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Recombinant Bacillus Calmette Guerin strain and preparation method and application thereof

A technology of recombinant BCG and BCG, which is applied in the field of vaccines and can solve problems such as poor protection

Pending Publication Date: 2020-11-24
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BCG can significantly reduce the risk of severe forms of tuberculosis (eg, tuberculous meningitis, miliary TB) in young children, but is poorly protected against pulmonary tuberculosis (PTB) or TB transmission[3]

Method used

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  • Recombinant Bacillus Calmette Guerin strain and preparation method and application thereof
  • Recombinant Bacillus Calmette Guerin strain and preparation method and application thereof
  • Recombinant Bacillus Calmette Guerin strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0053] Materials and methods

[0054] 1. Experimental animals

Embodiment 7

[0055] 6-8 week old female immunodeficiency mice (severe combined immuno-deficient, SCID) and BALB / c mice were purchased from Shanghai Slack. SCID mice are used for the toxicity analysis of embodiment 6, namely its survival experiment after tail vein injection of BCG related strains ( Image 6 (E)); BALB / c mice were used in Example 4 (i.e. after the mice were subcutaneously immunized with BCG-related bacterial strains in the abdomen, the number of colonies in the draining lymph nodes of the mice was analyzed. Fig. 4 (D)), Example 7 (i.e. the mice were immunized Mouse spleen T cell antigen PPD-specific cytokine expression experiment. Figure 8 ), embodiment 8 (being immunized mice anti-Mtb H37Rv ability experiment. Figure 9 ).

[0056] 2. Bacterial strains and their culture conditions

[0057] Escherichia coli (E.coli) Top10 used for molecular cloning was cultured in liquid or solid LB medium. Mycobacterium bovis BCG Pasteur (abbreviated as BCG) (American TypeCulture Colle...

Embodiment 1

[0102] Example 1: PPE17 1-177 can mediate ActA 27-612 Polarized distribution in BCG, and ActA mediated 27-612 Localized on the bacterial membrane and protruding outward

[0103] Plasmid construction: eg figure 2 As shown in (A), we constructed three plasmids: pMFA42-E expresses EGFP (E), and the resulting BCG strain is named BCG-E; pMFA42-PE expresses PPE17 1-177 -EGFP(PE), the resulting BCG strain was named BCG-PE; pMFA42-PAE expresses PPE17 1-177 -ActA 27-612 -EGFP(PAE), the resulting BCG strain was named BCG-PAE. In the excited state, E can emit green fluorescence, so it is convenient for fluorescence microscope observation and flow analysis; in addition, E-specific antibodies can also be used for Western hybridization analysis.

[0104] Fluorescence microscope observation: if figure 2 As shown in (B), under the observation of fluorescence microscope, E is uniformly distributed in BCG; PE is polarized in BCG, that is, although PE is distributed in all bacteria in B...

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Abstract

The present invention provides a recombinant Bacillus Calmette Guerin strain comprising a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises an N-terminal portion anda C-terminal portion, the N-terminal portion and the C-terminal portion are directly linked or linked by a linker with one or more amino acids, the N-terminal portion comprises an amino acid sequenceas shown in SEQ ID No. 1, and the C-terminal portion comprises an amino acid sequence as shown in SEQ ID No. 3. The invention also provides a vaccine comprising the recombinant Bacillus Calmette Guerin strain. In addition, the invention also provides application of the recombinant Bacillus Calmette Guerin strain or composition as a tuberculosis vaccine. The Bacillus Calmette Guerin strain disclosed by the invention is low in toxicity and can generate higher antigen-specific immune response.

Description

technical field [0001] Generally speaking, the present invention relates to the field of vaccines, more specifically to recombinant BCG strains, their preparation methods and uses. Background technique [0002] The main pathogenic bacteria of tuberculosis (Tuberculosis, TB) is Mycobacterium tuberculosis (Mycobacterium tuberculosis, Mtb). About 23% of the world's population is Mtb latent infection (latent TB infection, LTBI), that is, about 1.7 billion people [1]. Approximately 5-10% of the LTBI population is at risk of further progression to active TB. In 2017, there were approximately 10 million new cases of TB worldwide [2]. The number of TB deaths in 2017 was approximately 1.6 million [2]. The prevention of TB is by immunizing newborns with attenuated Mycobacterium bovis BCG (M.bovis Bacille Calmette-Guérin, BCG). The use of BCG can be traced back to 1921. BCG remains the only TB vaccine currently available. BCG can significantly reduce the risk of severe types of t...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K39/04A61P31/06C12N15/74C12R1/32
CPCC07K14/35C07K14/195C07K14/245C12N9/1241C12N15/74A61K39/04A61P31/06A61K2039/522
Inventor 范小勇吴康罗道良
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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