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Staphylococcus aureus SpA5 mutant, as well as preparation method and application thereof

A golden yellow, carrier technology, applied in the biological field, to achieve the effect of resisting Staphylococcus aureus infection, strong immunogenicity and controllable quality

Active Publication Date: 2014-04-02
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the ability of SpA to bind antibodies, using natural SpA as an antigen cannot achieve the desired goal, and it needs to be mutated to remove its antibody-binding activity while retaining its immunogenicity

Method used

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  • Staphylococcus aureus SpA5 mutant, as well as preparation method and application thereof
  • Staphylococcus aureus SpA5 mutant, as well as preparation method and application thereof
  • Staphylococcus aureus SpA5 mutant, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Sequence Synthesis

[0097] Using the MRSA252 genome (GI: 49240382) as a template to obtain the SpA base sequence and amino acid sequence (SpA (252), SEQ ID NO.5), the five EDABC domains (ie, 37aa-327aa of SEQ ID NO.5) , named as SpA5(252)) according to the preference of Escherichia coli on the website http: / / people.mbi.ucla.edu / sumchan / caltor.html for rare codon analysis and optimization, for each domain 4 amino acids were point mutated to synthesize the nucleotide sequences encoding amino acids 6-296 of the four proteins of SpA5(KKAA), SpA5(RRAA), SpA5(KKVV), and SpA5(RRVV), using BamHI and NotI as dual enzymes The cleavage site was loaded into the expression vector pGEX-6P-2, and then transformed into the host strain XL-1Blue (directly completed by Shanghai Jierui Biotechnology Co., Ltd.). The nucleotide sequences corresponding to the four SpA5 proteins are respectively shown in SEQ ID NO.9-12, wherein the 16th-891st nucleotides in the nucleotide sequence...

Embodiment 2

[0101] Example 2: Induced expression, purification and identification of expression forms of each protein in Escherichia coli

[0102] 1) Target protein induced expression

[0103] ① Take 100 μL of the culture solution of each recombinant engineered bacteria, add it to 10 mL of LB medium with an ampicillin concentration of 100 μg / mL, and culture overnight at 80 rpm at 37°C. Take 400 μL of the overnight cultured bacterial solution and add it to 20 mL of LB medium with an ampicillin concentration of 100 μg / mL, incubate at 37°C for 2 to 3 hours, and rotate at 220 rpm; The final concentration was 200 μM, and then placed on a shaker at 30° C. to induce expression for 3 hours, and then 16° C. to induce expression overnight.

[0104] ②Take out the bacterial solution after induced expression, centrifuge at 10,000rpm for 5min, discard the supernatant, add 1mL PBS to mix, ultrasonically (power 300 watts) for 10min (work for 6 seconds, rest for 9 seconds), then 14000×g at 4°C Centrifug...

Embodiment 3

[0110] Embodiment 3, the fermentation of engineering bacterium

[0111] 1. Determination of fermentation conditions

[0112] 1) To detect the effect of culture medium on the growth of engineered bacteria and the expression of target protein

[0113] Four media were tested in shake flasks by the culture method described in Example 2:

[0114] Plant-derived modified TB (potassium dihydrogen phosphate 2.3g, disodium hydrogen phosphate dodecahydrate 13g, glycerin 5ml, yeast extract 24g, soybean peptone 12g, magnesium sulfate 1g, add water to 1L);

[0115] Animal-derived modified TB (2.3g potassium dihydrogen phosphate, 13g disodium hydrogen phosphate dodecahydrate, 5ml glycerin, 24g yeast extract, 12g animal-derived tryptone, 1g magnesium sulfate, add water to 1L);

[0116] Plant-derived M9-CAA (disodium hydrogen phosphate 15.6g, potassium dihydrogen phosphate 4.3g, ammonium chloride 1g, magnesium sulfate 1g, sodium chloride 0.67g, glucose 5g, soybean peptone 3.6g, plant-derived...

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Abstract

The invention belongs to the technical field of biology and relates to a staphylococcus aureus SpA5 mutant protein, and a carrier, an engineering strain, a composition or a kit containing the mutant protein, as well as an application, a preparation method, a fermentation method and a purification method of the mutant protein. The SpA5 protein provided by the invention can effectively stimulate an organism to produce protective immune response and further resist staphylococcus aureus infection, and has the advantages of strong immunogenicity, safety, no toxicity, safety, effectiveness and controllable quality.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a SpA5 mutant protein of Staphylococcus aureus. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, SA), hereinafter referred to as Staphylococcus aureus, has another name of "flesh-loving bacteria". As a representative of Gram-positive bacteria, it is an important pathogenic bacteria causing nosocomial infection and community infection. Infection is characterized by acute, purulent inflammation. Local infection can cause purulent infection of the skin and soft tissues, which does not heal after a long time; systemic infection can lead to osteomyelitis, septic arthritis, endocarditis, pneumonia, and sepsis And other serious infections and complications, the mortality rate is as high as 20%. At the same time, the exotoxin of Staphylococcus aureus can also cause systemic fatal infections such as food poisoning, scalded skin syndrome and toxic shock syndrome. The...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K1/36C07K1/30C07K1/22C07K1/18C07K16/12C12N15/31C12N15/70C12N1/21C12P21/02A61K39/085A61K39/40A61P31/04G01N33/569
CPCA61K39/00A61K2039/505C07K14/31C07K16/1271G01N2333/31
Inventor 曾浩邹全明樊绍文卢陆冯强吴翼
Owner CHENGDU OLYMVAX BIOPHARM
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