Hairtail immune peptide FAFU-C for alleviating imidacloprid-induced immune damage and preparation method thereof

Abstract

The present invention belongs to the technical field of immune biological products, and specifically relates to a hairtail immune peptide FAFU-C for alleviating imidacloprid-induced immune damage and a preparation method thereof. The amino acid sequence of the hairtail immune peptide FAFU-C of the present invention is Asn-Gln-Val-Ala-Met-Asn-Pro-Thr-Asn-Thr-Val-Phe-Asp-Ala-Lys. When mice were intervened with imidacloprid, their immune function was damaged, indicating that imidacloprid has a certain feasibility in establishing an immune damage model mouse, and the hairtail immune peptide FAFU-C of the present invention can alleviate the immune damage of mice induced by imidacloprid.

Description

Technical Field

[0001] The invention belongs to the technical field of immune biological products, and particularly relates to a hairtail immune peptide FAFU-C for alleviating imidacloprid-induced immune damage and a preparation method thereof. Background Art

[0002] Immunomodulatory function is an important active function of active peptides. By extracting immunomodulatory peptides from natural food raw materials, health products or nutritional supplements can be made to improve human immunity. In recent years, there have been a lot of studies on the immune regulation function of biologically active peptides extracted from marine organisms. These marine peptides have shown different activities in the study, including cardioprotection, anti-hypertension, anti-cancer, anti-diabetes and antioxidant. For the above reasons, marine bioactive peptides are considered to be important candidates for the prevention and treatment of diseases. FangmiaoYu et al. extracted pentapeptides from green clams and acted on immunocompromised mice, and found that there was a significant immune effect; HouHu et al. extracted immunoactive peptides from cod skeletons using trypsin; ZhexinRen et al. extracted peptides from anglerfish eggs with good immunomodulatory effects.

[0003] Hairtail (Trichiurus lepturus), also known as knife fish or skirt fish, belongs to the class of bony fish, the order of Perciformes, the family of Trichiurus. It is narrow and long, silvery white, tender and delicious, and widely distributed in the Pacific coast and the Indian Ocean. Hairtail has become one of the most popular edible fish for consumers due to its high nutritional value and low price, but there are few reports on its use as a protein source for the extraction and preparation of immunoactive peptides.

[0004] Imidacloprid is a nicotinoid insecticide that has been widely used as a crop protection insecticide worldwide in the past decade due to its low soil persistence, low application rate and high insecticidal activity. The widespread use of imidacloprid may lead to the recycling of residues of this insecticide in the food chain. It has been reported that human exposure to imidacloprid may cause damage to the heart, kidneys and other organs, and animals exposed to residual concentrations of the pesticide may cause immunosuppression directly or through the involvement of stress mechanisms / neuroendocrine systems. Summary of the invention

[0005] The purpose of the present invention is to provide a hairtail immune peptide FAFU-C for alleviating the immune damage induced by imidacloprid and a preparation method thereof.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A hairtail immune peptide FAFU-C for alleviating imidacloprid-induced immune damage. The amino acid sequence of the hairtail immune peptide FAFU-C is Asn-Gln-Val-Ala-Met-Asn-Pro-Thr-Asn-Thr-Val-Phe-Asp-Ala-Lys.

[0008] Furthermore, the molecular weight of the above-mentioned hairtail immune peptide FAFU-C is 2002.2Da.

[0009] Furthermore, the above-mentioned hairtail immune peptide FAFU-C is derived from natural extraction or artificial synthesis of hairtail meat.

[0010] The method for preparing the above-mentioned hairtail immune peptide FAFU-C comprises the following steps:

[0011] 1) mincing the hairtail meat into minced meat and mixing it with isopropanol at a ratio of 1:1 w / w, incubating at 60° C. for 120 min to remove fat, then washing the hairtail meat minced meat with distilled water until there is no isopropanol smell, air-drying at 30-32° C. until there is no water droplet, and grinding it into powder to obtain defatted fish meat powder;

[0012] 2) Mix defatted fish meal and distilled water at a ratio of 1:4 w / v, add trypsin, the ratio of trypsin to defatted fish meal is 2000 U / g, perform enzymolysis at 35°C for 4 hours, and obtain an enzymolysis product;

[0013] 3) The enzymatic hydrolysate was inactivated by boiling water for 15 min, centrifuged at 8000 rpm and 4°C for 20 min, and the supernatant was collected to obtain the enzymatic hydrolysate;

[0014] 4) adjusting the pH of the enzymatic hydrolyzate to 7.0, performing ultrafiltration treatment using a 3 kDa ultrafiltration membrane, collecting a portion with a molecular weight less than 3 kDa, and freeze-drying to obtain a crude hairtail immune peptide;

[0015] 5) Add water to the crude hairtail immune peptide to prepare a solution, separate it by Sephadex G-25 gel chromatography, use ultrapure water as eluent, flow rate 1 mL / min, detection wavelength 280 nm, collect the elution peak with a retention time of 60 min, freeze-dry, and obtain the hairtail immune peptide FAFU-C.

