Helicobacter pylori polypeptide antigen group and immune whey prepared from same

Abstract

The invention relates to a helicobacter pylori polypeptide antigen group. The helicobacter pylori polypeptide antigen group comprises a polypeptide antigen CS-36 and a polypeptide antigen CI-33, the amino acid sequence of the polypeptide antigen CS-36 is as shown in SEQ ID No. 1, and the amino acid sequence of the polypeptide antigen CI-33 is as shown in SEQ ID No. 2. Polyclonal antibody whey prepared by immunizing dairy cows by using the mixed polypeptide antigen can prevent adhesion and colonization of helicobacter pylori, so that the effect of removing the helicobacter pylori is achieved, drug resistance is not generated, meanwhile, nutrition and double effects of whey can be obtained, and the mixed polypeptide antigen can be drunk for a long time.

Description

Technical Field

[0001] The invention relates to a Helicobacter pylori polypeptide antigen group and immune whey prepared therefrom. Background Art

[0002] Helicobacter pylori is classified as a Class I carcinogen by the World Health Organization and is an important risk factor for diseases such as gastric cancer.

[0003] Adhesion to host cells is the primary condition for establishing bacterial infection. Helicobacter pylori mainly uses the adhesion of outer membrane protein (OMP) to colonize gastric epithelial cells, causing chronic gastric infection and further damage to the gastric mucosa. OMP is a relatively conservative protein expressed on the surface of bacteria, including multiple families such as the Hop family, the Hor family and the Hof family, which first interact with the gastric epithelium. Among these OMPs, SabA, BabA, AlpA / B, OipA, HopQ, etc. are essential for Hp adhesion and colonization. Therefore, it is worth studying whether it is possible to screen out the abundantly expressed OMPs and obtain antibodies obtained by immunizing cows with OMP as antigens to prevent the adhesion and colonization of Helicobacter pylori and then eliminate Helicobacter pylori. Summary of the invention

[0004] The purpose of the present invention is to provide a polypeptide antigen group and its application which can prevent Helicobacter pylori from adhering and colonizing and then eliminate Helicobacter pylori.

[0005] The present invention adopts the following technical solution: A Helicobacter pylori polypeptide antigen group, comprising polypeptide antigen CS-36 and polypeptide antigen CI-33.

[0006] Among them, the amino acid sequence of the polypeptide antigen CS-36 is shown in SEQ ID No.1, and the amino acid sequence of the polypeptide antigen CI-33 is shown in SEQ ID No.2.

[0007] A gene encoding the above polypeptide antigen CS-36.

[0008] Furthermore, the coding gene of the polypeptide antigen CS-36 is shown in SEQ ID No.3.

[0009] A gene encoding the above polypeptide antigen CI-33.

[0010] Furthermore, the coding gene of the polypeptide antigen CI-33 is shown in SEQ ID No.4.

[0011] A polyclonal antibody prepared using the above Helicobacter pylori polypeptide antigen group.

[0012] A medicament comprising the above polyclonal antibody.

[0013] A vaccine prepared using the above-mentioned Helicobacter pylori polypeptide antigen group.

[0014] Furthermore, the vaccine comprises a mixed coupled polypeptide, wherein the mixed coupled polypeptide is obtained by mixing KLH coupled polypeptide antigen CS-36 and KLH coupled polypeptide antigen CI-33 in equal mass ratios.

[0015] Furthermore, the vaccine also includes an aluminum hydroxide adjuvant, and the mixed coupled polypeptide and the aluminum hydroxide adjuvant are mixed in an equal volume ratio.

[0016] An application of the above-mentioned Helicobacter pylori polypeptide antigen group in eliminating Helicobacter pylori.

[0017] Immune cow milk is obtained by immunizing cows with the above-mentioned Helicobacter pylori polypeptide antigen group.

[0018] The invention discloses immune whey, which is prepared by utilizing the immune cow's milk.

[0019] An application of the above-mentioned Helicobacter pylori polypeptide antigen group in preventing the adhesion and colonization of Helicobacter pylori.

