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MRSA (Methicillin Resistant Staphylococcus Aureus) vaccine recombinant protein antigen I1C 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., can solve the problems of unfavorable vaccine industrialization preparation, cumbersome methods, and great difficulty, so as to maintain spatial conformation and immunity The effect of originality, simple preparation method and simple steps

Active Publication Date: 2014-03-12
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the pathogenic factors of methicillin-resistant Staphylococcus aureus include dozens of capsular polysaccharides, ClfA, IsdB, enterotoxin, TSST-1, α-hemolysin, and coagulase, etc., and their contents are low, directly from the whole bacteria It is difficult to separate and purify the protective antigen, and the method is cumbersome, which is not conducive to the industrial preparation of vaccines
There is no report on the preparation of MRSA bivalent vaccines using ClfA and IsdB to select a fusion protein of an active functional fragment of a subunit as an immunogenic material

Method used

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  • MRSA (Methicillin Resistant Staphylococcus Aureus) vaccine recombinant protein antigen I1C and preparation method and application thereof
  • MRSA (Methicillin Resistant Staphylococcus Aureus) vaccine recombinant protein antigen I1C and preparation method and application thereof
  • MRSA (Methicillin Resistant Staphylococcus Aureus) vaccine recombinant protein antigen I1C and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1: The DNA sequence of I1C was synthesized by Shanghai Jierui Biotechnology Co., Ltd. and constructed on the vectors pGEX-6P-2 and pET22b, and the host bacteria XL-1blue, BL21(DE3), and HMS(174) were transformed respectively.

Embodiment 2

[0048] Example 2: Induced expression, purification and identification of expression forms of I1C multi-subunits in prokaryotic expression system-Escherichia coli

[0049] 1. Induced expression of target protein

[0050] 1) Take 100 μL of the pGEX-6P-2-I1C / 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 take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp-resistant bacterial solution TB medium (the rest of the bacterial solution was stored in a refrigerator at 4°C for later use), cultured at 37°C for 2-3 hours at a rotation speed of 200 rpm, and after secondary activation until the OD600 was 0.6-0.8, 40 μL of IPTG was added to make the final concentration 200 μM. Then placed on a shaker to induce expression overnight at 16°C.

[0051] 2) Take out the bacterial liquid after induced expression, centrifuge at 6000rpm for 15m...

Embodiment 3

[0058] Embodiment 3: Preparation of I1C antigen

[0059] 1. Scale up culture to obtain protein

[0060] Take 400 μL of the spare pGEX-6P-2-I1C / XL-1blue bacterial solution stored in the refrigerator at 4°C and add it to 20mL TB medium containing Amp resistance for one activation. After culturing at 200rpm and 37°C for 5~6h, take 8mL once Add the activated bacterial solution to 400mL TB medium containing Amp resistance for secondary activation, culture at 37°C for 3~4h until the OD600 is 0.8, add 80μl IPTG (final concentration is 200uM) and place in a shaker at 16°C overnight After induction, centrifuge at 6000rpm 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 of Glutathione Sepharose4B gel beads for binding to the GST fusion protein ( beads) combined processing.

[0061] 2. Use the enzyme digestion method to separate the target protein from the GST...

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Abstract

The invention relates to an MRSA (Methicillin Resistant Staphylococcus Aureus) vaccine recombinant protein antigen I1C and a preparation method and an application thereof. A fusion protein, namely the recombinant protein antigen I1C, is fused by an active fragment I1 which is positioned on the MRSA iron ion surface and used for determining protein IsdB (iron-regulated surface determinant B) and an active fragment C of C1fA (clumping factor A) antigen molecules. The fusion protein is high in purity and expression quantity, convenient to separate and purify and high in efficiency and safety. The preparation method is simple and rapid, easy to amplify and good in repeatability; the fusion protein can be used for preparing an MRSA-resistant submit vaccine after supplemented with an aluminum adjuvant and can also be used for preparing a detection kit for preparing MRSA; and animal experiments prove that the MRSA submit vacuum can be used for effectively stimulating organisms to generate higher humoral immune response and favorable MRSA infection resistant immunoprotection effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant protein antigen I1C of methicillin-resistant Staphylococcus aureus (MRSA) vaccine for humans, a preparation method and application thereof. 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 as 20%....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K39/085A61P31/04G01N33/68C12R1/19C12R1/445
Inventor 邹全明樊绍文曾浩冯强吴翼蔡昌芝郭鹰
Owner CHENGDU OLYMVAX BIOPHARM
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