Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof

A staphylococcus, recombinant protein technology, applied in the direction of biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of difficult control of the situation, few treatment methods, etc., and achieve simple steps, easy separation and purification, good The effect of immune protection

Active Publication Date: 2013-03-27
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And as the multi-antibiotic resistance of the bacteria makes the situatio

Method used

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  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof
  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof
  • Methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: The gene encoding HI2 was synthesized by Shanghai Jierui Biotechnology Co., Ltd. and cloned into pGEX-6P-2 and pET22b.

Embodiment 2

[0046] Example 2: Transformation and identification of recombinant plasmids into expression host bacteria.

[0047] 1. Conversion

[0048] Take 3 tubes of Escherichia coli XL1blue competent cells from the -80°C refrigerator, add pGEX-6P-2 plasmid to the first tube as a positive control; add synthetic pGEX-6P-2-HI2 to the second tube; Source DNA was used as a negative control. Ice bath for 50min, heat shock in 42℃ metal bath for 90s, rapid ice bath for 2min. Add 600 μl LB blank medium, mix well, and place in a shaker at 37°C at 220rp for 1h.

[0049] Each tube was centrifuged at 5000 rpm for 3 min at room temperature, 300 μl of the supernatant was discarded, and the bacteria were resuspended, and 200 μl was spread on an Amp-resistant LB plate. Plates were placed upside down in a 37°C incubator for 24 hours.

[0050] 2. Screening and identification of pGEX-6p-2-HI2 positive recombinant plasmid

[0051] ① There are no colonies on the negative control plate; the positive cont...

Embodiment 3

[0058] Example 3: Induced expression, purification and identification of expression form of recombinant fusion protein HI2 in prokaryotic expression system-Escherichia coli

[0059] 1. Induced expression of target protein

[0060] 1) Take 100 μL of the pGEX-6P-2-HI2 / XL-1blue bacterial solution that was 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, take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp-resistant (The rest of the bacterial solution is stored in a refrigerator at 4°C for later use), cultured at 37°C for 2-3 hours at a speed of 200rpm, and when the secondary activation reaches an OD600 of 0.8-1.0, add 4 μL of IPTG to a final concentration of 200 μM, and then placed on a shaking table to induce expression at 30°C for 3h, at 25°C for 5h, and at 16°C overnight to induce expression.

[0061] 2) Take out the bacterial solution after induced expression, centr...

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Abstract

The invention discloses a methicillin-resistant staphylococcus aureus (MRSA) vaccine recombinant protein antigen HI2, a preparation method and application thereof. The recombinant protein comprises a staphylococcus aureus (alpha) hemolysin (Hla) fragment and a ferrum surface determination protein (IsdB) fragment, wherein the Hla and the IsdB fragments are fused by a connecting peptide. The protein can be applied to preparation of a subunit vaccine and relative detection kits for resisting infection from methicillin-resistant staphylococcus aureus. According to the preparation method, the fused protein can be subjected to recombinant expression through the genetic engineering technology, therefore high expression quantity is gained, the separation and purification are convenient to carry out, and high efficiency and safety are ensured. As the animal experiment shown, the antigen can effectively stimulate an organism to perform higher humoral immune response and excellent immunoprotection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a recombinant fusion recombinant protein HI2 of a non-toxic mutant of methicillin-resistant Staphylococcus aureus alpha hemolysin and an active fragment of iron surface determinant protein (IsdB). The invention further relates to the recombinant protein The preparation method and its application in the preparation of vaccines and detection kits. Background technique [0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive coccus that can infect any part of the human body, and can cause a wide range of diseases in the human body, such as bacteremia, endocarditis, folliculitis , furuncle, furuncle, carbuncle, impetigo, cellulitis, staphylomycosis, toxic shock syndrome, central system infection, infectious eye inflammatory disease, osteomyelitis, bone and joint infection, respiratory system Infection, etc., pose a great threat to human health. And beca...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70C12N1/21A61K39/085A61P31/04G01N33/68C12R1/19
Inventor 邹全明樊绍文卢陆曾浩冯强左钱飞章金勇顾江鲁东水
Owner CHENGDU OLYMVAX BIOPHARM
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