CN109022522B - GFP-2 small peptide and preparation method and application thereof - Google Patents
GFP-2 small peptide and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a GFP-2 small peptide, a preparation method and application thereof, wherein the small peptide is prepared by the following method: (1) preparing a brown sugar culture medium, and sterilizing; (2) inoculating GFP-2 bacteria, inoculating glycerol bacteria and LB culture medium, and performing shake culture; (3) transferring the strain into a brown sugar culture medium, performing shaking table culture, centrifuging, collecting supernatant, and removing thalli; (4) salting out to separate out protein and small peptide, centrifuging again after salting out, removing supernatant, and collecting crude protein; (5) resuspending the crude protein in tris-HCl and membrane-passing the resuspended crude protein and dialyzing against tris-HCl; (6) ultrafiltering with ultrafiltering tube, collecting small peptide with molecular weight less than 10KD, cooling the obtained small peptide, and lyophilizing to obtain GFP-2 small peptide. The prepared small peptide can be used as an adjuvant and can greatly improve the immune efficacy.
Description
Technical Field
The invention relates to the technical field of biotechnology pharmaceutical engineering, in particular to a preparation method of a small peptide adjuvant derived from marine bacillus GFP-2 and application of the small peptide in improving the immune efficacy of a vaccine.
Background
Vaccination is the most cost-effective method for the prevention and treatment of infectious diseases, and adequate immunogenicity and safety of vaccines are the most important concerns in this field. Although 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. In addition, these vaccines are unstable and have low safety. Compared with the traditional vaccine, the novel vaccines such as DNA vaccine, recombinant vaccine and subunit vaccine are relatively safe. However, these vaccines have certain drawbacks, one of the biggest being that they are often less immunogenic and do not achieve the efficacy of the immune body, often requiring the addition of adjuvants or delivery vehicles to improve their immune efficacy. An immunoadjuvant is broadly defined as a modulator of the immune system that nonspecifically alters or enhances the body's specific immune response to the antigen, enhances the immunogenicity of the antigen or alters the type of immune response, and is not itself antigenic. It is therefore important to develop safe and effective adjuvants with high potential to boost specific immune responses.
Disclosure of Invention
Problems to be solved by the invention
The technical problems to be solved by the invention are that the immunogenicity is often low, the effect of an immune body cannot be achieved, the defect of an LB culture medium is overcome, a GFP-2 strain is subjected to enlarged culture by adopting a brown sugar culture medium, and the preparation method of the small peptide is provided.
Means for solving the problems
In order to solve the technical problems, the technical scheme adopted by the invention is to provide a preparation method of GFP-2 small peptide, which comprises the following steps:
a method for preparing a GFP-2 small peptide, which comprises the following steps:
(1) preparing a brown sugar culture medium, and sterilizing;
(2) inoculating GFP-2 bacteria, inoculating glycerol bacteria and LB culture medium, and performing shake culture;
(3) transferring the strain into a brown sugar culture medium, performing shaking table culture, centrifuging, collecting supernatant, and removing thalli;
(4) salting out to separate out protein and small peptide, centrifuging again after salting out, removing supernatant, and collecting crude protein;
(5) resuspending the crude protein in tris-HCl and membrane-passing the resuspended crude protein and dialyzing against tris-HCl;
(6) ultrafiltering with ultrafiltering tube, collecting small peptide with molecular weight less than 10KD, cooling the obtained small peptide, and lyophilizing to obtain GFP-2 small peptide.
Preferably, in the step (1), 0.8-1.2L of the glucose medium is prepared and sterilized at the temperature of 120 ℃ for 10-20 min; in the step (2), GFP-2 bacteria are inoculated, each tube is provided with 3-10mL of LB culture medium, and the ratio of glycerol bacteria to the culture medium is 1: inoculating 50-150 of strain, inoculating 2 tubes of GFP-2 strain liquid, and culturing by shaking for 8-15 h.
Preferably, in the step (3), transferring the cells into 0.5-1.5L of a brown sugar culture medium, and performing shake culture for 8-15 h; 2000-6000 rpm for 0.5-3h, collecting the supernatant, and discarding the cells.
Preferably, in step (4), the supernatant is mixed with a saturated ammonium sulfate solution according to a ratio of 2-5: 1, salting out to separate out protein and small peptide; centrifuging again after salting out, centrifuging at 10000-.
Preferably, in step (5), the crude protein is resuspended in tris-HCl and the resuspended crude protein is passed through a 0.45mm filter and dialyzed against tris-HCl for 3-8h, with the tris-HCl being exchanged every 0.5-2 h.
Preferably, in the step (6), ultrafiltration is carried out for 20-60min at 8000rpm and 4000-.
The invention also provides a GFP-2 small peptide, which is prepared by adopting the technical scheme.
Preferably, the small peptide has a molecular weight of less than 10 KD.
