RU2645070C2 - Method for antimicrobial peptides production from domestic chicken platelets - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to obtain antimicrobial peptides from the domestic chicken platelets, the following fresh blood of chicken is collected into plastic containers with addition of 3.8% sodium citrate solution (pH 3.5). The collected blood is centrifuged at 250 g for 30 minutes. The resulting supernatant is centrifuged at 1000 g for 30 minutes. The resulting precipitated platelets are washed three times with medium 199 with addition of 3.8% sodium citrate. The washed platelet mass is resuspended in 10% acetic acid and kept at -15…-20 °C for 24 hours. The product is thawed and centrifuged at 1000 g for 40 minutes. Antimicrobial substances are precipitated from the resulting supernatant with chilled acetone in a ratio of 1:7 (v/v) for 12 hours at 4 °C. The precipitate is centrifuged at 6000 g for 10 minutes, dried in air with vigorous stirring and crushed. The resulting acetone precipitate is desalted by reversed-phase high-performance liquid chromatography (RP-HPLC) in a stepwise gradient increasing the concentration of 80% aqueous acetonitrile in 0.1% aqueous trifluoracetic acid (TFA) to a value of 75%. After elution from the column, the total extract is removed from the excess content of acetonitrile on a vacuum centrifuge, followed by lyophilization. The dried desalted extract is diluted again in 0.1% TFA and separated by analytical RP-HPLC in a linear gradient of increasing the concentration of aqueous acetonitrile in aqueous TFA from 8 to 40% (basic) and 40-56% (optional). Antimicrobial peptides are collected and lyophilized.

EFFECT: obtaining of purified antimicrobial peptides from the domestic chicken platelets.

2 tbl, 1 ex

Description

The invention relates to microbiology and biochemistry, namely the production of antimicrobial peptides from platelets of chicken domestic, and can be used to obtain new drugs with antimicrobial activity.

Г.М., Шамова О.В. Катионные противомикробные пептиды как молекулярные факторы иммунитета: мультифункциональность. Журнал микробиологии, эпидемиологии и иммунобиологии. 2006. №2: 98]. Выделение и структурно-функциональное изучение антимикробных пептидов животного происхождения создает предпосылки для разработки и производства химически или биотехнологически синтезированных гомологов этих соединений [Кокряков В.Н. Очерки о врожденном иммунитете. СПб.: Наука, 2006: 108].In connection with the significant growth rate of the resistance of infectious agents to antibiotics [Vinogradova K.A., Bulgakova V.G., Polin A.N., Kozhevin P.A. The resistance of microorganisms to antibiotics: resistoma, its volume, diversity and development // Antibiotics and chemotherapy. 2013, v. 58. No. 5-6: 38-48], the search for alternative therapeutic agents, the resistance to which microorganisms will develop to a limited or completely absent [Ashby M., Petkova A., Hilpert K. Cationic antimicrobial peptides as potential, is relevant new therapeutic agents in neonates and children: A review // Current Opinion in Infectious Diseases. 2014.27 (3): 258-267]. Antimicrobial peptides may be such agents [Kokryakov V.N., Kovalchuk L.V.,

G.M., Shamova O.V. Cationic antimicrobial peptides as molecular factors of immunity: multifunctionality. Journal of Microbiology, Epidemiology and Immunobiology. 2006. No. 2: 98]. Isolation and structural-functional study of antimicrobial peptides of animal origin creates the prerequisites for the development and production of chemically or biotechnologically synthesized homologs of these compounds [Kokryakov V.N. Essays on innate immunity. St. Petersburg: Nauka, 2006: 108].

It is known that blood cells, in particular blood plates of humans and animals, are the source of various cationic peptides with pronounced antimicrobial activity [Tang Y.-Q., Yeaman M.R., Selsted M.E. Antimicrobial peptides from human platelets. Infection and Immunity. 2002, 70 (12): 6524-6533; Ivanov I.B., Gritsenko V.A. Comparative activities of cattle and swine platelet microbicidal proteins. Probiotics and Antimicrobial Proteins. 2009, 1 (2): 148-151]. A method for producing platelet cationic protein from human blood platelets is described [Bukharin OV, Chereshnev VA, Suleimanov K.G. Antimicrobial platelet protein. Yekaterinburg, 2000: 35-99]; platelets of pigs and cattle [RF patent No. 2278675, IPC A61K 35/14, A61K 38/02, A61P 31/04, 2005], human and horse platelets [Gorshkov NI, Malakhova II, Krasikov V.D., Zhurlov O.S., Ivanov Yu.B. Liquid chromatography of platelet proteins. Sorption and chromatographic processes. 2010, vol. 10. Vol. 5: 661-668], rabbit platelets [Yeaman M.R., Tang Y.-Q., Shen A.J., Bayer A.S., Selsted M. Purification and in vitro es of rabbit platelet microbicidal proteins. Infect. Immunol. 1997, 65: 1024-1025].

