Therapeutic mycobacterium tuberculosis DNA vaccine and preparation method and application thereof

A technology of Mycobacterium tuberculosis and DNA vaccine, which is applied in the field of therapeutic Mycobacterium tuberculosis DNA vaccine and its preparation, and can solve the problems of drug-resistant mutation of Mycobacterium tuberculosis and the reduction of the therapeutic effect of anti-tuberculosis drugs, etc.

Inactive Publication Date: 2015-06-10
中国人民解放军第四五八医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, taking these drugs has more side effects, and long-term use will lead to drug-resistant mutations in Mycobacterium tuberculosis, greatly reducing the therapeutic effect of anti-tuberculosis drugs

Method used

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  • Therapeutic mycobacterium tuberculosis DNA vaccine and preparation method and application thereof
  • Therapeutic mycobacterium tuberculosis DNA vaccine and preparation method and application thereof
  • Therapeutic mycobacterium tuberculosis DNA vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Therapeutic Mycobacterium tuberculosis DNA vaccine and its preparation method

[0053] 1. Experimental materials

[0054] 1. Strains

[0055] E.coli DH5ɑ, preserved for our laboratory.

[0056] The standard strain of Mycobacterium tuberculosis H37Rv, provided by Professor Tan Shouyong of Guangzhou Chest Hospital, has been inactivated at 121°C for 5 minutes.

[0057] 2. Eukaryotic expression vector

[0058] pVAX1 was purchased from Invitrogen.

[0059] 3. Kit

[0060] Bacterial DNA Extraction Kit (TIANamp Bacteria DNA Kit Bacterial Genomic DNA Extraction Kit, DP302).

[0061] Plasmid (miniature) extraction kit (Takara miniBEST Plasmid Purification kit ver 4.0, Code No.9760).

[0062] Gel extraction kit (Takara MinBEST Agarose Gel Extraction kit Ver3.0, D823A).

[0063] 4. Enzymes: pfu DNA polymerase (Tiangen), BamHI (Takara), XbaI (Takara), T4 DNA ligase (Takara).

[0064] 5. General reagents

[0065] Agarose, DNA Marker (Takara), Gelred TM Dye (BI...

Embodiment 2

[0142] Cell Transfection and Western Blot Detection of Example 2 Therapeutic TB DNA Vaccine

[0143] 1. Experimental materials

[0144] 1) RPMI 1640 medium (Hyclone)

[0145] 2) 10% fetal bovine serum: add 10% fetal bovine serum to the 1640 medium prepared in 1) at a volume ratio of 1:10. Fetal bovine serum was purchased from Hyclone Company.

[0146] 3) 0.25% trypsin: Asanced Technology & Industrial company.

[0147] 4) Cell line: African green monkey kidney cell COS-7, purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0148] 8.Lipofectamine TM 2000 transfection reagent was purchased from Guangzhou Yingwei Chuangjin Biotechnology Co., Ltd.

[0149] 2. Experimental method

[0150] 1. Cell transfection experiment

[0151] 1) Inoculate cells

[0152] One day before transfection, the well-grown COS-7 cells were taken, digested with trypsin, centrifuged at 1000r / min for 5min, counted, ...

Embodiment 3

[0171] Example 3 In vivo immune test in mice of therapeutic Mycobacterium tuberculosis DNA vaccine

[0172] 1. Experimental materials

[0173] Animal immunization dose, grouping and observation time: 50 SPF-grade healthy Balb / c mice, 18-20g, 6-8 weeks old, were randomly divided into 5 groups, 10 in each group: the first group was injected with normal saline; The third to fifth groups were injected with different doses of Ag85a-ESAT6 fusion gene vaccine in the bilateral tibialis anterior muscle of mice with in vivo electric pulse, respectively 10 μg / mouse, 20 μg / mouse, 50 μg / mouse. Only. Immunization was performed every 2 weeks for a total of 3 times. The BCG group was injected only once.

[0174] The Ag85a antigen used in the experiment was purchased from Prospec Company; the ELISPOT detection kit was purchased from BD Pharmingen Company of the United States; the lymphocyte separation liquid was purchased from Thermo Company.

[0175] 2. Animal immunity

[0176] (1) Get t...

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Abstract

The invention relates to the field of biotechnologies and DNA vaccine, in particular to therapeutic mycobacterium tuberculosis DNA vaccine and a preparation method and application thereof. The therapeutic mycobacterium tuberculosis DNA vaccine (for short TB DNA vaccine) is fusion gene expression vector containing encoded exogenous immunoprotective antigen protein Ag85a-ESAT6 obtained by inserting 5' end Ag85a-ESAT6 fusion gene with the human granulocyte-macrophage colony stimulating factor (GM-CSF) signal peptide into the pVAX1 carrier. The signal peptide can promote the secretion of antigen protein, and the immune effect is enhanced. According to the therapeutic mycobacterium tuberculosis DNA vaccine and the preparation method and application thereof, a gene recombination technology is adopted to build the fusion gene mycobacterium tuberculosis DNA vaccine containing a plurality of immunoprotective antigen, the DNA vaccine is injected into the tibialis anterior muscles on the two sides of a mouse through an in vivo electroporation (EP) technology, the Th1 type immune response and specific killing response can be induced for the tuberculosis antigen secreting IFNgamma of a high level, and the therapeutic mycobacterium tuberculosis DNA vaccine is the vaccine capable of preventing and treating the tuberculosis.

Description

technical field [0001] The invention relates to the field of DNA vaccines, in particular to a therapeutic Mycobacterium tuberculosis DNA vaccine and its preparation method and application. Background technique [0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB), which seriously endangers human health. BCG (Bacille Calmette Guerin, BCG) is currently the only commercially available vaccine for the prevention of tuberculosis. Since its discovery in 1921, it has attracted people's attention and is mainly used in the prevention of childhood tuberculosis. However, the protective efficiency of BCG gradually decreases over time, and for different regions and different populations, the protective rate varies from 0 to 80%. Studies have shown that the BCG strains currently in use lack the DNA fragments that encode certain protective immune effects in pathogenic Mycobacterium tuberculosis and Mycobacterium bovis. WHO declared in 1995 that BCG is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/00A61P31/06
Inventor 饶桂荣陈光明杨富强张换敬
Owner 中国人民解放军第四五八医院
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