[0020] The beneficial effect of the present invention is that the polyclonal antibody prepared by using the mixed polypeptide antigen of the present invention can prevent the adhesion and colonization of Helicobacter pylori, thereby achieving the effect of eliminating Helicobacter pylori. At the same time, the polyclonal antibody whey can be prepared by immunizing dairy cows with the mixed polypeptide antigen, which not only has the effect of preventing the adhesion and colonization of Helicobacter pylori, but also does not produce drug resistance, and at the same time can obtain the nutrition of whey, with dual effects in one, and can be drunk for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the mass spectrometry identification diagram of peptide CS-36.

[0022] Figure 2 This is the mass spectrometry identification diagram of peptide CI-33. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] 1. Artificial synthesis of peptides (1) Screen out OMPs with relatively abundant expression.

[0025] (2) The extracellular region was analyzed online using DeepTMHMM, and the B cell epitopes were predicted online using the ABCPred server.

[0026] (3) Screening the polypeptide sequences to obtain polypeptide CS-36 (amino acid sequence as shown in SEQ ID No. 1, nucleotide sequence as shown in SEQ ID No. 3) and polypeptide CI-36 (amino acid sequence as shown in SEQ ID No. 2, nucleotide sequence as shown in SEQ ID No. 4).

[0027] SEQ ID No. 1: CSGEGNDNKKSPSVTGVNNQNGKKDSIASGNTSGVS.

[0028] SEQ ID No. 2: CRPAIGQNVEFNVDYNSKKTQGAIRPSDGGYQI.

[0029] SEQ ID No. 3: TGCAGCGGCGAAGGCAACGATAACAAAAAAAGCCCGAGCGTGACCGGGCTGAACAACCAGAACGGCAAAAAAGATAGCATTGCGAGCGGCAACACCAGCGGCGTGAGC.

[0030] SEQ ID No. 4: TGCCGCCCGGCGATTGGCCAGAACGTGGAATTTAACGTGGATTATAACAGCAAAAAAACCCAGGGCCGATTCGCCCGAGCGATGGCGGCTATCAGATT.

[0031] (4) Artificially synthesize 20 mg of peptides. Mass spectrometry identification of the synthesized peptides is shown in Figure 1 and Figure 2 .

[0032] 2. Peptide Conjugation (1) Slowly open a bottle of maleimide-activated KLH to release the vacuum.

[0033] (2) Prepare maleimide-activated KLH at 5 mg / ml using ultrapure water.

[0034] (3) Reconstitute the coupling buffer with 10 ml of ultrapure water to obtain 20 mM sodium phosphate buffer, and add 100 mM EDTA and 80 mM sucrose, pH 6.6.

[0035] (4) Dissolve 4 mg (about 7000 Da) of cysteine-containing peptide in 0.5 ml of conjugate buffer.

[0036] (5) Immediately place the peptide solution and the maleimide-activated KLH mixed solution in a reaction bottle equipped with a stirring bar. Stir for 1-2 minutes under a gentle nitrogen flow while degassing the sample. Cover the reaction bottle and continue stirring at room temperature for 2 hours or at 2-8°C overnight, retaining 100 ml of the binding reaction solution. The coupled peptides of peptide CS-36 and KLH and the coupled peptides of peptide CI-33 and KLH were obtained respectively.

[0037] (6) Before immunization, the two KLH-coupled polypeptides were mixed in a 1:1 ratio (mass ratio m / m) to obtain a KLH-coupled mixed polypeptide, and then the coupled polypeptide was dialyzed in a dialysis bag and frozen at -20°C for future use.