М.В., Шейда Е.В., Карташова О.Л., Жуков А.П. Антимикробная активность тромбодефенсинов разных видов животных // Известия Оренбургского государственного аграрного университета. 2009, №4(24): 177-179].The authors first established the presence of antimicrobial substances in the acid extract of platelets of chicken domestic (Gallus gallus) to suppress the growth of strains of Bacillus subtilis, which are most sensitive to the action of platelet cationic protein [

M.V., Sheida E.V., Kartashova O.L., Zhukov A.P. Antimicrobial activity of thrombodefensins of different animal species // Bulletin of the Orenburg State Agrarian University. 2009, No. 4 (24): 177-179].

As a result of these studies, an assumption was made about the presence of home antimicrobial peptides in chicken platelets. However, the acid extract contains acetic acid, which excludes the possibility of its use in vivo, as well as components in the low molecular weight range, which does not allow us to evaluate the functional properties of the active compounds in an individual state.

The objective of the proposed technical solution is to create a method for producing purified antimicrobial peptides from platelets of chicken home.

To achieve the technical result in the claimed method for the production of antimicrobial peptides from platelets of chicken at home, fresh chicken blood is collected in plastic containers with the addition of a 3.8% sodium citrate solution (pH 3.5) in a ratio of 5 parts of blood: 1 part of 3.8% sodium citrate solution; centrifuging the collected blood at 250 g for 30 minutes to obtain a platelet-enriched supernatant; centrifuging the supernatant at 1000 g for 30 minutes to precipitate platelets; washing the precipitated platelets three times with 199 medium with the addition of 3.8% sodium citrate in a ratio of 1:10 (weight / weight); resuspension of the washed platelet mass in 10% acetic acid in a ratio of 1:10 (weight / volume) and keeping at -15 ... -20 ° C for 24 hours; thawing and centrifuging the obtained extract at 1000 g for 40 minutes; planting antimicrobial substances from the obtained supernatant with chilled acetone in a ratio of 1: 7 (volume / volume) for 12 hours at 4 ° C; centrifugation of the precipitate at 6000 g for 10 minutes, drying in air with vigorous stirring and grinding; desalting the obtained acetone precipitate by reverse phase high performance liquid chromatography (RP-HPLC) in a stepwise gradient to increase the concentration of 80% aqueous acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) to 75%; after elution from the column, stripping the total extract from the excess acetonitrile in a vacuum centrifuge followed by lyophilization; re-dissolving the dried desalted extract in 0.1% TFA and separation by analytical RP-HPLC in a linear gradient to increase the concentration of aqueous acetonitrile in aqueous TFA from 8 to 40% (basic) and 40-56% (optional); collection and lyophilization of antimicrobial peptides.

The technical result achieved during the implementation of the invention is that the claimed method for producing antimicrobial peptides from chicken platelets at home allows them to be produced in semi-preparative and preparative amounts (from milligrams to tens of milligrams), sufficient for the subsequent study of their functional properties, including stages preclinical trials.

Analogues of the invention in the patent and scientific literature are not found.

The method is as follows.

1. Fresh chicken blood is collected in plastic containers with the addition of a 3.8% sodium citrate solution (pH 3.5) in a ratio of 5 parts of blood: 1 part of a 3.8% sodium citrate solution.

2. The collected blood is centrifuged at 250 g for 30 minutes to obtain a platelet rich supernatant.

3. The supernatant is centrifuged at 1000 g for 30 minutes to precipitate platelets.

4. Precipitated platelets are washed three times with medium 199 with the addition of 3.8% sodium citrate in a ratio of 1:10 (weight / weight).

5. The washed platelet mass is resuspended in 10% acetic acid in a ratio of 1:10 (weight / volume) and kept at -15 ... -20 ° C for 24 hours.

