A Helicobacter pylori polypeptide antigenome and the immune whey prepared therefrom

Abstract

The present invention relates to a Helicobacter pylori polypeptide antigenome, which comprises a polypeptide antigen CS-36 and a polypeptide antigen CI-33; the amino acid sequence of the polypeptide antigen CS-36 is shown as SEQ ID No.1, and the amino acid sequence of the polypeptide antigen CI-33 is shown as SEQ ID No.2. The polyclonal antibody whey prepared by immunizing dairy cows with the mixed polypeptide antigen can prevent Helicobacter pylori from adhering and colonizing, thereby achieving the effect of clearing Helicobacter pylori, and will not produce drug resistance. At the same time, the nutrition of the whey can be obtained, achieving a dual effect, and it can be drunk for a long time.

Description

Technical Field

[0001] The present invention relates to a Helicobacter pylori polypeptide antigenome and an immune whey prepared therefrom. Background Art

[0002] Helicobacter pylori is listed 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 a prerequisite for establishing bacterial infection. Helicobacter pylori mainly colonizes gastric epithelial cells through the adhesion of outer membrane proteins (OMPs), causing chronic gastric infection and then damaging the gastric mucosa. OMPs are relatively conserved proteins expressed on the surface of the bacterial cell, including multiple families such as the Hop family, Hor family, and Hof family, and are the first to interact with gastric epithelium. Among these OMPs, SabA, BabA, AlpA / B, OipA, HopQ, etc. are crucial for the adhesion and colonization of Hp. Therefore, it is worthy of study whether antibodies obtained by immunizing dairy cows with OMPs as antigens, by screening out OMPs with rich expression, can prevent the adhesion and colonization of Helicobacter pylori and then eliminate Helicobacter pylori. Summary of the Invention

[0004] The object of the present invention is to provide a polypeptide antigenome that can prevent the adhesion and colonization of Helicobacter pylori and then eliminate Helicobacter pylori, and its application.

[0005] The present invention adopts the following technical solutions:

[0006] A Helicobacter pylori polypeptide antigenome, which comprises a polypeptide antigen CS-36 and a polypeptide antigen CI-33.

[0007] Wherein, 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.

[0008] A coding gene of the above-mentioned polypeptide antigen CS-36.

[0009] Furthermore, the coding gene of the polypeptide antigen CS-36 is as shown in SEQ ID No.3.

[0010] A coding gene of the above-mentioned polypeptide antigen CI-33.

[0011] Furthermore, the coding gene of the polypeptide antigen CI-33 is as shown in SEQ ID No.4.

[0012] A polyclonal antibody prepared by using the above-mentioned Helicobacter pylori polypeptide antigenome.

[0013] A drug containing the above-mentioned polyclonal antibody.

[0014] A vaccine prepared using the above-mentioned Helicobacter pylori polypeptide antigenome.

[0015] Furthermore, the vaccine includes a mixed conjugate polypeptide, which is obtained by mixing KLH-conjugated polypeptide antigen CS-36 and KLH-conjugated polypeptide antigen CI-33 in an equal mass ratio.

[0016] Furthermore, the vaccine also includes an aluminum hydroxide adjuvant, and the mixed conjugate polypeptide and the aluminum hydroxide adjuvant are mixed in an equal volume ratio.

[0017] An application of the above-mentioned Helicobacter pylori polypeptide antigenome in clearing Helicobacter pylori.

[0018] An immune milk, which is obtained by immunizing dairy cows with the above-mentioned Helicobacter pylori polypeptide antigenome.

[0019] An immune whey, which is prepared using the above-mentioned immune milk.

[0020] An application of the above-mentioned Helicobacter pylori polypeptide antigenome in preventing the adhesion and colonization of Helicobacter pylori.

[0021] The beneficial effects of the present invention are as follows: The polyclonal antibody prepared using the mixed polypeptide antigen of the present invention can prevent the adhesion and colonization of Helicobacter pylori, and thus achieve the effect of clearing Helicobacter pylori. At the same time, immunizing dairy cows with this mixed polypeptide antigen can prepare polyclonal antibody whey, which not only has the function of preventing the adhesion and colonization of Helicobacter pylori, but also does not produce drug resistance. At the same time, the nutrition of whey can be obtained, achieving a dual effect, and it can be consumed for a long time. Description of the Drawings

[0022] Figure 1 It is the mass spectrometry identification diagram of polypeptide CS-36.

[0023] Figure 2 It is the mass spectrometry identification diagram of polypeptide CI-33. Detailed Embodiments

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] I. Artificial Synthesis of Polypeptides

[0026] (1) Screen out the relatively abundant OMPs.

[0027] (2)Online analyze its extracellular region using DeepTMHMM, and simultaneously predict the B-cell epitopes online using the server of ABCPred.

[0028] (3)Screen 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).

[0029] SEQ ID No.1: CSGEGNDNKKSPSVTGVNNQNGKKDSIASGNTSGVS.

