CN112079915A

CN112079915A - Polypeptide and preparation method and application thereof

Info

Publication number: CN112079915A
Application number: CN202010957915.7A
Authority: CN
Inventors: 张智; 李昕阳
Original assignee: Xintai Sichuan Technology Co ltd
Current assignee: Xintai Sichuan Technology Co ltd
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2020-12-15

Abstract

The invention provides an amino acid sequence of a polypeptide and a preparation method thereof, which are used for solving the technical problems that the labor cost is increased due to the dependence of specific immunity on the conventional helicobacter pylori resistant egg yolk antibody, and the activity and the purification rate of an egg yolk antibody product are difficult to take into account. The polypeptide has anti-helicobacter pylori activity, and is obtained by dialyzing yolk produced by egg laying poultry without specific immunity to obtain yolk inner protein component; then, destroying the protein configuration by trypsin to obtain polypeptide; and finally, accurately separating and extracting the target polypeptide by using high performance liquid chromatography. Because the inherent antibody in the yolk is utilized, the poultry does not need to be subjected to specific immune treatment, thereby saving the related labor input. Compared with the yolk antibody, the polypeptide lacks space configuration, and the activity and the purification rate of the polypeptide can be ensured by the preparation process of the polypeptide.

Description

Polypeptide and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to an active polypeptide with helicobacter pylori resistance and a preparation method of the active polypeptide.

Background

Poultry eggs can complete normal hatchings in the absence of an intact immune mechanism, benefiting from the antimicrobial properties of egg white and egg yolk. In 1889, Klemperer found that egg yolk was rich in antibodies. With the continuous and deep research of the yolk antibody, the yolk antibody IgY is found to have the similar function to mammal IgG, but has a special structure and can not excite a complement system, thereby avoiding the generation of strong inflammatory reaction and having wide application prospect.

Currently, the application of yolk antibody to human disease treatment is more studied, and among them, the application against helicobacter pylori is a more recent hot spot. For example, Chinese patent CN109400705A discloses a duck egg yolk antibody for resisting helicobacter pylori and a preparation method thereof; chinese patent CN108440667A discloses a yolk antibody against helicobacter pylori lipopolysaccharide and a preparation method and application thereof; chinese patent CN106632672A discloses a preparation method and application of a yolk antibody containing anti-helicobacter pylori IgY; chinese patent CN109503710A discloses an anti-helicobacter pylori yolk antibody, a preparation method and application thereof and the like. In the technical scheme disclosed in the patent, the yolk is derived from the poultry body subjected to directional immunity. The egg-laying poultry is immunized with specific antigen for many times, so that the body of the poultry produces corresponding specific antibodies, the antibodies are transferred and stored in egg yolk, and then the egg yolk is extracted and purified to obtain corresponding egg yolk antibodies.

Considerable amount of yolk antibody can be obtained in a short time through the operation, but the antibody titer level is determined by the specific immune response condition of the poultry. Because the immune response condition is difficult to measure and control, the poultry needs to be subjected to multiple specific immune operations, thereby increasing the labor cost. And the biological activity of the egg yolk antibody product as a protein has strict requirements on purification conditions, so that the purification rate is influenced, and further, the use risk is generated.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a polypeptide and a preparation method thereof, and solves the technical problems that the labor cost is increased and the activity and the purification rate of a yolk antibody product are difficult to be considered simultaneously due to the dependence of the conventional helicobacter pylori resistant yolk antibody on specific immunity.

In order to achieve the purpose, the invention provides the following technical scheme:

the first technical scheme is as follows:

a polypeptide has an amino acid sequence shown as SEQ ID No. 1.

Furthermore, the polypeptide amino acid sequence also comprises a mutant sequence of any amino acid from 5 th position to 23 th position in SEQ ID No. 1.

Furthermore, the polypeptide amino acid sequence also comprises a combined mutation sequence of 5 th to 23 th amino acids in SEQ ID No. 1.

As shown in SEQ ID No. 1, the polypeptide has 28 amino acids. The polypeptides lack spatial configuration compared to proteins, and thus exhibit antagonistic properties against helicobacter pylori, which are mainly conferred by their amino acid sequences. The extraction process or the synthesis process of the polypeptide is an accurate screening process, so that the obtaining of the polypeptide has controllability and the purification rate of the polypeptide can be ensured.

The second technical scheme is as follows:

a method of producing a polypeptide comprising the steps of:

s1, preparing yolk dialysate;

s2, treating the yolk dialysate obtained in the step S1 by trypsin;

s3, separating and purifying the treatment liquid obtained in the step S2 by adopting high performance liquid chromatography;

wherein the yolk dialysate of step S1 is provided by egg-laying poultry not specifically immunized.

