CN104877009B - A red ray cartilage polypeptide angiogenesis inhibitor - Google Patents

Abstract

The invention relates to a dasyatis akajei cartilage polypeptide angiogenesis inhibiting factor, a rapid preparation method and an application thereof, wherein the amino acid sequence of the angiogenesis inhibiting factor is Gly-Tyr-Ile-Cys-Pro-Gln-Trp, and the molecular weight of ESI-MS detection is 865.98 Da. The angiogenesis inhibitor can effectively inhibit angiogenesis of chick chorioallantoic membrane (CAM), and has obvious inhibition effect on expression of three angiogenesis promoting factors, namely Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), of lung cancer tissues of Lewis lung cancer-bearing mice.

Description

A red ray cartilage polypeptide angiogenesis inhibitor

technical field

The invention relates to a marine biologically active polypeptide, in particular to a red ray cartilage polypeptide angiogenesis inhibitor.

Background technique

The growth and metastasis of tumors depend on the formation of new blood vessels, and the use of anti-angiogenic therapy to treat tumors has the advantages of high efficiency, low toxicity, and resistance to drug resistance. Therefore, the search for angiogenesis inhibitory factors with significant activity has become a research hotspot of anti-tumor drugs. Tumor angiogenesis is affected by a variety of factors, and vascular endothelial growth factor (VEGF) is the most important angiogenic factor and the main executor of VEGF signal transduction in endothelial cells, which is directly involved in the process of neovascularization. Studies have shown that VEGF must bind to the specific receptor VEGFR-2 located on the cell membrane to play its role. Therefore, drug screening with VEGFR-2 as a target has become an important strategy for anti-tumor angiogenesis.

The preparation and screening methods are very important in the research of anti-tumor drugs, while the conventional methods are time-consuming and the false positive rate of the obtained active substances is high. To maximize the success rate of anti-tumor drug development.

Cartilaginous fish cartilage is rich in a variety of anti-tumor active substances, mainly including angiogenesis inhibitory factors, anti-tumor factors and anti-invasion factors, but it has not been effectively utilized. Although it has been reported to prepare angiogenesis-inhibiting factors from cartilage such as red ray, stingray, etc., the obtained active substances are difficult to be developed because of their large molecular weight and low content.

Based on the above status quo, the present invention uses red ray cartilage as a material, adopts enzymatic hydrolysis and cell membrane chromatography technology to establish a rapid preparation method of tumor angiogenesis inhibitor, and provides a red ray cartilage polypeptide angiogenesis inhibitor with significant activity.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a red ray cartilage polypeptide angiogenesis inhibitory factor with significant angiogenesis inhibitory activity against the above-mentioned technical status. The amino acid sequence of the red ray cartilage polypeptide angiogenesis inhibitory factor is Gly-Tyr-Ile-Cys-Pro-Gln-Trp, and the molecular weight detected by ESI-MS is 865.98 Da.

The preparation method of the red ray cartilage polypeptide angiogenesis inhibitory factor is as follows:

1) Preparation of cell membrane suspension: Take tumor cells with high expression of vascular endothelial growth factor-specific receptor (VEGFR) and in logarithmic growth phase, use MEM medium (containing 10% fetal bovine serum), at 37°C , 5% CO ₂ incubator. When the cell count reached 10 ⁵ to 10 ⁷ , the cells were digested with trypsin, centrifuged at 4°C, 1000 r/min for 10 to 15 min, the precipitate was washed twice with normal saline, and then added to 50 mM Tris-HCl solution for sonication The cells were broken for 25-30min, the suspension was centrifuged at 1000r/min and 4°C for 5-10min, and the supernatant was centrifuged at 12000r/min and 4°C for 20-25min to obtain cell membrane precipitation, which was resuspended with normal saline. , after washing 2 to 3 times, add normal saline until the membrane protein content is 2.0 to 2.3 mg/mL, which is the cell membrane suspension;

2) Preparation of cell membrane stationary phase: Take the pretreated macroporous spherical silica gel and add it to a low temperature (4°C) reaction tube. Under shaking conditions, add the cell membrane suspension to the reaction tube according to the solid-to-liquid ratio of 1 g: 25-30 mL. , adsorbed for 15-20h, ultrasonically milled for 20-25min, centrifuged at 4000r/min for 10-15min, collected the precipitate, washed 2-3 times with normal saline, removed the unbound cell membrane, and obtained the cell membrane stationary phase;