[0030] SEQ ID No.2: CRPAIGQNVEFNVDYNSKKTQGAIRPSDGGYQI.

[0031] SEQ ID No.3: TGCAGCGGCGAAGGCAACGATAACAAAAAAAGCCCGAGCGTGACCGGCGTGAACAACCAGAACGGCAAAAAAGATAGCATTGCGAGCGGCAACACCAGCGGCGTGAGC.

[0032] SEQ ID No.4: TGCCGCCCGGCGATTGGCCAGAACGTGGAATTTAACGTGGATTATAACAGCAAAAAAACCCAGGGCGCGATTCGCCCGAGCGATGGCGGCTATCAGATT.

[0033] (4)Manually synthesize 20 mg of the polypeptide. For the mass spectrometry identification of the synthesized polypeptide, see Figure 1 and Figure 2 .

[0034] II. Conjugation of the Polypeptide

[0035] (1)Slowly open a bottle of maleimide-activated KLH to release the vacuum.

[0036] (2)Prepare maleimide-activated KLH into 5 mg / ml with ultrapure water.

[0037] (3)Re-prepare the conjugation buffer with 10 ml of ultrapure water to obtain 20 mM sodium phosphate buffer, and simultaneously add 100 mM EDTA and 80 mM sucrose, pH 6.6.

[0038] (4)Dissolve 4 mg (about 7000 Da) of the cysteine-containing peptide in 0.5 ml of the conjugate buffer.

[0039] (5) Immediately place the peptide solution and the maleimide-activated KLH mixed solution in a reaction flask equipped with a stir bar. Stir for 1 - 2 minutes under a gentle nitrogen stream while degassing the sample. Cover the reaction flask and continue stirring at room temperature for 2 hours or overnight at 2 - 8°C, reserving 100 ml of the conjugation reaction solution. Obtain the conjugated polypeptide of polypeptide CS-36 and KLH and the conjugated polypeptide of polypeptide CI-33 and KLH respectively.

[0040] (6) Before immunization, mix the two KLH-conjugated polypeptides in an equal ratio of 1:1 (mass ratio m / m) to obtain the KLH-conjugated mixed polypeptide, and then dialyze the conjugated polypeptide using a dialysis bag and store it frozen at -20°C for later use.

[0041] III. Immunizing Dairy Cows

[0042] Select healthy pregnant dairy cows at 2 - 3 weeks of age for immunization. Immunize the dairy cows in 4 separate times, with a 14-day interval between each immunization. Two months before parturition, mix 3 mg of the KLH-conjugated mixed polypeptide and aluminum hydroxide adjuvant in a ratio of 1:1 (volume ratio v / v) to make 2 ml, and conduct the first immunization on the pregnant dairy cows by injecting 1 ml at each of two points in the bilateral neck muscles; for the 2nd - 4th immunizations, mix 2 mg of the KLH-conjugated mixed polypeptide and aluminum hydroxide adjuvant in a ratio of 1:1 (volume ratio v / v) to make 2 ml, and inject 1 ml at each of two points in the bilateral neck muscles. Start collecting milk samples or blood samples every 1 week 1 week after the 2nd immunization, and detect the antibody titer using the ELISA method.

[0043] IV. Detection of Antibody Titer

[0044] (1) The negative control is pre-collected blood serum, and all are diluted starting from the initial concentration of the primary antibody (blocking diluent).

[0045] (2) Pipette 100 μl of 10 μg / ml antigen into each well of a 96-well plate, and incubate overnight at 4°C or for 2 hours at 37°C.

[0046] (3) Pour out the antigen solution, add 200 μl of blocking solution to each well, and incubate overnight at 4°C or for 2 hours at 37°C.

[0047] (4) Pour out the blocking solution, tap the plate on absorbent paper to drain as much residual liquid as possible, wash the plate three times with the washing solution, and pat dry the residual liquid as much as possible each time. If it needs to be stored for a period of time, dry it at 37°C, seal it with a sealable bag, and store it at -20°C.

[0048] (5) Take the coated 96-well plate. The first well is the blank control, the second well is added with 100 μl of the negative control solution, the third well is added with the test antiserum / milk diluted 1:200, and the subsequent wells are serially diluted based on this. Incubate at 37°C for 1 hour.

[0049] (6) Pour out the liquid, wash the plate three times with the washing solution, and pat dry. Add 100 μl of HRP-labeled goat anti-bovine IgG (diluted 1:5000) to each well and incubate at 37 °C for 45 minutes.

[0050] (7) Pour out the liquid, wash the plate three times with the 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 - 20 minutes (determine the color development time according to the color depth).

[0051] (8) Add 50 μl of stop solution (2N H2SO4) to each well and read the absorbance at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Collect the milk when the milk antibody titer detection reaches 1:1200 (both of the two polypeptides in the mixed polypeptide reach above 1:1200).

[0052] V. Preparation of Immune Whey Powder

[0053] (1) Filtration and Cooling

[0054] Filter and purify the immune milk, perform cold discharge treatment, and load it into a milk storage tank for standby. 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.

