Mycobacterium tuberculosis fusion proteins and their preparation method and use

A technology of mycobacterium tuberculosis and fusion protein, which is applied in the field of preparing tuberculosis subunit vaccines, can solve the problems of ineffective prevention of adult pulmonary tuberculosis, and achieve the effect of strengthening BCG immunity, lasting protection time, and strong immune protection

Inactive Publication Date: 2014-11-19
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BCG has been used for human vaccination since 1921. 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

Method used

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  • Mycobacterium tuberculosis fusion proteins and their preparation method and use
  • Mycobacterium tuberculosis fusion proteins and their preparation method and use
  • Mycobacterium tuberculosis fusion proteins and their preparation method and use

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Embodiment 1

[0054] 1. Construction and purification of fusion protein

[0055] 1.1 Construction of fusion protein LT70 expression plasmids pET30a(+)-LT70 and pET30a(+)-LT70 (plus linker)

[0056] (1) Construct the recombinant gene vector pET30a(+)-LT69(ESAT6-Ag85B-MPT64 190-198 -Mtb8.4-HspX).

[0057] A, Construction of pET30a(+)-EA (ESAT6-Ag85B):

[0058] Using clinical mycobacterium liquid as a template, the corresponding genes of ESAT6 and Ag85B were amplified by PCR and cloned into plasmid pET30a.

[0059]

[0060] Among them, the RCR reaction system:

[0061] Water 16.25μl

[0062] 5×PCR buffer 5μl

[0063] dNTP 2μl

[0064] Upstream primer P1 0.5μl

[0065] Downstream primer P2 0.5μl

[0066] DNA template 0.5μl

[0067] DNA polymerase (Primer STAR, takara company) 0.25μl

[0068] The total system is 25μl.

[0069] PCR reaction parameters: denaturation at 98°C for 15s, annealing at 60°C for 15 s, extension at 72°C for 50 s, a total of 30 cycles.

[0070] B. Design primers to amplify EA (ESAT6-Ag85B) gen...

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Abstract

The invention provides mycobacterium tuberculosis fusion proteins. The mycobacterium tuberculosis fusion proteins are shown in sequences 2 and 4 in the sequence table. The invention provides a preparation method and a use of the mycobacterium tuberculosis fusion proteins. The fusion protein LT70 and adjuvants of DDA and PolyI: C are combined and form a tuberculosis sigmasubunit vaccine. An experiment result shows that the vaccine can effectively induce immune response of tuberculosis antigen-specific cells and body fluid in mouse body and has a strong immune protection capacity, lasting immune protection time, immune protection effects better than that of BCG and BCG improvement effects in a mouse virulent strain attack protection efficiency test. The tuberculosis sigmasubunit vaccine is an effective tuberculosis vaccine candidate.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis fusion protein, and also relates to a preparation method of the fusion protein and its application in preparing a tuberculosis subunit vaccine. Background technique [0002] Tuberculosis, caused by Mycobacterium tuberculosis infection, is the most widely spread, longest lasting and most serious infectious disease in the world. Since the 1980s, with the emergence and prevalence of drug-resistant strains, especially multidrug-resistant tuberculosis, and the spread and prevalence of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS, AIDS), in The incidence of tuberculosis around the world shows a rising trend, and the morbidity and mortality remain high. As the pathogen of tuberculosis, one of the reasons why Mycobacterium tuberculosis is difficult to control is that Mycobacterium tuberculosis can exist in the immune cells of the human body as a latent infection state for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/116A61K39/04A61P31/06
Inventor 祝秉东刘勋雒艳萍牛红霞白春香彭金秀王倩胡丽娜王秉翔于红娟郭焱
Owner LANZHOU UNIVERSITY
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