CN1814775A - Method for preparing antiviral active oligomeric peptide - Google Patents

Abstract

A method for producing oligomeric peptides with antiviral activity by step-by-step directed enzymatic hydrolysis of oysters with complex enzymes, which is characterized in that Tris-HCl buffer solution is added to oyster meat for beating, and the liter of the buffer solution is 2 kg of oyster meat -3 times, add trypsin according to the ratio of 60-65U/g meat, stir at constant temperature, enzymolyze at 40-50°C for 3-4 hours, then inactivate the enzyme, cool down; use acid to adjust pH to 5-6, according to 70 Add bromelain at a ratio of -75U/g meat, stir, enzymolyze at a constant temperature of 45-55°C for 5-6 hours, inactivate the enzyme, cool, centrifuge, and take the supernatant through ultrafiltration and Sephadex G-25 gel column chromatography , DEAE Sephadex A-25 ion-exchange column chromatography, reversed-phase high-performance liquid chromatography for separation and purification, and freeze-drying. The product of the invention has high activity and has good inhibitory effect on influenza virus and herpes virus.

Description

A kind of preparation method of antiviral active oligomeric peptide

technical field

The invention relates to a method for preparing oligopeptides with antiviral activity by directional enzymolyzing oysters with complex enzymes.

Background technique

Viruses are a type of parasitic microorganisms closely related to human health. In recent years, the emergence and prevalence of viruses such as avian influenza, foot-and-mouth disease, mad cow disease, atypical pneumonia, AIDS, monkeypox, West Nile and Ebola have seriously threatened human health. Diseases caused by viruses are not only highly pathogenic, but also highly contagious. Therefore, the prevention and treatment of these diseases is a major and persistent problem faced by human beings.

Since the advent of penicillin in 1941, the rapid development of antibiotics has effectively controlled many bacterial infectious diseases, while the prevention and treatment of viral diseases is much more difficult than the former, and has become a prominent problem in infectious diseases. Because the structure and propagation mode of viruses are different from bacteria, they lack their own reproductive enzyme system, they must parasitize in the host cell, and they can grow and reproduce by synthesizing their own nucleic acid and protein with the help of the host cell's enzyme system, which makes the drug in the While acting on the virus, it can also kill the normal cells of the host, so the application of antiviral drugs is limited to a certain extent. In addition, clinical symptoms of viral infection often appear after the peak of viral growth, which also makes it difficult for drugs to work.

At present, most of the medicines for the treatment of viral diseases are western medicines, which have various side effects. There is no report on the preparation of antiviral oligopeptides from oyster protein.

Contents of the invention

The object of the present invention is to provide a method for producing oligomeric peptides with antiviral activity by step-by-step directed enzymatic hydrolysis of oysters with complex enzymes, which can make up for the above-mentioned shortcomings of the prior art.

A method for producing oligopeptides with antiviral activity by step-by-step directional enzymolysis of oysters with complex enzymes, characterized in that Tris-HCl buffer solution is added to oyster meat and beaten, and the number of liters of the buffer solution is the number of kilograms of oyster meat 2-3 times that of meat, add trypsin according to the ratio of 60-65U/g meat, stir at constant temperature, enzymolyze at 40-50°C for 3-4 hours, then inactivate the enzyme, cool down; adjust the pH to 5-6 with acid, Add bromelain according to the ratio of 70-75U/g meat, stir, enzymolyze at a constant temperature of 45-55°C for 5-6 hours, inactivate the enzyme, cool, centrifuge, and take the supernatant through ultrafiltration and Sephadex G-25 gel column Chromatography, DEAE Sephadex A-25 ion exchange column chromatography, reversed-phase high-performance liquid chromatography after separation and purification, freeze-dried.

The invention uses low-value oysters as raw materials, and uses trypsin and bromelain to hydrolyze to prepare the oligomeric peptide with antiviral activity. The obtained product has high activity and has better inhibitory effect on influenza virus and herpes virus.

Description of drawings

Accompanying drawing is the inhibitory effect diagram of active oligomeric peptide of the present invention to virus. In the figure, A is the cell control, B is the virus control, C is the 1# sample, and D is the 2# sample.

Detailed ways

Add 300ml of Tris-HCl damping solution to 100 grams of oyster meat, make a slurry, and the pH of the resulting slurry is 7.5-8.5, preferably 8.0. Add trypsin according to the proportion of 60-65U/g meat, preferably 60U/g meat, stir at constant temperature, and enzymolyze at 40-50°C, preferably 50°C for 3-4 hours, preferably 3 hours, then Inactivate the enzyme at 95-100°C, cool to 45-55°C, preferably 50°C; adjust the pH to 5-6 with hydrochloric acid, preferably 5.5, according to the ratio of 70-75U/g meat, preferably 700U/g meat Add bromelain, stir, and enzymatically hydrolyze at 45-55°C, preferably at 50°C, for 5-6 hours, preferably 5 hours, then inactivate the enzyme at 95-100°C, cool to room temperature, 9000-10000r/min Refrigerate and centrifuge at high speed, take the supernatant for ultrafiltration, collect the part with a molecular weight of 5-10K, and then separate it by Sephadex G-25 gel column chromatography, DEAE Sephadex A-25 ion exchange column chromatography, and reversed-phase high performance liquid chromatography. Purify and lyophilize to obtain the peptide with antiviral activity.

The concentration of the Tris-HCl buffer solution used in the present invention is 0.04-0.06.M, the pH is 7.5-8.5, and the number of liters of its consumption is 2-3 times of the number of kilograms of oyster meat.

The anti-influenza virus activity of samples 1# (oligomeric peptides with a molecular weight of 5-10k) and 2# (oligomeric peptides with a molecular weight of 1-5k) were detected by virus microneutralization assay, and the results are shown in the accompanying drawings. The experimental results show that compared with the virus control, the 1# sample (i.e. the peptide of the claimed molecular weight of the present invention at 5-10K) has a certain inhibitory effect on influenza virus, and it is kept intact by some cells after virus infection for 72 hours; Afterwards, the cells become completely diseased.

Claims (1)

1. A method for producing oligopeptides with antiviral activity by step-by-step directional enzymolysis of oysters with complex enzymes, characterized in that Tris-HCl buffer solution is added to oyster meat and beaten, and the liter of the buffer solution is equivalent to oyster meat 2-3 times the number of kilograms, add trypsin according to the ratio of 60-65U/g meat, stir at constant temperature, enzymolyze at 40-50°C for 3-4 hours, then inactivate the enzyme, cool; adjust the pH to 5- with acid 6. Add bromelain according to the ratio of 70-75U/g meat, stir, and enzymolyze at a constant temperature of 45-55°C for 5-6 hours, inactivate the enzyme, cool, centrifuge, and take the supernatant through ultrafiltration and Sephadex G-25 coagulation After separation and purification by gel column chromatography, DEAE SephadexA-25 ion exchange column chromatography, and reverse-phase high-performance liquid chromatography, it was freeze-dried.