[0038] 3. Immunization of dairy cows Healthy pregnant cows aged 2 to 3 weeks were selected for immunization. The cows were immunized 4 times, with an interval of 14 days each time. Two months before delivery, 3 mg of KLH-coupled mixed polypeptides were mixed with aluminum hydroxide adjuvant in a ratio of 1:1 (volume ratio v / v) to form 2 ml. The pregnant cows were immunized for the first time and injected at two points on the muscles on both sides of the cow's neck, with 1 ml at each point. For the second to fourth times, 2 mg of KLH-coupled mixed polypeptides were mixed with aluminum hydroxide adjuvant in a ratio of 1:1 (volume ratio v / v) to form 2 ml. They were injected at two points on the muscles on both sides of the cow's neck, with 1 ml at each point. Milk samples or blood samples were collected every week starting from one week after the second immunization, and the antibody titer was detected by ELISA.

[0039] IV. Antibody titer detection (1) The negative reference was pre-collected blood serum, which was diluted with 1 antibody starting concentration (blocking diluent).

[0040] (2) Add 100 µl of 10 µg / ml antigen to each well of a 96-well plate and incubate at 4°C overnight or at 37°C for 2 hours.

[0041] (3) Pour away the antigen solution, add 200 µl of blocking solution to each well, and incubate at 4°C overnight or at 37°C for 2 hours.

[0042] (4) Pour off the blocking solution and tap on absorbent paper to absorb as much residual liquid as possible. Wash the plate three times with detergent, patting as much residual liquid as possible each time. If the plate needs to be stored for a period of time, dry it at 37℃ and seal it in a sealing bag and store it at -20℃.

[0043] (5) Take the coated 96-well plate, add the blank control in the first well, add 100 μl of negative reference solution in the second well, and add 1:200 antiserum to be tested / milk in the third well. Dilute the subsequent wells in multiple ratios based on this, and incubate at 37°C for 1 hour.

[0044] (6) Pour off the liquid, wash the plate three times with washing solution, and pat dry. Add 100 μl of HRP-labeled goat anti-bovine IgG (1:5000 dilution) to each well and incubate at 37°C for 45 minutes.

[0045] (7) Pour off the liquid, wash the plate three times with washing solution, and pat dry. Add 100 μl of TMB substrate (Sigma single-component TMB) to each well and incubate at 37°C for 5 to 20 minutes (the color development time is determined by the color depth).

[0046] (8) Add 50ul of stop solution (2N H2SO4) to each well and read the absorbance at 450nm on a microplate reader. Collect the milk when the titer of the milk antibody reaches 1:1200 (both peptides in the mixed peptides reach above 1:1200).

[0047] 5. Preparation of Immune Whey Powder (1) Filtration and cooling After filtering and purification, the immune milk is cooled and placed in a milk storage tank for later use. The temperature of the milk storage tank should be maintained at 4-5°C, and the storage time of the immune milk in the milk storage tank should not exceed 24 hours.

[0048] (2) Standardization Before processing, the antibody titers of both polypeptide antigens in milk should be ≥1:1200.

[0049] (3) Preheating The temperature of the raw milk in the milk storage tank is raised to 45°C using a hot water circulating plate heat exchanger.

[0050] (4) Degreasing When using a disc centrifuge for centrifugal skimming, the fat content of the skimmed milk is required to be controlled below 0.1%.

[0051] (5) Low temperature pasteurization The skimmed immune milk was heated to 65°C using a plate heat exchanger and then placed in a heat preservation tank and kept at 62°C for 40 minutes for low-temperature pasteurization.

[0052] (6) Preparation of whey The pH of the milk was adjusted to 4.5-4.6 with 1 mol / L HCl, and then placed in a 40°C water bath for 1 hour to completely precipitate the casein. The milk was centrifuged again at 3000 r / min for 10 min, the supernatant was poured out, and the pH was restored to 6.8-7.0 with 1 mol / L NaOH to obtain bovine whey.

[0053] (7) Filtration and sterilization First filter with a 5 μm filter membrane, then filter with a 1 μm filter membrane, and finally filter with a 0.22 μm filter membrane.

[0054] (8) Ultrafiltration and concentration of whey Use external pressure hollow fiber ultra-rate concentration equipment to carry out circulating ultrafiltration concentration. During concentration, the material temperature is maintained between 50 and 55°C. When the material concentration reaches 20 to 23%, stop concentrating.

