; wherein the BCG polysaccharide And Bacillus nucleic acid were extracted from BCG culture by the following method:

(2) separating the BCG polysaccharide and the BCG nucleic acid from the BCG polysaccharide and the nucleic acid mixture by ion exchange chromatography;

(3) Mixing the BCG polysaccharide obtained in the step (2) with the BCG nucleic acid in proportion.

3. The composition comprising BCG polysaccharide and BCG nucleic acid according to claim 2, wherein the step (1) comprises the steps of:

(13) After the supernatant is extracted with an organic solvent, it is removed by dialysis or gel chromatography.

The composition comprising BCG and BCG nucleic acid according to claim 2, wherein the ion exchange chromatography described in the step (2) is column chromatography using an anion exchange medium. .

The composition comprising BCG polysaccharide and BCG nucleic acid according to claim 4, wherein the anion exchange medium is QS _e _{ar ar0Se} ^TM XL.

The composition comprising BCG polysaccharide and BCG nucleic acid according to claim 5, characterized in that the step (2) comprises the following steps:

(21) loading the BCG polysaccharide and nucleic acid mixture obtained in the step (1) simultaneously Collecting flow through;

(23) using the desalting column for desalting to obtain a BCG polysaccharide;

(24) Step (22) After the completion of the washing, elution is carried out by using a sodium chloride solution having a concentration of 0.1 to 2 mol/L to collect an elution peak;

(24) The collected eluate is desalted using a desalting column to obtain BCG nucleic acid.

7. Use of a composition comprising a BCG polysaccharide and a BCG nucleic acid according to claim 1 for the manufacture of a medicament for the treatment or prevention of a microbial infection, a tumor or an allergic disease.