Another object of the present invention is to provide a small peptide having immunopotentiator activity, which is a mixed small peptide secreted by GFP-2 and having a molecular weight of 10 kD.
The invention further aims to provide application of the small peptide in preparing vaccines, immunomodulators or therapeutic drugs.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention provides a GFP-2 small peptide and a preparation method thereof, and the small peptide can be used as an adjuvant, greatly improve the immune efficacy, strengthen the specific immune response and obviously improve the antibody titer.
Detailed Description
Firstly, the invention provides a preparation method of GFP-2 small peptide, which comprises the following steps:
(1) preparing a brown sugar culture medium, and sterilizing;
(2) inoculating GFP-2 bacteria, inoculating glycerol bacteria and LB culture medium, and performing shake culture;
(3) transferring the strain into a brown sugar culture medium, performing shaking table culture, centrifuging, collecting supernatant, and removing thalli;
(4) salting out to separate out protein and small peptide, centrifuging again after salting out, removing supernatant, and collecting crude protein;
(5) resuspending the crude protein in tris-HCl and membrane-passing the resuspended crude protein and dialyzing against tris-HCl;
(6) ultrafiltering with ultrafiltering tube, collecting small peptide with molecular weight less than 10KD, cooling the obtained small peptide, and lyophilizing to obtain GFP-2 small peptide.
In the context of the present invention, the term "GFP" is short for the Green fluorescent protein Green fluorescent protein, a protein consisting of about 238 amino acids, and GFP-2 is a marine Bacillus.
In the context of the present invention, the term "LB medium" means LB (Luria-Bertani) liquid medium, which can be prepared by conventional methods, for example by the following method (1L):
adding 10g of tryptone (tryptone) into 950ml of deionized water; 5g of yeast extract (yesat extract); 10g of NaCl. Adjusting pH to 7.0 with 1M NaOH, diluting to 1L with deionized water, sterilizing at 121 deg.C for 20min under high pressure.
In the context of the present invention, the term "tris-HCl" is a buffered solution of tris and HCl, wherein tris is tris-hydroxymethyl aminomethane.
The present invention will be described in further detail with reference to examples. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the technical field of bioengineering if no special description is provided.
In one embodiment, the invention provides a small GFP-2 peptide prepared by one or more of the above embodiments.
In a preferred embodiment, the small peptide has a molecular weight of less than 10 KD.
One embodiment of the present invention provides a small peptide with immunopotentiator effect, which is a mixed small peptide secreted by GFP-2 and having a molecular weight of 10 kD.
In another embodiment, the invention provides a use of the small peptide in preparation of a vaccine, an immunomodulator or a therapeutic drug.
The present invention will be described in further detail with reference to examples. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the technical field of bioengineering if no special description is provided.
Example 1
Preparation of GFP-2 Small peptides
(1) Mixing with 1L of brown sugar culture medium, and sterilizing at 110 deg.C for 15 min;
(2) inoculating GFP-2 bacteria, each tube is 5mL LB culture medium, and glycerol bacteria and culture medium are mixed according to the proportion of 1: inoculating bacteria at a ratio of 100. 2 tubes of GFP-2 bacteria liquid are inoculated in total, and shake culture is carried out for 12 hours;
(3) transferring the strain into a 1L of a brown sugar culture medium, culturing for 12h by using a shaking table, centrifuging for 1h at 4000 rpm, collecting supernatant, and discarding thalli;
(4) preparing saturated ammonium sulfate, and mixing the supernatant and the saturated ammonium sulfate according to the ratio of 4: 1, salting out to separate out protein and small peptide, centrifuging again after salting out, centrifuging at 12000rpm for 20min, removing supernatant, and collecting crude protein
(5) Resuspending the crude protein in tris-HCL, filtering the resuspended crude protein through a 0.45mm filter, dialyzing against tris-HCL for 5h, and changing the tris-HCL every 1 h;
(6) ultrafiltering at 6000rpm of 10KD ultrafiltering tube for 35 min, collecting small peptide with molecular weight less than 10KD, placing the collected liquid small peptide in-80 deg.C refrigerator for 1h, and freeze-drying the solid small peptide in freeze dryer to obtain lyophilized powder for storage.
Example 2
Research on improvement of antibody titer by GFP-2 small peptide
The experimental breed was Bab/c mice, all of which were vaccinated according to the normal immunization program during the experiment and managed in the conventional manner.
A total of 12 Bab/c mice were divided into 4 groups of 3 mice each, and the amount of vaccine used in the experiment was 20. mu.g/mouse.
(1) PBS group
Group of Cap (20. mu.g)
③ Cap (20 microgram) + Small peptide (20 microgram)
Cap (20 microgram) + small peptide (80 microgram)
The small peptide added above was the GFP-2 small peptide prepared in example 1.
The mice are adapted to the environment for one week after being bought back, then are immunized for the first time, the eyeballs are bled after two weeks of immunization, ELISA detection is carried out, and the titer of serum is measured, wherein the results are shown in Table 1.