[0016] The application of the above-mentioned hairtail immune peptide FAFU-C in the preparation of a drug for alleviating the immune damage induced by imidacloprid.

[0017] The beneficial effects of the present invention are:

[0018] 1) The present invention uses hairtail as raw material, prepares hairtail immune active peptide crude product by enzymatic hydrolysis, adopts ultrafiltration and SephadexG-25 gel column chromatography for separation and purification, and uses macrophage RAW264.7 cell proliferation ability and phagocytic ability as evaluation indicators to obtain immune peptide FAFU-C with high immune activity. The present invention adopts ultra-high performance liquid chromatography combined with electrospray-combined ion trap Orbitrap mass spectrometer for analysis and identification, and obtains a novel immune peptide sequence.

[0019] 2) The present invention successfully established an immune damage model mouse by using imidacloprid. The immune peptide FAFU-C prepared from hairtail as raw material has mild action conditions and high product safety. It can also produce specific peptides through the specificity of the enzyme, which can alleviate the immune damage of mice induced by imidacloprid, provide a reference for further research on its immune regulation mechanism, trigger people's thinking on the problem of pesticide residues, and pay attention to the hazards caused by pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Sephadex G-25 chromatograms of crude hairtail immune peptides. A, FAFU-A, retention time 20 min; B, FAFU-B, retention time 40 min; C, FAFU-C, retention time 60 min.

[0021] Figure 2 : Effects of each elution peak fraction on the proliferation and phagocytic ability of RAW264.7 cells.

[0022] Figure 3 : Secondary mass spectrum of the amino acid sequence of hairtail immune peptide FAFU-C.

[0023] Figure 4 :Schematic diagram of the establishment of imidacloprid-induced immune injury mouse model and dosing regimen.

[0024] Figure 5 :Effect of hairtail immune peptide FAFU-C on the leukocyte count in mice with imidacloprid-induced immune damage.

[0025] Figure 6 :Effects of hairtail immune peptide FAFU-C on organ indexes of mice with imidacloprid-induced immune injury.

[0026] Figure 7 :Effects of hairtail immune peptide FAFU-C on serum immune proteins in mice with imidacloprid-induced immune damage. DETAILED DESCRIPTION

[0027] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0028] In the present invention, the immunological activity determination method is as follows: (1) Determination of the proliferation rate of RAW264.7 cells: RAW264.7 cells in the exponential growth phase were digested into a single cell suspension and 1×10 4 Each well was inoculated in a 96-well plate and incubated at 37°C with 5% CO 2 After overnight culture in a cell culture incubator, the old culture medium was discarded, and 100 μL of each component solution at a concentration of 200 mg / mL was added. The cells were cultured normally for 24 hours, and the cell proliferation rate was detected by MTT method. (2) Determination of phagocytic rate of RAW264.7 cells Neutral red was dissolved in physiological saline to prepare a physiological saline solution containing 0.1% neutral red, which was stored at 4°C for use. RAW264.7 cells were cultured in a culture medium with 100 μL of each component solution at a concentration of 200 mg / mL for 24 hours, and then 100 μL of neutral red solution was added to each well. The cells were placed in an incubator and cultured for 1 hour. After being taken out, washed with PBS buffer for 3 times, 100.0 μL of cell lysis solution (1% glacial acetic acid: anhydrous ethanol = 1:1) was added, and the cells were left standing at room temperature for 2 hours. The cells were observed to be completely lysed under a microscope, and the OD value was measured at 540 nm to calculate the phagocytic rate.

[0029] Example 1 Preparation of immune peptide FAFU-C

[0030] A method for preparing hairtail immune peptide FAFU-C for alleviating imidacloprid-induced immune damage comprises the following steps:

[0031] 1) Pretreatment: Take fresh hairtail, remove the scales, fins, head, tail, viscera, and bones, mince the obtained fish meat into minced meat, mix it with isopropanol in a ratio of 1:1 (w / w), incubate it at 60°C for 120min to remove fat, then rinse the hairtail minced meat with distilled water until there is no isopropanol smell, air-dry it at 30-32°C until there is no water droplet, grind it into powder to obtain defatted fish meat powder, and store it at 4°C for later use.

[0032] 2) Enzymatic hydrolysis: Mix defatted fish meal and distilled water in a ratio of 1:4 (w / v), add trypsin, the ratio of trypsin to defatted fish meal is 2000 U / g, and perform enzymatic hydrolysis at 35°C for 4 hours to obtain an enzymatic hydrolysis product.

