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A strain of Rhodococcus rhodochrous and its application as an immune adjuvant in the preparation of vaccines

A technology of rhodococcus and vaccines, which is applied in the field of rhodococcus and its application as an immune adjuvant in the preparation of vaccines, and can solve the problems of unclear efficacy of inactivated vaccine immune adjuvants, low effect of slowly releasing antigens, human and animal Pathogenicity and other issues, to achieve the effect of good application prospects, short fermentation cycle, high immunostimulatory activity

Active Publication Date: 2021-11-26
北京利昂盛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The following problems generally exist in immune adjuvants at present: (1) inactivated Mycobacterium tuberculosis adjuvant, because natural Mycobacterium tuberculosis has high virulence, pathogenicity is arranged to human and animal
At present, attenuated BCG is used to prepare adjuvants, but there are still high requirements for follow-up processing, which is likely to cause incomplete inactivation and cause high animal health risks when used as adjuvants. In addition, it will cause strong inflammation and inflammation at the injection site. Ulcers, limited use
(2) Cytokines have the ability to adjust and rebuild the immune response, but they are expensive and not suitable for animal vaccines
(3) Double-stranded polyinosinic acid (polyinosinic acid) is a kind of artificially synthesized double-stranded ribonucleic acid. It is an interferon inducer and has antiviral and immune regulation functions. Hyperactivity, its utility as an immune adjuvant for inactivated poultry oil emulsion vaccines remains unclear
(4) Aluminum colloidal adjuvant is a commonly used adjuvant for animal vaccines at present. The main problems are: ①It mainly activates Th2 type immune cells, and only induces humoral immune response, which is suitable for disease vaccines mainly for antibody protection; ②Aluminum colloidal adjuvant Slow release of antigen at the injection site is less effective, not reliable for many antigens
Except for the extensive reports on the application of Rhodococcus rubella in environmental pollution control, there are no related reports on other applications

Method used

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  • A strain of Rhodococcus rhodochrous and its application as an immune adjuvant in the preparation of vaccines
  • A strain of Rhodococcus rhodochrous and its application as an immune adjuvant in the preparation of vaccines
  • A strain of Rhodococcus rhodochrous and its application as an immune adjuvant in the preparation of vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 (CGMCC NO.16640) Isolation and Identification of Rhodococcus ruber (Rhodococcus ruber) RDC-01

[0038] (1) Soil for testing: Soil samples were taken from the red soil in the suburbs of Guangzhou. The litter layer and roots on the soil surface were removed during sampling to avoid the influence of plants on the soil samples. The sampling depth is 5-10cm. The sampling time was late January 2017, passed through a 10-mesh sieve, and stored at 4°C.

[0039] (2) Strain screening: Take 10 g of fresh soil samples, put them into a conical flask filled with 90 mL of sterile water and glass beads, vibrate to make a bacterial suspension, and dilute it in a test tube. Choose three dilutions (10 -3 —10 -5 ) bacterial suspension was spread on LB medium, cultured at a constant temperature of 37°C, counted and observed the morphological characteristics of the colonies, and counted the types of autogenous bacteria in different soils. Select 6 well-growing strains, pick 5 si...

Embodiment 2

[0045] Embodiment 2 Neutral red method detects rhodococcus erythrococcus to mouse peritoneal macrophage Effects on Phagocytosis

[0046] Macrophages (abbreviated as ) are immune cells, It can activate and regulate lymphocytes or other immune cells to make them respond to pathogens, and can also secrete lymphokines, TNF, interferon, etc. to kill target cells. The neutral red method is to detect One of the commonly used detection methods for phagocytosis. The amount of neutral red phagocytized by macrophages can reflect its phagocytosis function, and the results are represented by A value.

[0047] (1) Experimental animals: 40 BALB / C mice, weighing 18-22 g, all male.

[0048] Medium: peptone 15g / L, yeast powder 15g / L, NaCl 10g / L, glucose 15g / L, NH 4 SO 4 2g / L, pH7.3.

[0049] 0.09% neutral red: Weigh 90 mg of neutral red biological dye, dissolve in 100 mL of normal saline, and sterilize under high pressure at 121°C.

[0050] Cell lysate: 0.1mol / L acetic acid: ethan...

Embodiment 3

[0058] The preparation of embodiment 3 H5 subtype avian influenza oil emulsion inactivated vaccine

[0059] (1) Adjuvant sample processing and quantification

[0060] Take the suspension of Rhodococcus erythrococcus RDC-01 made of sterile normal saline, shake it well, accurately measure 10mL into a glass petri dish, dry it in a blast drying oven at 80°C for 24h, and then weigh it every 1h , until the weight of the two weighings before and after is consistent, calculate the dry weight of the bacteria in the suspension, prepare the suspension with sterile saline to make an adjuvant solution of 100mg / mL (dry weight of the bacteria), and store it at 4°C for future use .

[0061] Take 10 mL of the above-mentioned Rhodococcus erythrococcus RDC-01 adjuvant solution, dissolve it in 10 mL of normal saline, prepare an adjuvant solution with a concentration of 50 mg / mL, and store it at 4°C for future use.

[0062] (2) Preparation of antigen (water phase):

[0063] ①Unadjuvanted contro...

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Abstract

The invention discloses a rhodococcus strain and its application as an immune adjuvant in vaccine preparation. The Rhodococcus ruber of the present invention is also called Rhodococcus ruber (Rhodococcus ruber) RDC-01, and the preservation number is CGMCC NO.16640. The activity of cells can induce interferon and other cytokines. After inactivating the bacteria, adding it as an immune adjuvant to the oil emulsion inactivated vaccine can significantly promote the production of vaccine-induced animal antibodies. Compared with using the oil emulsion inactivated vaccine alone It can produce high-titer antibodies, is safe to use, has no short-term and long-term toxic and side effects, and has a good application prospect in the field of animal vaccine preparation.

Description

technical field [0001] The invention belongs to the field of microbiology and immunology, and in particular relates to a rhodococcus rubococcus and its application as an immune adjuvant in preparing vaccines. Background technique [0002] Inactivated vaccines for various infectious diseases in poultry have played an important role in the prevention and control of these diseases. The use of inactivated vaccines is expected to achieve eliminative immunity, that is, to completely protect the body from the infection of such diseases after being immunized by a certain vaccine, or not to shed the virus after infection, and not to spread the virus to other animals. However, after the existing inactivated vaccine immunizes the body, the antibody is produced slowly, the duration of the antibody is short, and the uniformity of the antibody level is poor. In order to make the antibody level uniform, on the one hand, multiple immunizations are generally required to maintain a high anti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K39/39A61P37/04C12R1/01
CPCA61K39/39A61K2039/552A61K2039/55594A61P37/04C12N1/20C12N1/205C12R2001/01
Inventor 詹少春孙超廖想
Owner 北京利昂盛生物技术有限公司
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