3) Preparation and enzymatic hydrolysis of red ray cartilage protein: add the crushed and homogenized red ray cartilage into a 1.0 mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1 g: 10 to 15 mL, and after 44 to 48 hours of shaking extraction at 4°C , centrifuge at 4°C and 10000r/min for 15-20min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1kDa, dialyze it with double distilled water below 4°C for 22-24h, and freeze-dry the dialysate to obtain red ray cartilage. Protein; add red ray cartilage protein into barbital sodium-hydrochloric acid buffer (pH 7.0) at a solid-to-liquid ratio of 1 g: 15-20 mL, and add neutral protease (enzyme activity) according to 1.5-2.5% of the red ray cartilage protein content. ≥1.0×10 ⁵ U/g), enzymolysis temperature 55～65℃, enzymolysis 3～4h, the solution is heated to 90～95℃, and kept at this temperature for 10～15min, the temperature drops to 35～40℃, Adjust the pH to 7.5～8.0 with NaOH, add trypsin (enzyme activity ≥1.9×10 ⁴ U/g) according to 1.5～2.5% of the cartilage protein of red ray, the enzymolysis temperature is 35～40℃, and the enzymolysis is carried out for 3～4h , the solution was heated to 90-95°C, and kept at this temperature for 10-15min, centrifuged at 10000r/min and 4°C for 15-20min, and the supernatant was taken, and the supernatant was subjected to ultrafiltration treatment with a 1kDa ultrafiltration membrane , collect the part with molecular weight less than 1kDa, place it in a dialysis bag with a molecular weight cut-off of 100, dialyze it with three distilled water for 24h, freeze-dry it, and obtain the active component of the enzymolysate;

4) Preparation of red ray cartilage polypeptide angiogenesis inhibitory factor: the active components of the enzymatic hydrolyzate are prepared into a solution of 0.03-0.05 mg/mL with three-distilled water, and the cell membrane stationary phase is added to the active component according to the solid-liquid ratio of 1 g: 10 mL. In the component solution, after incubation at 35～40℃ for 2～3h, centrifuge at 4000r/min for 10～15min, take the supernatant, rinse the precipitate with triple distilled water for 8～10 times, combine the washing solution and the supernatant, Freeze-drying is the cell membrane adsorption residue; RP-HPLC was used to establish the fingerprints of the active components of the enzymatic hydrolysate and the cell membrane adsorption residue, and the difference peaks with disappearance or significant reduction in peak area were purified and prepared, and the chicken embryo villi were analyzed. Allantoic membrane (CAM) angiogenesis inhibition assay, the amino acid sequence of the most active peak was determined by protein/peptide sequence analyzer as Gly-Tyr-Ile-Cys-Pro-Gln-Trp (GYICPQW), and the molecular weight detected by ESI-MS was 865.98Da.

Preferably, the tumor cells in 1) are liver cancer cells Bel-7402;

)中加入适量HCl溶液(1mol/L)，超声处理15～20min，转移至圆底烧瓶瓶中搅拌回流水解2～3h后，将硅胶移至烧杯中，加双蒸水洗涤3～5次，每次25～30min。将洗涤处理好的大孔球形硅胶减压抽干，于120℃下活化6～8h。Preferably, the pretreatment method of the macroporous spherical silica gel in 2) is as follows: the macroporous spherical silica gel (specification: 5 μm,

), add an appropriate amount of HCl solution (1mol/L), ultrasonically treat it for 15 to 20 minutes, transfer it to a round-bottomed flask, stir and reflux for 2 to 3 hours and hydrolyze it for 2 to 3 hours, then move the silica gel to the beaker, add double distilled water to wash 3 to 5 times, 25 ~ 30min each time. The washed macroporous spherical silica gel was vacuum-dried and activated at 120°C for 6-8 hours.

Preferably, the RP-HPLC conditions in 4) are: the injection volume is 5-10 μL; the chromatographic column is Amethyst C18 (250 mm×4.6 mm, 5 μm); to 50%; flow rate 0.4mL/min; UV detection wavelength 215nm.

The technical solution adopted by the present invention to solve the above-mentioned third technical problem is: an application of red ray cartilage polypeptide angiogenesis inhibitory factor, which is characterized in that Gly-Tyr-Ile-Cys-Pro-Gln-Trp (GYICPQW) Gly-Tyr-Ile-Cys-Pro-Gln-Trp (GYICPQW) has the advantages of safety, no toxic side effects and strong activity.

Compared with the prior art, the present invention has the advantages that the extraction and purification processes are advanced, rapid and accurate, and the prepared angiogenesis inhibitory factor has significant activity.

Description of drawings

Fig. 1 is the RP-HPLC fingerprint of the enzymatic hydrolyzate active component (A) and the cell membrane adsorption residue (B) of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Example:

A preparation method of red ray cartilage polypeptide angiogenesis inhibitory factor, the preparation process is as follows: red ray cartilage-tissue fragmentation-guanidine hydrochloride extraction-ultrafiltration-cell membrane stationary phase adsorption-HPLC fingerprint comparative analysis-angiogenesis inhibitory factor.