6. Thawing is carried out and the resulting extract is centrifuged at 1000 g for 40 minutes.

7. Antimicrobial substances from the obtained supernatant are planted with chilled acetone in a ratio of 1: 7 (vol / vol) for 12 hours at 4 ° C.

8. The precipitate is centrifuged at 6000 g for 10 minutes, dried in air with vigorous stirring and grinding.

9. Solid phase extraction (desalting) of the obtained acetone precipitate is carried out by reverse phase high performance liquid chromatography (RP-HPLC) in a stepwise gradient to increase the concentration of 80% aqueous acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) to 75%.

10. After elution from the column, the total extract is evaporated from the excess acetonitrile in a vacuum centrifuge, followed by lyophilization (in order to remove residual TFU),

11. The dried desalted extract is redissolved in 0.1% TFA and separated by analytical RP-HPLC in a linear gradient of increasing the concentration of aqueous acetonitrile in aqueous TFA from 8 to 40% (basic) and 40-56% (optional).

12. Collect and lyophilize antimicrobial peptides.

Examples of specific performance:

Example 1

At the Orenburg Broiler poultry farm, fresh chicken blood was collected in plastic containers with the addition of a 3.8% sodium citrate solution (pH 3.5) in a ratio of 5 parts of blood: 1 part of a 3.8% sodium citrate solution.

From 5000 ml of fresh citrated chicken blood by centrifugation at 250 g for 30 minutes, a platelet-rich supernatant was obtained. Platelets were pelleted by centrifugation at 1000 g for 30 minutes. 5 ml of precipitated platelets was washed three times with 199 medium with addition of 3.8%) sodium citrate in a ratio of 1:10, after which they were resuspended in 45 ml of 10% acetic acid in a ratio of 1:10 and kept at -15 ... -20 ° С in 24 hours. Then the platelet mass was thawed and centrifuged at 1000 g for 40 minutes. Antimicrobial substances from the obtained supernatant were landed with chilled acetone in a ratio of 1: 7 (v / v) for 12 hours at 4 ° C. After centrifugation (6000 g for 10 minutes), the precipitate was dried in air with vigorous stirring and grinding. Then, the obtained acetone precipitate was redissolved in 0.1% aqueous trifluoroacetic acid (TFA) and desalted by RP-HPLC on an Aquapore C ₈ cartridge column (Applied Biosystems, USA). After leaving the column of all unbound components, the sorbed fraction was eluted with a stepwise gradient (75%) of a concentration of 80% acetonitrile in 0.1% TFA. The absorption was detected at a wavelength of 214 nm. Subsequently, after evaporation of the organic solvent in a vacuum centrifuge (Lab-conco, USA), the desalted extract was separated by analytical RP-HPLC in a linear gradient of increasing the concentration of 80% acetonitrile in 0.1% TFA on a Luna C ₁₈ column 4.6 × 250 mm (Phenomenex, USA) with detection of absorption at a wavelength of 214 nm. The collected fractions were lyophilized to remove organic solvent and residual amounts of TFA.

As a result of chromatographic separation, 13 fractions of antimicrobial peptides were obtained.

The antimicrobial activity of the obtained peptide fractions was tested on test cultures of Escherichia coli K 12, Bacillus subtilis, Staphylococcus aureus P 209, Bacillus cereus, Micrococcus luteus, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter cloacae, Candida albican K., Hancock REW Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature protocols. 2008, 3 (2): 163-175].

For this, test cultures were grown on Müller-Hinton agar at 37 ° C for 18 hours, and suspension was prepared with a density of 0.181; 0.184 and 0.228, respectively. The optical density was determined on a STAT FAX 2100 spectrophotometer at a wavelength of 630 nm. Peptide fractions were resuspended in a solution of 0.01% acetic acid containing 0.2% bovine serum albumin (SIGMA, Germany), and serial double dilutions of the peptides were prepared in wells of a sterile 96-well polypropylene plate.

100 μl of bacterial and fungal suspension was added to the wells of a 96-well polypropylene plate from the 1st to 11th row and 11 μl of double dilutions of the tested peptides were added to each well from the 1st to the 10th row. 11 row - control the growth of culture. In the 12th row of the tablet, sterile Mueller-Hinton broth was added (sterility control and a form for scanning cells). 11 μl of a solution of 0.01% acetic acid containing 0.2% bovine serum albumin was added to the wells of the 11th and 12th rows. The plates were incubated at 37 ° C. After 24 hours of incubation, 10 μl of the contents were plated from the wells on Mueller-Hinton agar. The number of colonies grown at 37 ° C for 24 hours was taken into account.

