Methicillin-resistant Staphylococcus aureus (MRSA) vaccine recombinant proteantigen I12C, and preparation method and application thereof

A recombinant protein and protein technology, applied in biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., to achieve the effect of simple steps, high expression, and good immune protection effect

Active Publication Date: 2013-04-24
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this study, the active functional fragments of ClfA and IsdB were screened out by bioinformatics technology, and the active functional fragments were combined. There is no fusion protein that uses ClfA and IsdB to select the active functional fragments of the two subunits as immunogenic materials. Preparation MRSA Bivalent Vaccine Report

Method used

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  • Methicillin-resistant Staphylococcus aureus (MRSA) vaccine recombinant proteantigen I12C, and preparation method and application thereof
  • Methicillin-resistant Staphylococcus aureus (MRSA) vaccine recombinant proteantigen I12C, and preparation method and application thereof
  • Methicillin-resistant Staphylococcus aureus (MRSA) vaccine recombinant proteantigen I12C, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Cloning of expression vector fused with two active fragments of methicillin-resistant Staphylococcus aureus iron surface determinant protein IsdB and one active fragment of ClfA

[0065] 1. Take out the MRSA-252 strain of methicillin-resistant Staphylococcus aureus from the freezer at -80°C and spread it on the special solid medium for MRSA-252, cultivate it overnight at 37°C, and then pick a single colony and inoculate it on MRSA- 252 special liquid culture medium for 8 hours, referring to the bacterial genome extraction kit to extract the MRSA genome.

[0066] 2. Using the PCR method to self-synthesize template I 12 Amplification I 12 -Linker-gene fragment, the amplification steps are as follows:

[0067] 1) Design PCR primers P1 and P2.

[0068]

[0069] 2) Using Synthetic Template I 12 PCR amplification of ClfA 97-639 Gene fragment.

[0070] 3) PCR system:

[0071] Template (50ng / μl)

2.5μl

P1 (50μM)

1μl

P2 (50μM) ...

Embodiment 2

[0112] Embodiment 2: I 12 Induced expression, purification and identification of expression form of C multi-subunit in prokaryotic expression system-Escherichia coli

[0113] 1. Induced expression of target protein

[0114] 1) Take double enzyme digestion to identify the correct pGEX-6P-2-I 12 Add 100 μL of C / XL-1blue bacterial solution to 10 mL of Amp-resistant TB medium, 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 Amp-resistant TB medium (the rest of the bacterial solution should be stored in a refrigerator at 4°C) Incubate at 37°C for 2~3h, rotate at 200rpm, reactivate to OD600 of 0.6~0.8, add 40μL of IPTG to make the final concentration 200μM, then place on a shaker to induce expression overnight at 16°C.

[0115] 2) Take out the bacterial solution after induced expression, centrifuge at 6000rpm for 15min, discard the supernatant, add 1mLlysis buffer to mix, ultrasonically lyse for 3min (sonicat...

Embodiment 3

[0122] Embodiment 3: I 12 Preparation of C antigen

[0123] 1. Amplify culture to obtain protein

[0124] Take the spare pGEX-6P-2-I stored in the refrigerator at 4°C 12 Add 400μL of C / XL-1blue bacteria solution to 20mL TB medium containing Amp resistance for primary activation. After incubating at 200rpm at 37°C for 5~6h, take 8mL of primary activated bacterial solution and add it to 400mL TB medium containing Amp resistance. Secondary activation was carried out at 37°C for 3~4h until OD600 was 0.8, 80μl IPTG (final concentration 200uM) was added and placed in a shaker at 16°C for overnight induction, and the cells were collected by centrifugation at 6000rpm for 15min, and then 20mL lysis buffer was added After the bacteria were resuspended, the bacterial solution was ultrasonically lysed for 3 minutes (200V), and the supernatant was collected and combined with 800 μL of Glutathione Sepharose4B gel beads (beads) for binding to the GST fusion protein; then SDS-PAGE gel elect...

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Abstract

The invention relates to a preparation method and application of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine recombinant protein I12C. The fusion protein is composed of two active segments of MRSA iron ion surface determination protein IsdB and an active segment of ClfA antigen molecule, and the segments are used by connecting peptides. The fusion protein has the advantages of high purity, high expression quantity, high efficiency and high safety, and is convenient for separation and purification. The preparation method is simple and easy to amplify, and has favorable repetitiveness. After using the aluminum adjuvant, the fusion protein can be used for preparing an anti-MRSA subunit vaccine and preparing a detection kit for MRSA. The animal experiment proves that the fusion protein can effectively stimulate the mechanisms to generate high humoral immune response and favorable immunoprotection function of resisting MRSA infection.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and in particular relates to a recombinant protein antigen I for human methicillin-resistant Staphylococcus aureus (MRSA) vaccine 12 C and preparation method and application. Background technique [0002] Methicillin-resistant Staphylococcus aureus refers to Staphylococcus aureus resistant to oxazole penicillins such as methicillin, oxacillin and flucloxacillin. Gram-positive bacteria in the perineum, perineum, and intestinal tract usually cause skin and soft tissue infections, bacteremia, and metastatic complications such as pneumonia, endocarditis, septic arthritis, and osteomyelitis. Since it was first discovered by British scholar Jevons in 1961, it has become one of the pathogenic bacteria with the highest infection rate in ICU wards, burns, and war wounds in the world. The local infection caused by it lasts for a long time, and the mortality rate of systemic infection is as high a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K39/085A61P31/04G01N33/569C12R1/19
Inventor 曾浩邹全明冯强樊绍文卢陆蔡昌芝吴翼顾江章金勇
Owner CHENGDU OLYMVAX BIOPHARM
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