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Purifying method of methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C

A methicillin- and staphylococcus-resistant technology is applied in the field of biopharmaceuticals to achieve the effects of high humoral immune response, simple process and good immune protection.

Inactive Publication Date: 2013-10-02
CHONGQING YUANLUN BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no recombinant dual-subunit genetically engineered protein I targeting (MRSA) 1 Report on Purification Method of C Vaccine

Method used

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  • Purifying method of methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C
  • Purifying method of methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C
  • Purifying method of methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 autoclave, centrifugation

[0032] The recombinant double-subunit genetic engineering protein I constructed by the applicant to express soluble methicillin-resistant Staphylococcus aureus 1 The Escherichia coli engineering bacteria of C (refer to the invention patent application of 201310021212.3, the antigen I 1 The nucleotide sequence of C is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2). Through high-density fermentation, the expression rate of the target protein was 13%, and the cells were collected by centrifugation for later use.

[0033] 200-500 g of bacterial cells were mixed with PBS (10-20 mM, pH 7.0-7.5) buffer solution according to the weight: volume ratio of 1:10, and pre-cooled at 4°C.

[0034] High-pressure homogenizer: Use distilled water to flush the pipeline of the high-pressure homogenizer, and turn on the low-temperature circulation system to pre-cool to 1-4°C for later use.

[0035] Add the pre-cooled su...

Embodiment 2

[0037] Embodiment 2 ammonium sulfate step-by-step precipitation, redissolving:

[0038] Under the condition of stirring at 4°C, slowly add ammonium sulfate with a final concentration of 30% to the supernatant, stir for more than half an hour, centrifuge at 10000-15000g for more than 20 minutes, and collect the supernatant; continue to slowly add ammonium sulfate with a final concentration of 40% to the supernatant Ammonium sulfate, stirred for more than half an hour, centrifuged at 10000-15000g for more than 20 minutes at high speed, and collected the precipitate;

[0039] Precipitation redissolution: Weigh the wet weight of the precipitate and add buffer A (10-20mM Na 2 HPO 4 -NaH 2 PO 4 , 0.5M NaCl, 2mM EDTA, 0.5% Poloxamer188, pH7.0-7.5), stirred and mixed for 10-15 minutes, centrifuged at 10000-15000g for more than 20 minutes, and collected the supernatant; the electrophoresis results of the reconstituted samples were as follows: figure 1 As shown, I 1 The purity of p...

Embodiment 3G

[0040] Embodiment 3 GST affinity purification:

[0041] Choose GST affinity chromatography filler for preliminary purification, GST affinity filler is one of GST-Sepharose4B, GST-Sepharose6B, GST-Sepharose FastFlow, GST-Sepharose HP, and the amount of filler per 100g wet weight of broken bacteria is 100ml.

[0042] Prescission Protease (PP enzyme) for enzyme digestion and elution: the PP enzyme used has a GST tag to facilitate the removal of PP enzyme and obtain I 1 C target protein, electrophoresis results are as follows figure 2 shown. After GST primary purification, the purity of the target protein is further improved, reaching about 85%, and further purification is still needed to remove trace impurities.

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Abstract

The invention discloses a purifying method of a methicillin-resistant staphylococcus aureus MRSA recombinant protein vaccine I1C, wherein the I1C protein is obtained by recombining and fusing active functional fragments of two antigen molecules of IsdB and ClfA of methicillin-resistant staphylococcus aureus and expressing escherichia coli genetically engineered bacteria. The high purity MRSA recombinant dual subunit genetic engineering protein I1C is obtained by technologies on the genetically engineered bacteria such as high pressure breaking, salting out, GST (Glutathione S Transferase) affinity chromatography, PP (Prescission Protease) digestion, adhere chromatography, gel filtration chromatography and the like. The method disclosed by the invention is simple and rapid in purifying process, easiness in application and good in repeatability. The target protein obtained is high in purity. Animal tests verify that the vaccine can effectively stimulate the organism to generate higher humoral immune response and good immunoprotection.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to methicillin-resistant Staphylococcus aureus (MRSA) recombinant protein vaccine I 1 C purification method. Background technique [0002] Methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus, MRSA) is a gram-positive coccus that can infect any part of the human body. Local infection persists for a long time, and the mortality rate of systemic infection is as high as 20%. One of the pathogenic bacteria with the highest infection rate in burns and war wounds. Recent studies have proved that among all the pathogenic factors of MRSA, Staphylococcus aureus iron element determinant B protein (Factor B of surface Iron from Staphylococcus aureus, IsdB) is not only an important outer membrane rivet protein of MRSA, but also plays a role in the colonization and adhesion of MRSA. It plays an important role, and it is also a main tool for MRSA to obtain i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/36C07K1/30C07K1/22C07K1/16
Inventor 敬海明郭鹰邹全明章金勇董衍东曾浩
Owner CHONGQING YUANLUN BIOTECH
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