[0055] (2) Standardization

[0056] Before processing, the antibody titers of the two polypeptide antigens in the milk should both be ≥ 1:1200.

[0057] (3) Preheating and Temperature Rising

[0058] Use a hot water circulation plate heat exchanger to raise the temperature of the raw milk in the milk storage tank to 45 °C.

[0059] (4) Defatting

[0060] Use a disc centrifuge for defatting, and require the fat content of the defatted milk to be controlled below 0.1%.

[0061] (5) Low-temperature Pasteurization

[0062] Use a plate heat exchanger to raise the temperature of the defatted immune milk to 65 °C, then input it into a holding tank, and keep it at 62 °C for 40 min for low-temperature pasteurization.

[0063] (6) Preparation of Whey

[0064] Adjust the pH of the milk to 4.5 - 4.6 with 1 mol / L HCl, then place it in a 40 °C water bath for 1 h to completely precipitate the casein. Centrifuge again at 3000 r / min for 10 min, pour out the supernatant, and restore the pH to 6.8 - 7.0 with 1 mol / L NaOH to obtain milk whey.

[0065] (7)Filter sterilization

[0066] First, filter with a 5μm filter membrane, then with a 1μm filter membrane, and finally with a 0.22μm filter membrane.

[0067] (8)Ultrafiltration concentration of whey liquid

[0068] Use an external pressure type hollow fiber ultrafiltration concentration equipment for cyclic ultrafiltration concentration. During concentration, the material temperature is maintained between 50~55°C. When the material concentration reaches 20~23%, stop concentration.

[0069] (9)Low-temperature spray drying

[0070] Low-temperature spray drying: Inlet air temperature 120°C, fluidized bed temperature 65°C; Outlet temperature 62°C, cyclone separator outlet temperature 70°C, negative pressure in the tower 0.5 mmHg2O, cyclone separator pressure difference 50 mmHg2O, fluidized bed pressure difference 45 mmHg2O.

[0071] (10)Obtain immune whey powder

[0072] Pack the immune whey powder after low-temperature spray drying as needed.

[0073] VI. Animal experiments

[0074] (1) Purchase 20 4-week-old male SPF-grade BALB / c mice and divide them into 2 groups with 10 mice in each group. Place the mice in a trexler-type flexible film plastic isolator for 12 hours each day and night, and they can freely eat sterile food and drink water.

[0075] Gavage the mice with freshly prepared Helicobacter pylori (1×10 9 CFU) once a day for 3 consecutive days. Three weeks after confirming that the mice are infected with Helicobacter pylori, dissolve 20mg of whey powder in 0.5ml of PBS and gavage once a day for 4 consecutive weeks. The control group mice are only given PBS.

[0076] (2)Preparation of Columbia agar plate

[0077] Weigh 3.9g of Columbia agar medium and dissolve it in 100ml of ultrapure water. After high-pressure cooling to 50°C, add 10ml of defatted sheep blood, and then pour it into a 9cm petri dish. The thickness of the medium is about 4mm.

[0078] (3)Helicobacter pylori plate counting

[0079] To count the number of Helicobacter pylori adhering to the gastric epithelium, the stomach of the mouse was removed. First, the stomach was cut into two halves along the greater curvature of the stomach. After weighing, one half was placed into a 15 ml plastic centrifuge tube containing 10 ml of PBS, and then homogenized with a homogenizer for 10 seconds. The stomach was removed, and then 100 μL of the homogenate was aspirated and evenly spread on a Columbia agar medium culture dish.

[0080] (4)Result observation

[0081] After culturing the plate in a microaerophilic incubator for 72 hours, the plate was taken out and counted with a bacterial counter. After taking the logarithm, the total number of bacteria in the PBS control group was 5.1 ± 0.2 Log 10 CFU / g, and that in the whey antibody experimental group was 3.9 ± 0.06 Log 10 CFU / g. The difference between the two groups was significant ( p <0.05).

[0082] The above results showed that the prepared polyclonal antibody whey could prevent the adhesion and colonization of Helicobacter pylori. Therefore, oral administration of such whey antibody could eliminate Helicobacter pylori in vivo and obtain the nutrition of whey at the same time.

Claims

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

2.

2. A polyclonal antibody prepared by using the Helicobacter pylori polypeptide antigen group as described in claim 1.

3. The polyclonal antibody according to claim 2, wherein, It is obtained by immunizing dairy cows after mixing KLH-conjugated polypeptide antigen CS-36 and KLH-conjugated polypeptide antigen CI-33 in an equal mass ratio.

4. A drug comprising the polyclonal antibody as described in claim 2 or 3.

5. An immune milk, characterized in that, It is obtained by immunizing dairy cows with the Helicobacter pylori polypeptide antigen group as described in claim 1.

6. An immune whey, characterized in that, It is prepared from the immune milk as described in claim 5.

7. An application of the Helicobacter pylori polypeptide antigen group as described in claim 1 in the preparation of a drug for preventing the adhesion and colonization of Helicobacter pylori.

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