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Construction and expression purification method of mycobacterium tuberculosis fusion protein LT29 and application of mycobacterium tuberculosis fusion protein LT29

A technology of Mycobacterium tuberculosis and Mycobacterium tuberculosis, applied in the field of bioengineering, can solve problems such as functions that have not yet been studied clearly, and achieve the effect of strong cellular immune response and promoting the generation of memory T cells

Inactive Publication Date: 2021-06-18
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Rv2626c is one of the protective antigens predominantly expressed in the latent period of Mycobacterium tuberculosis, but its function has not yet been studied clearly. Studies have found that this gene can induce high levels of IFN-γ in the mononuclear cells of patients with active tuberculosis

Method used

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  • Construction and expression purification method of mycobacterium tuberculosis fusion protein LT29 and application of mycobacterium tuberculosis fusion protein LT29
  • Construction and expression purification method of mycobacterium tuberculosis fusion protein LT29 and application of mycobacterium tuberculosis fusion protein LT29
  • Construction and expression purification method of mycobacterium tuberculosis fusion protein LT29 and application of mycobacterium tuberculosis fusion protein LT29

Examples

Experimental program
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Embodiment 1

[0037] Example 1 Construction, expression and purification of Mycobacterium tuberculosis fusion protein LT29

[0038] In the present invention, firstly, the Mycobacterium tuberculosis active stage antigen EAST6 and the Mycobacterium tuberculosis latent stage antigen Rv2626c are fused at the gene level to construct the recombinant vector pET30a(+)-LT29. First construct the pET30a-Rv2626c plasmid, and then insert the ESAT6 gene in front of the Rv2626c gene. In order to prevent ESAT6 and Rv2626c from interacting with each other in terms of spatial structure, a linker (GGGGS) was added to the downstream primer of ESAT6 3 , constituted the recombinant plasmid pET30a(+)-LT29 (pET30a(+)-ESAT6-linker-Rv2626c).

[0039] Then, the pET30a(+)-LT29 plasmid vector was transformed into Escherichia coli E.coli to express the fusion protein LT29. Finally, according to the protein properties of the protein, the fusion protein was successfully purified through the combination of salting out, h...

Embodiment 2

[0054] The preparation of embodiment 2 fusion protein LT29 subunit vaccine

[0055] The fusion protein LT29 was diluted to 0.2mg / ml or 0.04mg / ml with PBS; PolyI:C (acting on the agonist of Toll-like receptor 3) was dissolved to 0.5mg / ml with phosphate buffer; cationic liposome— 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 LT29 protein solution, and let stand at room temperature for 1 min. 100 μL of DDA solution was added dropwise to the mixed solution, and then fully emulsified to make the vaccine in a uniform creamy state to obtain the subunit vaccine LT29-DDA / PolyI:C. When the vaccine is used for immunization, the dosage is 200 μL / mouse.

Embodiment 3

[0056] Example 3 Detection of Immunological Activity of Fusion Protein LT29 Subunit Vaccine

[0057] 1. Experimental materials: subunit vaccine LT29-DDA / PolyI:C; BCG (BCG); phosphate buffered saline (PBS).

[0058] 2. Experimental animals: C57BL / 6 mice;

[0059] 3. Grouping of experimental animals (six groups in total):

[0060] (1) PBS; (2) BCG; (3) DDA / PolyI:C; (4) EAST6-DDA / PolyI:C; (5) Rv2626c-DDA / PolyI:C; (6) LT29-DDA / PolyI:C; C

[0061] 4. Immunization of animals:

[0062] 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). Animals in the experimental group were immunized twice with the same dose at week 0 after immunization at week 2 and week 4 respectively.

[0063] 5. Immune Index Determination Method

[0064] (1) ELISA method to detect the level of antigen-specific IFN-γ secreted by mi...

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Abstract

The invention discloses an expression purification method of a mycobacterium tuberculosis fusion protein LT29. An active-period antigen EAST6 of mycobacterium tuberculosis and an incubation-period antigen Rv2626c of the mycobacterium tuberculosis are fused and connected by a flexible peptide fragment so as to construct the fusion protein ESAT6-linker-Rv2626c. The fusion protein is subjected to expression purification in escherichia coli B21 engineering bacteria, and is combined with an adjuvant DP to construct a tuberculosis subunit vaccine. The fusion protein is successfully expressed and purified in escherichia coli; strong specific cellular immunoreaction aiming at the specific antigens (ESAT6 and Rv2626c) of the mycobacterium tuberculosis can be induced to be generated in a mouse body, the generation of long-term memory T cells is promoted, and the fusion protein has strong immunogenicity. According to the invention, the novel fusion protein LT29 without a label is successfully constructed, expressed and purified, the protein fuses the active-period antigen and the incubation-period antigen of tubercle bacillus, antigen-specific cellular immunity and humoral immunity can be induced to be generated, and the novel fusion protein LT29 is expected to become an effective candidate vaccine for preventing and treating tuberculosis.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to the construction of a novel tuberculosis subunit vaccine using a genetic engineering method. Background technique [0002] Mycobacterium tuberculosis (MTB), the pathogen of tuberculosis, is one of the most adaptable human pathogens to the human environment. It can survive in human macrophages in a latent state for a long time. It is estimated that one-third of the world's population has latent infection with Mycobacterium tuberculosis, and 10% of this population progresses from latent TB to active TB when they are immunocompromised. Control strategies for TB rely heavily on diagnosis of the disease and long-term treatment with at least three different anti-tuberculosis drugs. But this has led to the continued development of multidrug-resistant TB. In addition, due to the prevalence of HIV, concurrent Mycobacterium tuberculosis infection has gradually become an urgent problem to be...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C07K1/20C07K1/18C07K1/14A61K39/04A61K39/39A61P31/06
CPCC07K14/35C12N15/70A61K39/04A61K39/39A61P31/06C07K2319/00A61K2039/575A61K2039/57A61K2039/55555
Inventor 祝秉东马岩林张艺凡石新彤杜秀芬谭大权牛红霞李菲龚洋
Owner LANZHOU UNIVERSITY
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