[0055] (9) Low temperature spray drying Low-temperature spray drying: inlet air temperature 120°C, fluidized bed temperature 65°C; discharge temperature 62°C, cyclone separator outlet temperature 70°C, tower negative pressure 0.5mmH2O, cyclone separator pressure difference 50mmH2O, fluidized bed pressure difference 45mmH2O.

[0056] (10) Get immune whey powder The low-temperature spray-dried immune bovine whey powder is packaged as needed.

[0057] VI. Animal Testing (1) Purchase 20 4-week-old, male SPF-grade BALB / c mice and divide them into 2 groups, 10 mice in each group. Place the mice in a Trexler-type flexible film plastic isolator for 12 hours a day and 12 hours a night, with free access to sterile food and water.

[0058] Freshly prepared Helicobacter pylori (1 × 10 9 CFU) was gavaged into mice once a day for 3 consecutive days. After 3 weeks of confirmation of H. pylori infection in mice, 20 mg of whey powder dissolved in 0.5 ml of PBS was gavaged into mice once a day for 4 consecutive weeks. The control group mice were only given PBS.

[0059] (2) Preparation of Columbia agar plates Weigh 3.9 g of Columbia agar medium and dissolve it in 100 ml of ultrapure water. After high-pressure cooling to 50°C, add 10 ml of defatted sheep blood and pour into a 9 cm culture dish. The thickness of the medium is about 4 mm.

[0060] (3) Helicobacter pylori plate count In order to count the number of Helicobacter pylori adherent to the gastric epithelium, the stomach of the mouse was removed and first cut into two halves along the greater curvature of the stomach. After weighing, one half was placed in a 15 ml plastic centrifuge tube containing 10 ml PBS and then homogenized with a homogenizer for 10 seconds. The stomach was removed and 100 μL of the homogenate was aspirated and evenly spread on a Columbia agar culture dish.

[0061] (4) Observation of results After the plates were placed in a microaerobic incubator and incubated for 72 hours, the plates were taken out and counted using a bacteria counter. After taking the logarithm, the total number of bacteria in the PBS control group was 5.1±0.2 Log 10 CFU / g, whey antibody experimental group 3.9±0.06 Log10 CFU / g, the difference between the two groups was significant ( p <0.05).

[0062] The above results show that the prepared polyclonal antibody whey can prevent the adhesion and colonization of Helicobacter pylori. Therefore, oral administration of this whey antibody can eliminate Helicobacter pylori in the body and obtain the nutrition of whey at the same time.

Claims

1. A Helicobacter pylori polypeptide antigen group, characterized in that: It comprises polypeptide antigen CS-36 and polypeptide antigen CI-33; the amino acid sequence of the polypeptide antigen CS-36 is shown in SEQ ID No.1, and the amino acid sequence of the polypeptide antigen CI-33 is shown in SEQ ID No.

2.

2. A gene encoding the polypeptide antigen CS-36 or polypeptide antigen CI-33 as claimed in claim 1.

3. A polyclonal antibody prepared using the Helicobacter pylori polypeptide antigen group as claimed in claim 1.

4. A vaccine prepared using the Helicobacter pylori polypeptide antigen group according to claim 1.

5. The vaccine according to claim 4, characterized in that The mixed coupling polypeptide comprises a mixed coupling polypeptide, wherein the mixed coupling polypeptide is obtained by mixing a KLH coupling polypeptide antigen CS-36 and a KLH coupling polypeptide antigen CI-33 in equal mass ratios.

6. Use of the Helicobacter pylori polypeptide antigen group as claimed in claim 1 in eliminating Helicobacter pylori.

7. An immune milk, characterized in that: The antigen is obtained by immunizing dairy cows with the Helicobacter pylori polypeptide antigen group as claimed in claim 1.

8. An immune whey, characterized in that Prepared using the immune milk as described in claim 7.

9. Use of the Helicobacter pylori polypeptide antigen group as claimed in claim 1 in preventing the adhesion and colonization of Helicobacter pylori.

10. A medicament comprising the polyclonal antibody according to claim 3.

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