The results are presented in table 1, which shows that 1, 2 and 13 peptide fractions suppressed the growth of B. subtilis, 5 peptide fraction - S. aureus P 209; 4 peptide fraction - B. subtilis and S. aureus P 209; 6 peptide fraction - S. aureus P 209 and E. coli K 12; 11 peptide fraction - E. coli K 12 and B. subtilis; 3, 7, 8, 9, and 10 peptide fractions are all three studied microorganisms.

Peptide fractions 1, 2, 4, 5, 6, 10 inhibited the growth of B. cereus and M. luteus; 3 peptide fraction - B. cereus; 13 peptide fraction - B. cereus and K. pneumoniae; 7, 8, 9, 11, 12 peptide fractions - inhibited the growth of all three test cultures.

Peptide fractions 5 and 13 inhibited the growth of E. faecalis; 1 peptide fraction - C. albicans; peptide fractions 6 and 12 - E. cloacae and C. albicans; peptide fraction 9 — E. faecalis and E. cloacae; peptide fractions 2, 3, 4, 7, 8, 10, 11 - inhibited the growth of all studied test cultures.

The data obtained indicate that all the obtained peptide fractions in one way or another suppress the growth of not only bacteria, but also Candida albicans fungi, that is, they are characterized by antimicrobial activity and are antimicrobial peptides.

Example 2

Orenburgskaya Poultry Farm CJSC collected fresh chicken blood in plastic containers with the addition of a 3.8% sodium citrate solution (pH 3.5) in a ratio of 5 parts of blood: 1 part of a 3.8% sodium citrate solution.

According to example 1, 13 fractions of antimicrobial peptides were obtained from chicken platelets.

The antimicrobial activity of the obtained peptide fractions was tested on test cultures of Escherichia coli K 12, Bacillus subtilis, Staphylococcus aureus P 209, Bacillus cereus, Micrococcus luteus, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter cloacae, Candida albicans with microtiter. The results obtained are presented in table 2, which shows that all the obtained peptides had antimicrobial activity against the studied microorganisms, inhibiting their growth.

Thus, the inventive method for producing antimicrobial peptides from platelets of chicken home provides peptides with high inhibitory activity against both gram-positive and gram-negative microorganisms, as well as Candida albicans fungi, which allows us to consider them in the future as the basis for the creation of antimicrobial agents.

Claims (13)

A method for producing antimicrobial peptides from platelets of chicken domestic, comprising the following steps: - collection of fresh chicken blood in plastic containers with the addition of a 3.8% sodium citrate solution (pH 3.5) in a ratio of 5 parts of blood: 1 part of a 3.8% sodium citrate solution; - centrifuging the collected blood at 250 g for 30 minutes to obtain a platelet-enriched supernatant; - centrifugation of the supernatant at 1000 g for 30 minutes to precipitate platelets; - washing the precipitated platelets three times with medium 199 with the addition of 3.8% sodium citrate in a ratio of 1:10 (weight / weight); - resuspension of the washed platelet mass in 10% acetic acid in a ratio of 1:10 (weight / volume) and keeping at -15 ... -20 ° C for 24 hours; - Thawing and centrifugation of the obtained extract at 1000 g for 40 minutes; - planting antimicrobial substances from the obtained supernatant with chilled acetone in a ratio of 1: 7 (volume / volume) for 12 hours at 4 ° C; - centrifugation of the precipitate at 6000 g for 10 minutes, drying in air with vigorous stirring and grinding; - desalting the obtained acetone precipitate by reverse phase high performance liquid chromatography (RP-HPLC) in a stepwise gradient to increase the concentration of 80% aqueous acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) to 75%; - after elution from the column, pairing of the total extract from the excess acetonitrile content in a vacuum centrifuge followed by lyophilization; - re-dissolution of the dried desalted extract in 0.1% TFA and separation by analytical RP-HPLC in a linear gradient of increasing the concentration of aqueous acetonitrile in aqueous TFA from 8 to 40% (basic) and 40-56% (optional); - collection and lyophilization of antimicrobial peptides.