[0033] 3) Inactivation of enzyme: Heat the enzymatic hydrolysate in a boiling water bath for 15 min to inactivate the enzyme, centrifuge at 8000 rpm and 4°C for 20 min, collect the supernatant and obtain the enzymatic hydrolysate.

[0034] 4) Ultrafiltration: The pH value of the enzymatic hydrolyzate is adjusted to 7.0 with NaOH solution, and ultrafiltration is performed using a 3 kDa ultrafiltration membrane. The portion with a molecular weight less than 3 kDa is collected and freeze-dried to obtain crude hairtail immune peptide.

[0035] 5) Gel column chromatography: The crude hairtail immune peptide was added to distilled water to prepare a solution with a concentration of 40 mg / mL, and separated by Sephadex G-25 gel chromatography, with ultrapure water as the balance solution and ultrapure water as the eluent. The flow rate was 1 mL / min, the sample volume was 5 mL, and the absorbance of the eluent was detected at a wavelength of 280 nm. The elution curve was drawn as shown in Figure 1 As shown, the eluate from each peak was collected, freeze-dried and then tested for immunoreactivity. Figure 2 As shown, the elution peak FAFU-C has the strongest immune activity, and after freeze-drying it, the hairtail immune peptide FAFU-C was obtained.

[0036] 6) Amino acid sequence determination: Ultra-high performance liquid chromatography coupled with electrospray-combined ion trap Orbitrap mass spectrometry was used to identify the amino acid sequence of hairtail immune peptide FAFU-C. The chromatographic conditions were as follows: chromatographic column: nanocolumn filled with reversed phase ReproSil-Pur C18-AQ resin; mobile phase: 0.1% formic acid (dissolved in water) (A) and 0.1% formic acid (dissolved in acetonitrile) (B); flow rate: 300 nL / min; gradient elution program: 0-5 min, 6%-9% B; 5-25 min, 9%-14% B; 25-60 min, 14%-30% B; 60-70 min, 30%-40% B; 70-75 min, 40%-95% B; 75-80 min, 95% B. The primary mass spectrometry parameters are as follows: resolution: 70000; automatic gain control target: 3e6; maximum cycle time: 40ms; scanning range: 350-1800m / z. The secondary mass spectrometry parameters are as follows: resolution: 75000; automatic gain control target: 1e5; maximum cycle time: 60ms; TopN: 20; NCE / steppedNCE: 27. Figure 3 As shown, after identification and analysis by ultra-high performance liquid chromatography coupled to electrospray ionization-combined ion trap Orbitrap mass spectrometry, the amino acid sequence of the obtained hairtail immune peptide FAFU-C was Asn-Gln-Val-Ala-Met-Asn-Pro-Thr-Asn-Thr-Val-Phe-Asp-Ala-Lys (NQVAMNPTNTVFDAK), which is an undiscovered new immune peptide segment.

[0037] Example 2 Hairtail immune peptide FAFU-C can alleviate the immune damage induced by imidacloprid in mice

[0038] Experimental methods:

[0039] The hairtail immune peptide FAFU-C (NQVAMNPTNTVFDAK) obtained in Example 1 was subjected to solid phase synthesis, and the synthesized polypeptide was fed to immune-damaged mice.

[0040] Thirty-two 8-week-old clean-grade C57BL / 6J male mice (21-25g) were raised in an environment with a temperature of 25°C ± 1°C, a humidity of 55% ± 5%, and a 12-h light / dark cycle. Food and water were provided to the mice for free intake. After one week of adaptation, 8 mice were randomly selected as the control group (Control), and the remaining 24 mice were subjected to immune injury modeling: the mice were gavaged with 30 mg / kg imidacloprid (IMI) once a day for 5 consecutive days. The immune-damaged mice were randomly divided into 3 groups (n = 8), including the model group (Model), the positive group (Positive), and the experimental group (Peptide). All mice were treated by gavage. The control group was gavaged for 20 consecutive days with normal saline; the model group was gavaged for 15 consecutive days with normal saline; the positive group was gavaged for 15 consecutive days with 10 mg / kg levamisole hydrochloride; the experimental group was gavaged for 15 consecutive days with 100 mg / kg solid-phase synthetic hairtail immune peptide FAFU-C. The specific scheme is as follows Figure 4 .