The technical proposal adopts the yolk produced by the egg-laying poultry which is not subjected to specific immunity to obtain the protein component in the yolk through dialysis; then, destroying the protein configuration by trypsin to obtain polypeptide; and finally, accurately separating and extracting the target polypeptide by using high performance liquid chromatography.

The technical scheme utilizes the inherent antibody in the yolk, and does not need to carry out specific immune treatment on the poultry, thereby saving the related labor input.

Further, step S1 includes the steps of:

s11, mixing the yolk solution and a NaOH solution with the concentration of 0.1-0.2M in a volume ratio of 4-9: 10;

s12, adjusting the pH value of the mixed solution obtained in the step S11 to 6-9, and carrying out water bath at 40 ℃ for 1.5-2.4 h;

s13, centrifuging the mixed solution obtained in the step S12 at the rotation speed of 10000 rpm-12000 rpm, taking the supernatant, and dialyzing for 6-12 h at the temperature of 4 ℃.

Through the above operation, the protein component in the yolk is sufficiently precipitated to ensure the extraction amount of the target polypeptide.

In the step S2, the trypsin is immobilized to improve the enzymolysis effect and facilitate preservation, and the treatment process includes the following steps:

s21, mixing chitosan and 0.8-1.2% acetic acid solution uniformly according to the mass volume ratio of 1-1.5: 100;

s22, adding a NaOH solution with the concentration of 4M-5M into the mixed solution obtained in the step S21 according to the volume ratio of 20-28: 500, uniformly mixing, and filtering to obtain a precipitate;

s23, washing the precipitate in the step S22 to be neutral, putting the precipitate into 1.6-2.2% glutaraldehyde solution for carrier activation, and taking out the precipitate to be washed to be neutral;

and S24, based on the chitosan obtained in the step S21, preparing the prepared activated carrier and trypsin liquid with the concentration of 5 mg/mL-12 mg/mL according to the mass-volume ratio of 1-2: 80, mixing, performing low-temperature crosslinking adsorption for 4-12 h, washing, and performing suction filtration.

The immobilized trypsin obtained by the operation can be stored in a refrigerator at the temperature of-20 ℃ for later use.

Further, the trypsin in the step S2 and the yolk dialysate in the step S1 are disposed in a mass-to-volume ratio of 2: 15-30. The trypsin is fully utilized to carry out enzymolysis on the protein in the yolk dialysate, so that the formation of the target polypeptide is ensured.

Further, the treatment temperature in the step S2 is 40 ℃, and the treatment time is 200-260 min. The treatment temperature and time determine the amount of the target polypeptide extracted.

Further, a single peak appearing when the retention time of the high performance liquid chromatography in step S3 was 2.76min was collected. As can be seen from FIG. 1, 8 peak-to-peak substances were collected by liquid chromatography. The substances are prepared into a test solution with the working concentration of 0.1-1.0 mg/mL, and the helicobacter pylori cultured according to a standard method is subjected to antibacterial experimental verification.

The third technical scheme is as follows:

a method for preparing polypeptide comprises chemically synthesizing amino acid sequence shown in SEQ ID No. 1.

The chemical synthesis method is favorable for saving the production cost and can control the purity to be more than 95 percent.

The technical scheme is as follows:

use of a polypeptide in treating or preventing helicobacter pylori infectious diseases.

The polypeptide is prepared into an oral preparation by an effective treatment amount and acceptable pharmaceutic adjuvants.

The above polypeptide can also be added into milk powder, trehalose, starch, etc. as adjuvants to prepare prophylactic health preparation.

Drawings

FIG. 1 is a high performance liquid chromatogram obtained in example 1 of the present application.

Detailed Description

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the claimed embodiments. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

Example 1

The embodiment of the application of the invention provides a preparation method of polypeptide, which comprises the following steps:

1. mixing yolk solution and NaOH solution with concentration of 0.2M at volume ratio of 7:10, adjusting pH of the mixture to 8 with phosphoric acid, bathing in water at 40 deg.C for 2 hr, centrifuging at 12000rpm for 10min, and collecting supernatant, and dialyzing at 4 deg.C for 8 hr.

2. Mixing chitosan and 1% acetic acid solution according to the mass volume ratio of 1:100 uniformly, slowly adding NaOH solution with the concentration of 5M into the mixed solution of chitosan and acetic acid according to the volume ratio of 24:500, uniformly mixing, and filtering by using gauze to collect the generated white flocculent precipitate.

3. Washing the white flocculent precipitate with PBS until neutral, filtering again, adding 2% glutaraldehyde solution with the same volume as 1% acetic acid solution, activating the carrier in a shaker at room temperature at 120rpm for 5h, and repeatedly washing the activated carrier with PBS.

4. Based on the amount of chitosan, preparing an activation carrier and a trypsin solution with the concentration of 8 mg/mL (the enzyme activity of each gram of enzyme is more than 10 ten thousand units) according to the mass-volume ratio of 1.2:80, placing the activation carrier and the trypsin solution in a shaking table at 4 ℃ and 60rpm for cross-linking adsorption for 8 hours, washing with ultrapure water, performing suction filtration to obtain immobilized trypsin, and placing the immobilized trypsin in a refrigerator at-20 ℃ for later use.

