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Thymine auxotrophic bacillus anthracis and application thereof

A technology of Bacillus anthracis and thymine, which can be used in bacteria, antibacterial drugs, bacterial antigen components, etc., and can solve problems such as non-compliance with requirements

Inactive Publication Date: 2010-08-25
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But such expression often requires antibiotics to provide resistance pressure and does not meet today's requirements for vaccine-like biologics

Method used

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  • Thymine auxotrophic bacillus anthracis and application thereof
  • Thymine auxotrophic bacillus anthracis and application thereof
  • Thymine auxotrophic bacillus anthracis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Acquisition of auxotrophic attenuated Bacillus anthracis without resistance markers

[0033] 1. Targeting plasmid construction

[0034] According to the LoxP sequence reported in the literature, two long primers loxpF and LoxpR were designed to anneal directly to synthesize a fragment containing two LoxP sequences and a Sal I site, and the fragment was ligated into the pEASY-T1 vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd. company) obtained a new vector pT-loxP. Wherein, primers loxpF and LoxpR sequences are:

[0035] loxpF: CCGCGG ATAACTTCGTATAATGTATGCTATACGAAGTTAT GTC GAC AT

[0036] AACTTCGTATA (sequence 1 in the sequence listing)

[0037] loxpR: GAATTC ATAACTTCGTATAGCATACATTATACGAAGTTAT GTC GAC AT

[0038] AACTTC (SEQ ID NO: 2 in the SEQUENCE LISTING).

[0039] Then use the plasmid pSET4s (Daisuke Takamatsu, Makoto Osaki, and Tsutomu Sekizaki. Thermosensitive Suicide Vectors for Gene Replacement in Streptococcus suis. Plasm...

Embodiment 2

[0069] Example 2. Functional verification of auxotrophic attenuated Bacillus anthracis without resistance markers

[0070] 1. Phenotype analysis of defective bacteria

[0071] Use two parts of liquid LB and two parts of solid LB to culture the defective strain Bacillus anthracis (Bacillus anthracis) AP425, one part of liquid LB or solid LB with thymine added, the other part of liquid LB or solid LB without thymine, culture at 37°C After 14 hours, observe its growth condition. The results showed that, in the case of adding thymine, no matter whether it was liquid culture or solid culture, the bacteria grew well, but the bacteria grew very slowly without adding thymine (as shown in Figure 8). A in Fig. 8 is a photo of Bacillus anthracis (Bacillus anthracis) AP425 cultured in liquid LB (left) with thymine added and liquid LB without thymine (right), and B in Fig. 8 is solid LB with thymine added (left ) and Bacillus anthracis AP425 cultured on solid LB without added thymidine (...

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Abstract

The invention discloses a thymine auxotrophic strain and application thereof. The strain is obtained by inactivating the thyA gene and the thyX gene of bacillus anthracis, which is specifically bacillus anthracis CGMCC No.3405. The strain can serve as a host to build a balanced lethal expression system for studying relative bacillus anthracis vaccines and laying a foundation for the study of novel vaccines.

Description

technical field [0001] The invention relates to a thymidine auxotroph phenotype Bacillus anthracis and its application. Background technique [0002] Bacillus anthracis is a G + Bacteria, which cause anthrax is a severe infectious disease, which is listed as a Class B infectious disease in my country and has been included in the national immunization plan. The existing anthrax vaccine A16R strain still retains the large plasmid pXO1, which has certain residual toxicity. Therefore, it is very necessary to study a safer and more effective new Bacillus anthracis vaccine. [0003] The combination of bacterial components and protective antigens is currently a widely recognized research strategy for anthrax vaccines, and research on the expression of protective antigens using attenuated Bacillus anthracis as a host has also been reported. But such expression often requires antibiotics to provide resistance pressure, which does not meet today's requirements for vaccine-like biol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12N15/75A61K39/07A61P31/04C12R1/07
Inventor 王艳春展德文刘纯杰陶好霞袁盛凌王令春王芃
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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