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Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein FnbA1 and preparation method and application thereof

A methicillin-resistant and recombinant protein technology, applied in the field of biotechnology and pharmaceuticals, can solve the problems of complex antigenic components of methicillin-resistant Staphylococcus aureus, unfavorable vaccine industrialization preparation, cumbersome methods, etc., and achieve good resistance to MRSA infection , good immune protection effect, simple steps

Active Publication Date: 2013-04-03
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The antigen components of methicillin-resistant Staphylococcus aureus are complex and the content is low. It is difficult to directly isolate and purify the protective antigen from the whole bacteria, and the method is cumbersome, which is not conducive to the industrial production of vaccines.

Method used

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  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein FnbA1 and preparation method and application thereof
  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein FnbA1 and preparation method and application thereof
  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein FnbA1 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cloning of Methicillin-resistant Staphylococcus aureus Fibronectin Binding Protein A Active Fragment FnBA1 Active Fragment

[0050] 1. First, according to the full-length gene sequence of MRSA-252 FnBA protein, use bioinformatics software for structural analysis to determine the FnBA1 target gene fragment that needs to be amplified.

[0051] 2. According to the analysis results, the PCR method was used to amplify the FnBA1 target gene fragment from the MRSA-252 genome, and the amplification steps were as follows:

[0052] 1) Design the PCR primers as follows, which are SEQ ID NO: 5-6 (the base sequence of the restriction site is underlined)

[0053] FnBA1-F: SEQ ID NO: 5

[0054] 5′-CGC GGATCC ATG GGACAAGATAAAGAAGCTGCA-3′

[0055] BamH Ⅰ

[0056] FnBA1-R: SEQ ID NO: 6

[0057] 5′-TTTTCCTTTT GCGGCCGC CTAT CCATTATCCCATGTTAATGTAT-3′

[0058] Not Ⅰ

[0059] 2) The methicillin-resistant Staphylococcus aureus MRSA-252 strain was taken out of the -80°C fre...

Embodiment 2

[0086] Example 2: MRSA-252 FnBA subunit active fragment FnBA1 induced expression, purification and expression form identification in prokaryotic expression system-Escherichia coli

[0087] 1. Induced expression of target protein

[0088] 1) Take 100 μL of the pGEX-6P-2-FnBA1 / XL-1blue bacterial solution that has been correctly identified by double enzyme digestion and add it to 10 mL of Amp-resistant TB medium, cultivate overnight at 80 rpm at 37°C, and add 400 μL of the overnight cultured bacterial solution to 20 mL of Amp In the resistant TB culture medium (the rest of the bacterial solution is stored in a 4°C refrigerator for later use), culture at 37°C for 2~3h, rotate at 200rpm, and reactivate until the OD600 is 0.8-1.0, add 4μL of IPTG to make the final concentration 200 μM, then placed on a shaker to induce expression at 30°C for 3h, at 25°C for 5h, and at 16°C overnight to induce expression.

[0089] 2) Take out the bacterial liquid after induced expression, centrifuge...

Embodiment 3

[0096] Example 3: Preparation of FnBA1 antigen

[0097] 1. Amplify culture to obtain protein

[0098]Take 400 μL of the spare pGEX-6P-2-FnBA1 / XL-1blue bacterial solution stored in the refrigerator at 4°C and add it to 20mL TB medium containing Amp resistance for one activation. Add the primary activated bacterial solution to 400mL TB medium containing Amp resistance for secondary activation, culture at 37°C for 3~4h until OD600 is 1.0, add 80ul IPTG (final concentration is 200uM) and place in a shaker at 16°C After overnight induction, centrifuge at 12000rpm for 15min to collect the bacteria, add 20mL of lysis buffer to resuspend the bacteria, then ultrasonically lyse the bacteria for 3min (200V), collect the supernatant and 800μL Glutathione Sepharose 4B gel for binding to GST fusion protein Beads (beads) binding treatment; then SDS-PAGE gel electrophoresis.

[0099] 2. Use the enzyme digestion method to separate the target protein from the GST tag to obtain the FnBA1 targe...

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Abstract

The invention discloses a methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen FnbA1 and a preparation method and application thereof. The invention also discloses a method for establishing an expression vector of the recombinant protein and converting host bacteria to prepare the recombinant protein and application of the recombinant protein in preparing a subunit vaccine resistant to MRSA and a related detection kit. Through the invention, the recombinant protein prepared by the method is convenient to separate and purify and efficient and safe, and the expression amount is large. Animal experiments prove that the prepared recombinant protein can effectively stimulate the organism to generate relatively high humoral immune response and good immune protection effect.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and relates to a recombinant protein of methicillin-resistant Staphylococcus aureus fibronectin-binding protein A active fragment FnBA1. The invention further relates to the preparation method of the recombinant protein and its use in the preparation of vaccines and detection kits in the application. Background technique [0002] Methicillin-resistant Staphylococcus aureus or Multiple-resistant Staphylococcus aureus (MRSA) is a unique strain of Staphylococcus aureus that is resistant to all penicillins, including methicillin and other β-lactamase-resistant penicillin. First discovered in the UK in 1961, MRSA is now widespread and is even known as a "superbug" in hospitals. [0003] At present, MRSA has become one of the pathogenic bacteria with the highest infection rate in ICU wards, burns, and war wounds in the world. In 2005, MRSA infection surveillance data in the United States sh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/63C12N1/21A61K39/085A61P31/04G01N33/569C12R1/19
Inventor 邹全明樊绍文卢陆曾浩冯强吴翼蔡昌芝董衍东鲁东水
Owner CHENGDU OLYMVAX BIOPHARM
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