5. Preparing the immobilized trypsin prepared in the step 4 and the yolk dialysate prepared in the step 1 according to the mass-to-volume ratio of 2:15, performing enzymolysis for 220-260 min at 40 ℃, centrifuging, collecting precipitates, and freeze-drying an enzymolysis polypeptide mixed product;

wherein, the immobilized trypsin after refrigeration is used after being resuspended by phosphate buffer with the mass volume ratio of 1:5 and the pH value of 8.0.

6. Adopting analytical and semi-preparative High Performance Liquid Chromatography (HPLC), WELCHAQ-C18 chromatographic column, and Dikma EasyGuard protective column; mobile phase: acetonitrile-0.2% (volume fraction) glacial acetic acid (volume ratio 60: 40); flow rate: 1.0 mL/min; column temperature: 30 ℃; evaporation photodetector carrier gas flow rate: 2.2 mL/min; temperature of the drift tube: at 90 ℃. The obtained liquid chromatogram is shown in FIG. 1.

7. Respectively collecting the peak-producing products with retention time of 1.61min, 1.84min, 2.39 min to 2.48min, 2.76min, 4.2min, 6.0min and 8.74min in the graph 1, respectively preparing the peak-producing products into preparations with working concentration of 0.1 mg/mL to 1.0 mg/mL, and carrying out antibacterial experimental verification on helicobacter pylori cultured according to a standard method.

8. According to the result of the antibacterial experiment verification, a peak product with retention time of 2.76min is selected, and an amino acid sequence shown as SEQ ID No. 1 is obtained through sequencing.

9. And (4) freeze-drying the peak product with retention time of 2.76min, and storing at 0-4 ℃ or-20 ℃.

Example 2

1. Through chemical synthesis, the amino acid sequence shown in SEQ ID No. 1 is directly obtained, and the purity requirement is more than 95%.

2. The preparations are respectively prepared into preparations with working concentration of 0.1 mg/mL-1.0 mg/mL, and the helicobacter pylori cultured according to a standard method is subjected to antibacterial experimental verification.

Example 3

1. Mixing yolk solution and 0.1M NaOH solution at a volume ratio of 4:10, adjusting pH of the mixed solution to 6 with phosphoric acid, bathing with water at 40 deg.C for 1.5 hr, centrifuging at 10000rpm for 12min, collecting supernatant, and dialyzing at 4 deg.C for 6 hr.

2. Mixing chitosan and 1% acetic acid solution uniformly according to the mass volume ratio of 1.5:100, slowly adding NaOH solution with the concentration of 4M into the mixed solution of chitosan and acetic acid according to the volume ratio of 20:500, uniformly mixing, and filtering by using gauze to collect the generated white flocculent precipitate.

Sequence listing

<110> Sichuan Xintai science and technology Limited

<120> polypeptide and preparation method and application thereof

<130> 2019.8.13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> PRT

<213> Unknown (Unknown)

<400> 1

Lys Val Cys Thr Ser Ser Asn Val Met Glu Glu Arg Ala Glu Glu Arg

1 5 10 15

Tyr Pro Ile Leu Pro Glu Tyr Leu Tyr Gly Ile Leu

20 25

Claims

1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide further comprises a mutated sequence of any one of amino acids 5 to 23 of SEQ ID No. 1.

3. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide further comprises a combined mutation of amino acids 5 to 23 of SEQ ID No. 1.

4. A method for producing the polypeptide of claim 1, comprising the steps of:

s1, preparing yolk dialysate;

s2, treating the yolk dialysate obtained in the step S1 by trypsin;

5. The production method according to claim 4,

step S1 includes the following steps:

s13, centrifuging the mixed solution obtained in the step S12 at the rotation speed of 10000 rpm-12000 rpm, taking the supernatant, and dialyzing for 6-12 h at the temperature of 4 ℃;

in step S2, the trypsin is immobilized, and the treatment process includes the following steps:

6. The preparation method according to claim 5, wherein the trypsin in step S2 and the yolk dialysate in step S1 are disposed in a mass-to-volume ratio of 2:15 to 30.

7. The method according to claim 4, wherein the treatment temperature in step S2 is 40 ℃ and the treatment time is 200 to 260 min.

8. The production method according to claim 4, wherein a single peak appearing when the retention time of the high performance liquid chromatography in step S3 is 2.76min is collected.

9. The method for producing the polypeptide of claim 1, wherein the amino acid sequence of SEQ ID No. 1 is chemically synthesized.

10. Use of the polypeptide of any one of claims 1-3 for treating or preventing helicobacter pylori infectious diseases.