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Protective antigen mutant of Bacillus anthracis

A Bacillus anthracis, protective antigen technology, applied in the field of Bacillus anthracis protective antigen gene and its encoded protein, can solve the problems of allergic reactions in patients, special groups that are not suitable for antibiotic allergies, and limited serum sources

Inactive Publication Date: 2009-05-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many defects in these traditional medicines and treatment methods: first, antibiotic treatment can only kill the Bacillus anthracis cells infected in the body, but it has no ability to spores with huge potential harm; second, for people in the late stage of infection, Because a large number of exotoxins have been secreted into the blood, antibiotics cannot remove them; third, because the current use of a large number of antibiotics has led to strong drug resistance in pathogenic bacteria; fourth, for the treatment of anthrax, these antibiotics Both require large doses and are not suitable for special populations who are allergic to antibiotics
Although antiserum therapy can remove the anthrax exotoxin secreted into the blood, there are still many problems such as limited serum sources and allergic reactions after injection.

Method used

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  • Protective antigen mutant of Bacillus anthracis
  • Protective antigen mutant of Bacillus anthracis
  • Protective antigen mutant of Bacillus anthracis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Cloning of Bacillus anthracis protective antigen gene and preparation of protein

[0021] The present invention relates to the cloning of the Bacillus anthracis protective antigen gene Pag and the cloning of related mutants thereof, and its main operations include:

[0022] (1) Primer design and synthesis

[0023] According to the nucleotide sequence of the pag gene published by NCBI GENEBANK (accession number: YP_016495), the following primer pair was designed and synthesized: Forward primer (PA-F'): 5'-ATT GGATCC GAA GTT AAA CAG GAG AAC-3', including BamHI restriction site (the underline is the restriction site); reverse primer (PA-R'): 5'-ACCG CTCGAG TTA TCC TAT CTC ATA GCC-3', including the XhoI restriction enzyme site (the underline is the enzyme site). The above primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0024] (2) Template preparation

[0025] Veterinary Bacillus anthracis spore vaccine (the strain was purc...

Embodiment 2

[0052] Example 2 Expression, Purification and Analysis of Bacillus anthracis Protective Antigen Protein

[0053] (1) Expression of recombinant protein

[0054] The recombinant plasmid pGEX-PA was transformed into the host cell E. coli BL21(DE3) (for the operation steps, refer to the transformation part in Example 1). Transfer the overnight activated transformants to 1L LB liquid medium containing 100μg / mL ampicillin at an inoculum size of 1%, culture at 37°C for 2-3hrs, and make the OD600 reach 0.6-0.7, add isopropyl-B-D- Thiogalactoside (IPTG) was grown to a final concentration of 0.2 mM and cultured at 15° C. at 200 rpm for 8 hours. Collect the bacteria by centrifugation, then wash once with PBS buffer (140.0mM sodium chloride, 2.7mM potassium chloride, 10.0mM disodium hydrogen phosphate, 1.8mM dipotassium hydrogen phosphate, pH 7.4), and then suspend with 50mL PBS buffer , and then break the cells with a high-pressure cell breaker (purchased from THERMO Company, USA). Th...

Embodiment 3

[0089] Example 3 Cell culture and cytotoxicity detection

[0090] Mouse mononuclear cell line RAW264.7 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) was used in DMEM high-glucose medium (purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.) containing 10% fetal bovine serum (purchased from from Promega, USA) at 37°C with 5% CO 2, cultivated in an incubator with 95% air (purchased from Japan Sanyo Company). The cells were subcultured with trypsin diethylaminetetraacetic acid (EDTA) digestion solution (purchased from Beyentian Institute of Biotechnology) to digest the cells, and then seeded in 96-well culture plates (purchased from CORNING Company) until the cells grew to 1.5 × 10 per well. 4 When the number of cells is 1, discard the culture solution, add 100 μL of DMEM high-glucose medium containing the sample to be tested, continue to cultivate for three hours, and then add the fluorescent dye ALamar...

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Abstract

The invention belongs to the technical field of microorganism genetic engineering and particularly relates to a Bacillus anthracis protective antigen mutant and encoding protein thereof. The sequence of the mutant gene and the amino acid sequence of the encoding mutant protein are shown in SEQ ID NO of 1 in the sequence table. Through the verification of biology, the mutant protein has complete privation of cytotoxicity, and can inhibit the cytotoxicity of wild lethal toxin.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular to a gene of a mutated bacillus anthracis protective antigen and its encoded protein. Background of the invention [0002] Anthrax is an acute, febrile, septic infectious disease that is caused by Gram-positive Bacillus anthracis (Bacillus anthracis). Because it can cause black carbon-like necrosis of skin and other tissues, it is called "anthrax". In 1849, Kock, a famous German scholar, first discovered Bacillus anthrax. Since then, anthrax has been distributed or broke out many times around the world. Anthrax not only poses a threat to human life and health, but also brings huge economic losses to the world's animal husbandry. [0003] The pathogenic factors of anthrax mainly include the bacterial capsule encoded by the endogenous plasmid pOX-1 and the exotoxin encoded by the plasmid pOX-2. The former is composed of negatively charged poly-D-glutamic acid, which can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/32
Inventor 刘子铎吴高兵洪玉枝郭爱珍封纯芳曹莎
Owner HUAZHONG AGRI UNIV
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