[0041] Determination of organ index: 24 hours after the last administration of the drug, the weight of the mice was recorded and the mice were killed by cervical dislocation. The thymus and spleen of the mice were separated and removed, the blood on the organs was washed clean with saline, and the water on the surface of the organs was absorbed with filter paper to prevent errors caused by weighing. The organ index (mg / g) was calculated according to the following formula:

[0042] Organ index (mg / g) = organ weight (mg) / body weight (g)

[0043] Determination of white blood cell count: draw blood from the eyeball, quickly place the whole blood in an anticoagulant tube, shake it up and down to mix the blood and anticoagulant evenly, and use a complete blood cell analyzer to detect the white blood cell count.

[0044] Serum IgA and IgG concentration detection: Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of IgA and IgG in serum. According to the kit instructions and instrument operation procedures, the absorbance was measured at 450nm. The logistic curve model in the ELISA Calc software was used to determine the serum concentration of IgA and IgG according to the standard calibration curve.

[0045] Experimental results:

[0046] 1. No obvious toxic and side effects

[0047] During the experimental drug intervention, no abnormal behavior was observed in the mice, the mice survived normally, and no mice died during the experiment.

[0048] 2. Determination of white blood cell count

[0049] The number of white blood cells in the blood is tested using a blood cell analyzer. Figure 5 As shown, the number of white blood cells in the blood of mice in the model group was lower than that in the control group, and the total number of white blood cells in the blood of mice treated with the positive group and hairtail immune peptide FAFU-C was higher than that in the model group, indicating that hairtail immune peptide FAFU-C can increase the number of white blood cells in the blood of immune-damaged mice.

[0050] 3. Organ index determination

[0051] The thymus and spleen are important immune organs for the growth, differentiation, maturation and immune response of immune cells. Their immune regulation is closely related to the increase of organ indexes. Each organ index reflects the functional status of the immune organs to a certain extent. Figure 6 It can be seen that the spleen and thymus indexes of the model group mice induced by imidacloprid were significantly reduced, indicating that the immune damage mice were successfully modeled; from the positive group and the experimental group, it can be seen that the spleen index and thymus index of the immunosuppressed mice induced by imidacloprid were significantly improved. It is preliminarily shown that the hairtail immune peptide FAFU-C can alleviate the function of imidacloprid-induced immune damage.

[0052] 4. Serum IgA and IgG concentration determination

[0053] Immunoglobulins are proteins produced by B cells after proliferation and differentiation in response to specific antigens. Secreted IgG and IgM are the main antibody components in serum and are important effector molecules of humoral immune response. Figure 7 As shown in the figure, the IgA and IgG concentrations of the model group were significantly reduced after induction by imidacloprid, and its immune function was severely damaged, indicating that imidacloprid can induce immune damage. Compared with the model group, the IgA and IgG concentrations in the serum of the experimental group were increased after treatment with the hairtail immune peptide FAFU-C, indicating that the hairtail immune peptide FAFU-C can effectively alleviate the immune damage induced by imidacloprid.

[0054] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing hairtail immune peptide FAFU-C, characterized in that: The following steps are involved: 1) Grind the hairtail meat into minced meat and mix it with isopropanol in a ratio of 1:1 w / w, incubate at 60°C for 120 minutes to remove fat, then rinse the hairtail meat with distilled water until there is no isopropanol smell, air-dry at 30-32°C until there is no water droplet, and grind it into powder to obtain defatted fish meat powder; 2) Mix defatted fish meal and distilled water at a ratio of 1:4 w / v, add trypsin, the ratio of trypsin to defatted fish meal is 2000 U / g, and perform enzymolysis at 35°C for 4 hours to obtain an enzymolysis product; 3) The enzymatic hydrolysate was inactivated by boiling water for 15 min, centrifuged at 8000 rpm and 4°C for 20 min, and the supernatant was collected to obtain the enzymatic hydrolysate; 4) The pH of the enzymatic hydrolysate was adjusted to 7.0, and ultrafiltration was performed using a 3 kDa ultrafiltration membrane to collect the fraction with a molecular weight less than 3 kDa, and freeze-dried to obtain crude hairtail immune peptide; 5) Add water to the crude hairtail immune peptide to prepare a solution, separate it by Sephadex G-25 gel chromatography, use ultrapure water as eluent, flow rate 1 mL / min, detection wavelength 280 nm, collect the elution peak with a retention time of 60 min, freeze-dry, and obtain hairtail immune peptide FAFU-C; The amino acid sequence of the hairtail immune peptide FAFU-C is Asn-Gln-Val-Ala-Met-Asn-Pro-Thr-Asn-Thr-Val-Phe-Asp-Ala-Lys.

2. The use of hairtail immune peptide FAFU-C in the preparation of a drug for alleviating the immune damage induced by imidacloprid, characterized in that: The amino acid sequence of the hairtail immune peptide FAFU-C is Asn-Gln-Val-Ala-Met-Asn-Pro-Thr-Asn-Thr-Val-Phe-Asp-Ala-Lys.

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