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Lactic acid bacteria extracellular polysaccharide and immune adjuvant

An extracellular polysaccharide and immune adjuvant technology, applied in bacteria, biochemical equipment and methods, microorganisms, etc., can solve problems such as side effects and can not meet the requirements of new vaccine adjuvants

Active Publication Date: 2019-01-11
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the increase of vaccine research and development varieties, it is found that there are certain limitations in its use, and there will be certain side effects, which are far from meeting the requirements of new vaccine adjuvants

Method used

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  • Lactic acid bacteria extracellular polysaccharide and immune adjuvant
  • Lactic acid bacteria extracellular polysaccharide and immune adjuvant
  • Lactic acid bacteria extracellular polysaccharide and immune adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the acquisition of WXD030

[0058] 1. Isolation and purification of strains

[0059] 1. Dilute the dairy product with sterile water, then take a small amount of liquid and spread it on the MRS solid medium, place it upside down in an anaerobic tank, and incubate at 37°C for 48 hours under anaerobic conditions.

[0060] 2. After step 1 is completed, isolate and mark the strains with the same colony shape, and repeatedly line the isolated strains to purify them. One strain is screened out and named as strain WXD030.

[0061] 2. Identification of strains

[0062] 1. Strain WXD030 is round on MRS solid medium, with medium-sized colonies, raised, slightly white, moist, with neat edges, such as Figure 13 shown. Gram-positive, as in Figure 14 shown.

[0063] 2. The 16S rDNA sequence of the strain WXD030 was amplified and sequenced, and the sequence result was shown in sequence 1 of the sequence table.

[0064] After the above identification, it was determ...

Embodiment 2

[0067] Example 2, Exopolysaccharide extraction, purification and identification of WXD030

[0068] 1. Extraction of WXD030 Extracellular Crude Polysaccharide

[0069] 1. Inoculate WXD030 in MRS liquid medium, and culture it statically at 37°C for 30 hours to obtain fermentation broth (bacterial concentration is 1×10 9 CFU / mL).

[0070] 2. Centrifuge the fermentation broth obtained in step 1 at 5000 rpm at 4°C for 15 minutes, and take the supernatant.

[0071] 3. Slowly add trichloroacetic acid to the supernatant in step 2 until the concentration of trichloroacetic acid in the supernatant is 40 mg / mL. After standing at 4°C for 8 hours, centrifuge at 10,000 rpm for 10 minutes at 4°C to collect the supernatant.

[0072] 4. Add 4 parts by volume of absolute ethanol to 1 part by volume of the supernatant obtained in step 3, alcohol precipitation overnight at 4° C., centrifuge at 10,000 rpm for 10 minutes at 4° C., and collect the precipitate.

[0073] 5. Dissolve the precipitate...

Embodiment 3

[0113] Example 3. Application research of WXD030 exopolysaccharide as immune adjuvant

[0114] 1. Cytokine secretion of mouse bone marrow-derived cells (BMDCs) stimulated by WXD030 exopolysaccharide

[0115] 1. Follow the steps below to prepare mouse bone marrow-derived cells (BMDCs):

[0116] (1) BALB / c female mice (SPF grade, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) aged 6-8 weeks were killed by neck dislocation and soaked in 75% alcohol for 5-10 minutes;

[0117] (2) Aseptically take the femur, tibia and humerus, and place them in PBS for soaking;

[0118] (3) Carefully remove the muscle tissue, connective tissue and cartilage tissue attached to the bone, wash in PBS for soaking, and transfer to fresh PBS for soaking;

[0119] (4) Clamp the bone with sterile tweezers, cut off the epiphysis at both ends, use a 1mL sterile syringe to absorb the RPMI1640 basal medium containing double antibodies, wash repeatedly from one end of the epiphysis until the ...

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Abstract

The invention discloses a lactic acid bacteria extracellular polysaccharide and an immune adjuvant. The Lactobacillus casei (Lactobacillus casei) WXD030 protected by the present invention is depositedunder the accession number CGMCC No. 12415. The Lactobacillus casei WXD030 is deposited under the accession number CGMCC No. 12415. Extracellular polysaccharide can significantly enhance the secretion of cytokines from bone marrow-derived dendritic cells in mice. Immunization of mice with exopolysaccharide and OVA antigen could significantly increase the titers of IgG antibody and IgG subclass specific for OVA in mice serum, while immunization of mice with exopolysaccharide and OVA antigen could increase the titers of IgG antibody subclass specific for OVA in mice serum. Extracellular polysaccharides could also significantly promote splenic T lymphocyte proliferation and INF-Gamma and IL-4 secretion; Significant increase in spleen IL-4 + CD4 + T cells, IFN-Gamma + CD4 + T cells and IFN-Gamma + CD8 + T cells; In addition, extracellular polysaccharide could significantly enhance the titer of specific antibody induced by inactivated FMD vaccine in mice.

Description

technical field [0001] The invention relates to a lactic acid bacteria exopolysaccharide and an immune adjuvant. Background technique [0002] Vaccines are the core products for the prevention and control of infectious diseases, and play an important role in the prevention and control of animal infectious diseases, the protection of human health, and the sustainable development of animal husbandry and agriculture. Although a lot of efforts have been made in the development and preparation of vaccines, the immune effect of some vaccines is not satisfactory at present. To overcome the shortcomings of single immune response effect and weak immunogenicity caused by existing vaccines, it is necessary to supplement them with more effective vaccines. Adjuvants are used to enhance the immune response to vaccines. The application of adjuvants in vaccines can improve the immunogenicity of antigens, enhance the effect of vaccines, reduce the amount of antigens used, and make vaccines ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/04A61K39/39A61P37/04C12R1/245
CPCA61K39/39A61P37/04C12P19/04A61K2039/55583C12R2001/245C12N1/205
Inventor 王潇修磊杜瑞平
Owner INNER MONGOLIA UNIVERSITY
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