Research and application of Mycobacterium extract

A technology of mycobacteria and bacteria, applied in the direction of antibacterial drugs, bacterial antigen components, medical raw materials derived from bacteria, etc., can solve safety problems and other problems, achieve the effect of shortening the course of treatment, reducing side effects, and improving curative effect

Inactive Publication Date: 2009-09-02
中国人民解放军总医院第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The animal experiments of the adjuvants reported above are very effective, but the main problem is the safety of clinical use

Method used

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  • Research and application of Mycobacterium extract
  • Research and application of Mycobacterium extract
  • Research and application of Mycobacterium extract

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of mycobacterial preparation

[0038] According to the mycobacterium cell composition, cell wall structure and active ingredients, the extraction process was formulated. Taking Mycobacterium light flavinus as an example, BCG, Mycobacterium light yellow, Mycobacterium vaccae, etc. cultured on the improved Sutong medium were collected by filtration, washed and centrifuged twice with normal saline, and the wet weight of the bacteria was accurately weighed , add 3 times (V / W) acetone by volume and weight, extract for 2-10 hours, and extract 2 times in total. Wash twice with normal saline and once with pH8.0 20mmol / L Tris.Cl. pH8.0 20mmol / LTris.Cl suspended bacteria, 1mg / ml lysozyme digested at 37°C for 4-10 hours. Centrifuge, wash the cells with PBS, and dilute the suspended cells with PBS to 20mg / ml.

[0039] Preparation of mycobacterium preparations by high-pressure crushing: the above-mentioned bacterial suspension is homogenized and crus...

Embodiment 2

[0060] Example 2: Effects of Mycobacterium Preparations Immunizing Mice on the Production of Cytokines by Mouse Spleen Lymphocytes

[0061] Mice were immunized with different doses, and an equal volume of normal saline was injected as a control. Two weeks after the last immunization, the spleen was aseptically taken and grinded to adjust the splenocyte concentration to 5×10 6 / ml, dispense into 96-well plates, 100 μl per well, 37°C, 5% CO 2 After culturing for 72 hours, the supernatant was collected by centrifugation and stored at -20°C. The mouse IL-12, IL-4, IFN-γELISA kit produced by BD company detects the cytokines in the culture supernatant of splenic lymphocytes, and the results are shown in Figure 1-3 .

[0062] Cytokines results showed that mycobacterial preparations could stimulate the secretion of Th1 cytokines IL-12 and IFN-γ, and reduce the expression of Th2 cytokines IL-4. This has important implications for TB prevention.

Embodiment 3

[0063] Embodiment 3: the effect of the mycobacterium preparation of the present invention immunizing mice on the production of NO by mouse macrophages

[0064] Nitric oxide (NO), synthesized by nitric oxide synthase (NO synthase), is a very important physiological intracellular and intercellular signaling molecule. The increase of its level is conducive to the body's response to parasitic cells. Injury and removal of bacteria and viruses. Nitric oxide itself is extremely unstable, and there is no good way to directly measure it.

[0065] NaNO 2 It is a stable product formed by the metabolism of nitric oxide, so the determination of NaNO 2 Equivalent to the determination of the cumulative production of nitric oxide.

[0066] In vitro culture of macrophages can induce the production of NO, and the produced NO is rapidly oxidized to NO 2 - , by detecting NO 2 - Can reflect the level of NO production.

[0067] Immunize mice with different doses, and inject an equal volume ...

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Abstract

The invention relates to a method for extracting effective components of Mycobacterium, aims at removing lipid components, reducing side effects, remaining effective components thereof and leading to a function of immunological adjuvant, and comprises the strain of Mycobacterium and application of the extract thereof on the prevention and treatment of tuberculosis.

Description

technical field [0001] The invention relates to the field of biotechnology and the field of pharmaceutical application of microbial products, an active ingredient of mycobacteria extracted by biochemistry, and the application of the preparation in new tuberculosis vaccine prevention tuberculosis and tuberculosis immunotherapy. Background technique [0002] Mycobacterium tuberculosis (abbreviated as Mycobacterium tuberculosis) is the pathogenic bacterium of tuberculosis, and about three million people in the world suffer from tuberculosis caused by Mycobacterium tuberculosis every year. my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of patients ranks second in the world, second only to India. In recent years, the incidence of tuberculosis in my country has been on the rise. The fourth national epidemiological survey of tuberculosis in 2000 showed that the number of infected people in my country was large (as many as 550 ...

Claims

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Application Information

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IPC IPC(8): A61K35/74A61K39/04A61K9/19A61P31/06
Inventor 何秀云汪海滨庄玉辉张小刚王艳军黄香玉王春美李新荣
Owner 中国人民解放军总医院第二附属医院
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