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GFP-2 nano oligopeptide collagen and a preparing method and application thereof

A technology of GFP-2 and culture medium, which is applied in the preparation of small peptide adjuvants derived from marine bacillus GFP-2, and in the application field of immune efficacy, which can solve the problems of low immunogenicity and inability to achieve immune body efficacy, etc.

Active Publication Date: 2018-12-18
杭州洪晟生物技术股份有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is that the immunogenicity is often low, and the effect of the immune body cannot be achieved. To overcome the deficiency of LB medium, brown sugar medium is used to expand and cultivate GFP-2 bacteria, and a preparation of small peptide is provided. The method, the medium can increase the expression of small peptides, which is more conducive to industrial production, and the small peptides are successfully expressed and secreted into the medium, which is beneficial to the preparation and purification of small peptides, and is conducive to maintaining the activity of small peptides

Method used

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  • GFP-2 nano oligopeptide collagen and a preparing method and application thereof

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preparation example Construction

[0025] First of all, the present invention provides a preparation method of GFP-2 small peptide, the preparation method comprising the following steps:

[0026] (1) Prepare brown sugar medium and sterilize it;

[0027] (2) Inoculate GFP-2 bacteria, glycerol bacteria and LB medium, and culture on a shaking table;

[0028] (3) Transfer to brown sugar culture medium, carry out shaking table culture, then carry out centrifugation, collect supernatant, and discard bacteria;

[0029] (4) Salt out to precipitate proteins and small peptides, centrifuge again after salting out, remove supernatant, and collect crude protein;

[0030] (5) Resuspend the crude protein with tris-HCl, pass the resuspended crude protein through the membrane, and dialyze with tris-HCl;

[0031] (6) Perform ultrafiltration with an ultrafiltration tube to collect small peptides with a molecular weight of less than 10KD, cool the obtained small peptides, and freeze-dry to obtain GFP-2 small peptides.

[0032] ...

Embodiment 1

[0045] Preparation of GFP-2 Small Peptide

[0046] (1) Prepare 1 L of brown sugar medium and sterilize at 110°C for 15 minutes;

[0047] (2) Inoculate GFP-2 bacteria, 5mL LB medium per tube, and inoculate glycerol bacteria and medium at a ratio of 1:100. A total of 2 tubes of GFP-2 bacterial solution were connected and cultured on a shaking table for 12 hours;

[0048] (3) Transfer to 1L brown sugar medium, culture on a shaker for 12 hours, centrifuge at 4000 rpm for 1 hour, collect the supernatant, and discard the bacteria;

[0049] (4) Prepare saturated ammonium sulfate. Salt out the supernatant and saturated ammonium sulfate at a ratio of 4:1 to precipitate proteins and small peptides. After salting out, centrifuge again at 12,000 rpm for 20 minutes, remove the supernatant, and collect crude protein.

[0050] (5) Resuspend the crude protein with tris-HCL, pass the resuspended crude protein through a 0.45mm filter membrane, dialyze with tris-HCL for 5 hours, and change the...

Embodiment 2

[0053] Study of GFP-2 Small Peptide on Improving Antibody Titer

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Abstract

The invention relates to GFP-2 nano oligopeptide collagen and a preparing method and application thereof. The GFP-2 nano oligopeptide collagen is prepared by (1) preparing a brown sugar medium and performing sterilization; (2) inoculating GFP-2 bacteria, a bacterial glycerol stock and an LB medium and performing shake culture; (3) transferring a product to the brown sugar medium, performing shakeculture, and then performing centrifugation, with supernatant liquid being collected and bacterial cells being abandoned; (4) salting out to precipitate protein and nano oligopeptide collagen, then performing centrifugation again, removing supernatant liquid and collecting crude protein; (5) resuspending the crude protein with tris-HCl, allowing the resuspended crude protein to pass through a membrane and performing dialysis with tris-HCl; and (6) performing ultrafiltration with an ultrafiltration tube, collecting nano oligopeptide collagen the molecular weight of which is less than 10 KD, cooling the nano oligopeptide collagen and freeze-drying the nano oligopeptide collagen to obtain the GFP-2 nano oligopeptide collagen. The prepared GFP-2 nano oligopeptide collagen can be used as an adjuvant and can greatly boost immunity.

Description

technical field [0001] The invention relates to the technical field of biotechnology and pharmaceutical engineering, in particular to a method for preparing a small peptide adjuvant derived from marine bacillus GFP-2, and the application of the small peptide in improving the immune efficacy of vaccines. Background technique [0002] Vaccination is the most cost-effective way to prevent and treat infectious diseases, and adequate immunogenicity and safety of vaccines are the most important concerns in this field. While traditional vaccines such as live, attenuated organisms and killed or inactivated organisms are generally more immunogenic, there is a limited potential risk of reversion to virulence. Additionally, these vaccines are unstable and have low safety profiles. Compared with traditional vaccines, new vaccines such as DNA vaccines, recombinant vaccines and subunit vaccines are relatively safe. However, these vaccines also have certain defects. One of the biggest de...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/32C07K1/34A61K39/39A61P37/04C12R1/07
CPCA61K39/39A61K2039/55516A61P37/04C07K14/32C12P21/02
Inventor 吕正兵舒建洪王丹
Owner 杭州洪晟生物技术股份有限公司
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