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Mycobacterium tuberculosis fusion protein (EAMMH) and constructing, expressing and purifying method and application thereof

A technology of Mycobacterium tuberculosis and fusion protein, which is applied in chemical instruments and methods, biochemical equipment and methods, peptide preparation methods, etc., can solve problems such as inability to effectively prevent adult pulmonary tuberculosis, and no new anti-tuberculosis vaccines have been developed.

Active Publication Date: 2014-10-15
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have confirmed that it can effectively prevent severe meningeal tuberculosis and systemic miliary tuberculosis in newborns and children, but it cannot effectively prevent the occurrence of pulmonary tuberculosis in adults
Following the application of BCG vaccine for more than 80 years, no effective new anti-TB vaccine has been developed in the world

Method used

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  • Mycobacterium tuberculosis fusion protein (EAMMH) and constructing, expressing and purifying method and application thereof
  • Mycobacterium tuberculosis fusion protein (EAMMH) and constructing, expressing and purifying method and application thereof
  • Mycobacterium tuberculosis fusion protein (EAMMH) and constructing, expressing and purifying method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Construction, expression and purification of Mycobacterium tuberculosis fusion protein EAMMH:

[0097] The fusion antigen EAMM and the single antigen HspX were fused at the gene level to construct the recombinant vector pET30a(+)-EAMMH. Since the terminal antigen M gene fused in the existing pET30a(+)-EAMM plasmid vector (ie Mtb8.4) has a base sequence corresponding to the stop codon UAA, HspX cannot be directly fused behind EAMM.

[0098] First construct pET30a(+)-MPT64 190-198 -Mtb8.4-HspX (MMH) vector, then PCR amplify the ESAT6-Ag85B (EA) gene fragment, and insert the EA fragment into the pET30a (+)-MMH vector by restriction endonuclease digestion and ligase connection The front end of the MMH fragment was successfully constructed into the pET30a(+)-EAMMH plasmid vector. Then, the pET30a(+)-EAMMH plasmid vector was transformed into Escherichia coli E.coli to express the fusion protein EAMMH. Finally, according to the protein properties of the protein, the fusion ...

Embodiment 2

[0121] Preparation of fusion protein EAMMH subunit vaccine:

[0122] Dilute the fusion protein EAMMH with PBS (phosphate buffered saline) to 0.2mg / ml or 0.04mg / ml; PolyI:C (acting on the agonist of Toll-like receptor 3) is dissolved in PBS to 0.5mg / ml; cationic lipid Plasmid—dimo-thylidioctyl ammonium bromide (DDA) was prepared with sterile distilled water to a concentration of 2.5 mg / ml, placed in a water bath at 80°C for 10 minutes, and cooled to room temperature. Take 50 μl of PolyI:C solution and mix well with an equal amount of EAMMH protein solution, and let stand at room temperature for 1 min. Add 100 μL of DDA solution dropwise to the mixed solution, and then fully emulsify to make the vaccine in the form of a uniform cream to obtain the subunit vaccine EAMMH-DDA / PolyI:C. When the vaccine is used for immunization, the dosage is 200 μL / mouse.

Embodiment 3

[0124] Detection of immunological activity of fusion protein EAMMH subunit vaccine:

[0125] 1. Experimental materials: subunit vaccine EAMMH-DDA / PolyI:C; BCG (BCG); phosphate buffered saline (PBS);

[0126] 2. Experimental animals: C57BL / 6 mice;

[0127] 3. Grouping of experimental animals (four groups in total):

[0128] (1) PBS; (2) BCG; (3) (EAMM+MH)-DDA / PolyI:C; (4) 2 μg EAMMH-DDA / PolyI:C; (5) 10 μg EAMMH-DDA / PolyI:C; ( 6) 50 μg EAMMH-DDA / PolyI:C;

[0129] 4. Immunized animals:

[0130] In the 0th week, subcutaneously immunize the animals in the experimental group once with the prepared subunit vaccine inguinal (200 μl / animal), and at the same time immunize the control group PBS and BCG groups (5×10 6 CFU / only), the animals in the experimental group were immunized twice with the same dose in the 2nd and 4th weeks after immunization in the 0th week;

[0131] 5. Determination method of immune index:

[0132] (1) ELISPOT method to detect the level of antigen-specific I...

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Abstract

The invention discloses mycobacterium tuberculosis fusion protein EAMMH and a constructing, expressing and purifying method and application thereof. The fusion protein is expressed in a soluble form, and greatly improves EAMM inclusion body form expression weakness. Tuberculosis subunit vaccine (LT69) constructed by combination of the fusion protein and an adjuvant has strong protective immunity, is superior to traditional BCG (Bacillus Calmette-Guerin) vaccine and EAMM+MH combined vaccine; the vaccine as an enhanced vaccine can significantly enhance the BCG initial immune immunity and protection effect of anti tuberculosis, and to a certain extent, reduces the pathological injury of the lung; in addition, the subunit vaccine contains a wide variety of antigens of growth period and latency period of mycobacterium tuberculosis, can induce strong specific cellular immune and humoral immune response aiming at each period of tubercle bacillus antigen, has protective effect on the tubercle bacillus in different metabolic states, is long in protection time, and is expected to become an effective vaccine for clinical tuberculosis prevention.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to mycobacterium tuberculosis fusion protein EAMMH, its construction, expression and purification methods and its application. Background technique [0002] Mycobacterium tuberculosis is the pathogen of tuberculosis, and it is one of the most adaptable human pathogens to the human environment. It can exist in human macrophages in a latent state for a long time. It is estimated that 2 billion people in the world are latently infected by Mycobacterium tuberculosis, and 10% of this population progresses from latent tuberculosis to active tuberculosis when the immune function is low. Control strategies for tuberculosis rely heavily on long-term treatment with at least three different anti-tuberculosis drugs. As a result, the continuous development of multi-drug resistance poses a huge obstacle to the control of tuberculosis. In countries with the highest rates of tuberculosis, acces...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/36C07K1/30C07K1/20C07K1/18C12N15/62C12N15/70C12N1/21A61K39/04A61P31/06C12R1/19
Inventor 祝秉东牛红霞彭金秀白春香胡丽娜刘勋王秉翔雒艳萍于红娟王鸿亢鸿飞李菲
Owner LANZHOU UNIVERSITY
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