Construction and application of mycobacterium tuberculosis fusion protein

A Mycobacterium tuberculosis and fusion protein technology, which is applied in the field of fusion protein construction, can solve the problems of low antigen expression, no immune protection of Mycobacterium tuberculosis, and inability to produce immune responses in dormant bacteria

Inactive Publication Date: 2010-02-24
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the body is immunized with BCG, due to the low expression of antigens in the dormant phase, an immune response cannot be produced against the dormant bacteria, resulting in no immune protection against dormant Mycobacterium tuberculosis

Method used

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  • Construction and application of mycobacterium tuberculosis fusion protein
  • Construction and application of mycobacterium tuberculosis fusion protein
  • Construction and application of mycobacterium tuberculosis fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Fusion protein AMH (Ag85B-Mpt64 190-198 - Construction of HspX)

[0024] 1. Materials and methods:

[0025] 1.1 Main reagent materials:

[0026] 1.1.1 Main materials: Mycobacterium tuberculosis virulence standard strain H37Rv was provided by Shanghai Pulmonary Hospital; DDA was purchased from Sigma-Aldrich; recombinant plasmids pET28a-AMM and pET28a-Ag85B were constructed by the Institute of Genetics, Fudan University. MPT64 190-198 The polypeptide fragments were synthesized by Jill Biochemical (Shanghai) Co., Ltd. with a purity of 90.45%; Ni-NTA His·Bind Resins were purchased from Novagen (EMD Chemicals Inc); the rest of the reagents were domestic or imported analytically pure.

[0027] Plasmid extraction kits and PCR product purification kits were purchased from Huashun Company, endonucleases and ligases were purchased from Biolab Company; IFN-γ ELISPOT kits were products of U-Cytech Company (Netherlands).

[0028]1.1.2 Main instruments and equipment: SC...

Embodiment 2

[0048] The preparation of embodiment 2 subunit vaccine:

[0049] BCG-PSN was dissolved to 0.6 mg / ml with physiological saline, and the fusion protein AMH was diluted to 0.5 mg / ml with PBS. DDA was prepared with water for injection to 2.5 mg / ml, placed in a water bath at 80°C for 10 minutes, and cooled to room temperature. Take 50 μl of BCG-PSN solution and mix well with an equal amount of AMH protein solution, and let stand at room temperature for 1 min. Add 100 μl of DDA dropwise to the mixed solution, and then fully emulsify it so that the vaccine is in the form of a uniform cream.

Embodiment 3

[0050] Example 3 Animal Immunization Test

[0051] 1. Grouping of experimental animals (six groups in total)

[0052] A. AMH (20ug / time / piece) + DDA (250ug / time / piece) + BCG polysaccharide nucleic acid (BCG-PSN, 30ug / time / piece)

[0053] B.Ag85B (20ug / time / piece)+DDA (250ug / time / piece)+BCG polysaccharide nucleic acid (30ug / time / piece)

[0054] C.DDA (250ug / time / piece) + BCG polysaccharide nucleic acid (30ug / time / piece)

[0055] D.AMM (Ag85B-Mpt64 190-198 -Mtb8.4, 20ug)+DDA(250ug / time / piece)+BCG-PSN(30ug / time / piece)

[0056] E. Physiological saline (NS, 200μl / time / only)

[0057] F.BCG (5×10 6 CFU / only)

[0058] 2. Immunization method:

[0059] In the 1st, 4th, and 7th weeks, the animals were subcutaneously immunized with the prepared protein vaccine in the groin (200 μl / only), and BCG 5×10 6 Animals were immunized with CFU subcutaneously in the groin once in the first week. Blood was collected 5 weeks after the last protein vaccine immunization to detect humoral immune...

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Abstract

The invention discloses construction and application of a mycobacterium tuberculosis fusion protein, and relates to construction of fusion proteins. Specific antigens of bacteria in a dormant period are omitted in the prior mycobacterium tuberculosis fusion protein; and the mycobacterium tuberculosis fusion protein provided by the invention contains a protective antigen HspX, a segment of amino-peptides from 190 position to 198 position of Mpt64 of CD8<+>T-cell epitopes and a mycobacterium tuberculosis antigen 85 complex Ag85B. The protective antigens in the dormant period are screened; the protective antigens in the dormant period and antigens in the exponential phase are combined to construct a vaccine for attempting to create immunoreaction aiming at bacterial communities of the mycobacterium tuberculosis in different growth states so as to effectively eliminate or monitor multiple kinds of mycobacterium tuberculosis including latent infection and prevent the occurrence of tuberculosis of adults.

Description

technical field [0001] The present invention relates to the construction field of fusion protein. Background technique [0002] About 1 / 3 of the world's population is infected with tuberculosis, and more than 90% are in a latent state. Attenuated Mycobacterium bovis - BCG (Bacille Calmette-Guerin, BCG) is effective in preventing severe tuberculosis infection in children, but has little protective effect on tuberculosis in adults, so subunit vaccine booster immunization strategy is important to improve the immune protection level of tuberculosis in adults of great significance [1-4] . Previous studies on tuberculosis subunit vaccines mainly selected antigens expressed in the early growth phase and logarithmic growth phase, ignoring the specific antigens of bacteria in the dormant phase. However, dormant Mycobacterium tuberculosis is ubiquitous in the human body. Mycobacterium tuberculosis that infects the body may have mycobacterium tuberculosis in a dormant state. After...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35A61K39/39A61K39/04A61P31/06
Inventor 祝秉东李青姜雯雯王秉翔雒彧于红娟傅林锋宋楠楠郄亚卿王洪海
Owner LANZHOU UNIVERSITY
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