TABLE 1 ELISA assay for potency of the immunized two-week sera
The OD values given above give:
cap group serum titers were less than 1: 25600
Cap + small peptide (20) group serum titers were in 1: 25600-1: 51200
Cap + small peptide (80) group serum titers were greater than 1: 51200.
therefore, the antibody titer of the GFP-2 small peptide group added with 20 micrograms is obviously improved, and the antibody titer of the GFP-2 small peptide group added with 80 micrograms is obviously improved, so that the GFP-2 small peptide has the effect of an immunologic adjuvant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Claims (8)
1. The application of a GFP-2 small peptide in preparing an immunologic adjuvant comprises the following steps:
(1) preparing a brown sugar culture medium, and sterilizing;
(2) inoculating GFP-2 bacteria, inoculating glycerol bacteria and LB culture medium, and performing shake culture;
(3) transferring the strain into a brown sugar culture medium, performing shaking table culture, centrifuging, collecting supernatant, and removing thalli;
(4) supernatant and saturationAmmonium sulfateThe solution is prepared according to the following steps of 2-5: 1, salting out to separate out protein and small peptide, centrifuging again after salting out, removing supernatant, and collecting crude protein;
(5) resuspending the crude protein in tris-HCl and membrane-passing the resuspended crude protein and dialyzing against tris-HCl;
(6) ultrafiltering with ultrafiltering tube, collecting small peptide with molecular weight less than 10KD, cooling the obtained small peptide, and lyophilizing to obtain GFP-2 small peptide.
2. The use of a small GFP-2 peptide according to claim 1 for the preparation of an immunoadjuvant, wherein: in the step (1), 0.8-1.2L of the glucose culture medium is prepared and sterilized at the temperature of 120 ℃ for 10-20 min; in the step (2), GFP-2 bacteria are inoculated, each tube is provided with 3-10mL of LB culture medium, and the ratio of glycerol bacteria to the culture medium is 1: inoculating 50-150 of strain, inoculating 2 tubes of GFP-2 strain liquid, and culturing by shaking for 8-15 h.
3. The use of a small GFP-2 peptide according to claim 1 for the preparation of an immunoadjuvant, wherein: in the step (3), transferring the culture medium into 0.5-1.5L of a brown sugar culture medium, and performing shake culture for 8-15 h; 2000-6000 rpm for 0.5-3h, collecting the supernatant, and discarding the cells.
4. The use of a small GFP-2 peptide according to claim 1 for the preparation of an immunoadjuvant, wherein: in the step (4) of the preparation method, the supernatant is saturatedAmmonium sulfateThe solution is prepared according to the following steps of 2-5: 1, salting out to separate out protein and small peptide; centrifuging again after salting out, centrifuging at 10000-.
5. The use of a small GFP-2 peptide according to claim 1 for the preparation of an immunoadjuvant, wherein: in the preparation method, in step (5), the crude protein is resuspended by tris-HCL, and the resuspended crude protein is filtered through a 0.45mm filter membrane and dialyzed against tris-HCl for 3-8h, and the tris-HCl is replaced every 0.5-2 h.
6. The use of a small GFP-2 peptide according to claim 1 for the preparation of an immunoadjuvant, wherein: in the step (6), ultrafiltration is carried out for 20-60min at 8000rpm and 4000-.
7. A GFP-2 small peptide produced according to any one of claims 1 to 6.
8. The small GFP-2 peptide of claim 7, wherein: the molecular weight of the small peptide is less than 10 KD.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103908668A (en) * | 2014-04-03 | 2014-07-09 | 浙江大学 | Bots antibacterial peptide adjuvant and preparation method thereof, and vaccine formulation containing adjuvant and use thereof |
| CN104910262A (en) * | 2014-11-27 | 2015-09-16 | 沈阳化工研究院有限公司 | Bacillus subtilis antibacterial peptide, separation and purification method, and application thereof |
| CN107446019A (en) * | 2016-07-01 | 2017-12-08 | 四川大学 | Antibacterial peptide derivatives and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103908668A (en) * | 2014-04-03 | 2014-07-09 | 浙江大学 | Bots antibacterial peptide adjuvant and preparation method thereof, and vaccine formulation containing adjuvant and use thereof |
| CN104910262A (en) * | 2014-11-27 | 2015-09-16 | 沈阳化工研究院有限公司 | Bacillus subtilis antibacterial peptide, separation and purification method, and application thereof |
| CN107446019A (en) * | 2016-07-01 | 2017-12-08 | 四川大学 | Antibacterial peptide derivatives and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Jia Wu et al..Isolation and characterization of Bacillus sp. GFP-2, a novel Bacillus strain with antimicrobial activities, from Whitespotted bamboo shark intestine.《AMB Expr.》.2018,第8卷(第84期), * |
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