1) Preparation of cell membrane suspension: Take Bel-7402 liver cancer cells with high expression of vascular endothelial growth factor specific receptor (VEGFR) and in logarithmic growth phase, and use MEM medium (containing 10% fetal bovine serum), Incubate in a 37°C, 5% CO ₂ incubator, when the cell count reaches 10 ⁷ , digest the cells with trypsin, centrifuge at 4°C, 1000 r/min for 10 min, take the precipitate and wash it twice with normal saline, add The cells were disrupted by sonication in 50 mM Tris-HCl solution for 30 min. Centrifuge the suspension at 1000 r/min and 4 °C for 5 min, take the supernatant and centrifuge it at 12000 r/min and 4 °C for 25 min to obtain cell membrane pellets, resuspend with normal saline, wash 3 times, add normal saline to The membrane protein content is 2.2mg/mL, which is the cell membrane suspension;

2) Preparation of cell membrane stationary phase: Take the pretreated macroporous spherical silica gel and add it to a low temperature (4°C) reaction tube. Under shaking conditions, add the cell membrane suspension to the reaction tube according to the solid-to-liquid ratio of 1 g: 25 mL, and adsorb it. After 15h, ultrasonic grinding for 25min, centrifugation at 4000r/min for 15min, the precipitate was collected, washed three times with normal saline, and the unbound cell membrane was removed to obtain Bel-7402 cell membrane stationary phase.

3) Preparation and enzymatic hydrolysis of red ray cartilage protein: add the broken and homogenized red ray cartilage into 1.0 mol/L guanidine hydrochloride solution at a solid-to-liquid ratio of 1 g: 15 mL, and extract at 4 °C for 48 hours with shaking. , Centrifuge at 10000r/min for 15min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1kDa, dialyze it with double distilled water for 24h at below 4°C, and freeze-dry the dialysate to obtain red ray cartilage protein; The solid-to-liquid ratio of 1g:20mL was added to sodium barbital-hydrochloric acid buffer (pH 7.0), and neutral protease (enzyme activity ≥1.0×10 ⁵ U/g) was added according to 2% of the cartilage protein of red ray, and enzymatic hydrolysis The temperature was 60 °C, enzymatic hydrolysis was carried out for 4 h, the solution was heated to 95 °C, and kept at this temperature for 10 min, the temperature was lowered to 37 °C, the pH was adjusted to 7.8 with NaOH, and trypsin (enzyme activity) was added according to 2% of the cartilage protein of red ray. ≥1.9×10 ⁴ U/g), the enzymatic hydrolysis temperature is 37°C, enzymolysis is performed for 4h, the solution is heated to 95°C, maintained at this temperature for 15min, centrifuged at 10000r/min, 4°C for 20min, and the supernatant is taken. The supernatant was subjected to ultrafiltration treatment with a 1kDa ultrafiltration membrane, and the fraction with molecular weight less than 1kDa was collected, placed in a dialysis bag with a molecular weight cutoff of 100, dialyzed with distilled water for 24 hours, and freeze-dried to obtain the active component of the enzymatic hydrolysate.

4) Preparation of red ray cartilage polypeptide angiogenesis inhibitory factor: the active component of the enzymatic hydrolyzate is made into a solution of 0.05 mg/mL with three-distilled water, and the cell membrane stationary phase is added to the active component according to the solid-to-liquid ratio of 1 g: 10 mL. In the solution, after incubation at 37°C for 2 h, centrifuge at 4000 r/min for 15 min, the supernatant was collected, the precipitate was rinsed 8 times with triple distilled water, the washing liquid and the supernatant were combined, and freeze-dried, which was the cell membrane adsorption residue; RP-HPLC was used (the injection volume was 5-10 μL; the chromatographic column was Amethyst C18 (250 mm×4.6 mm, 5 μm); the elution conditions were that the acetonitrile concentration increased from 10% to 50% at a constant speed within 0-40 min; the flow rate was 0.4 mL/ min; UV detection wavelength is 215 nm) to establish the fingerprints of the active components of the enzymatic hydrolysate and the adsorption residues of the cell membrane, purify and prepare the difference peaks with disappearance or significant reduction in peak area, and perform the chicken embryo chorioallantoic membrane (CAM) analysis on them. The angiogenesis inhibitory effect was measured, and the red ray cartilage polypeptide angiogenesis inhibitory factor P3 was obtained (Fig. 1).

5) Structure detection: The peptide P3, which has the strongest inhibitory effect on angiogenesis in chick embryo chorioallantoic membrane (CAM), was collected and detected as a single peak. The amino acid sequence determined by protein/peptide sequence analyzer was Gly-Tyr-Ile-Cys -Pro-Gln-Trp(GYICPQW), the molecular weight detected by ESI-MS is 865.98Da.

The prepared Gly-Tyr-Ile-Cys-Pro-Gln-Trp (GYICPQW) was tested for its angiogenesis inhibitory activity in chick chorioallantoic membrane (CAM). The results showed that the red ray cartilage polypeptide angiogenesis inhibitor GYICPQW significantly inhibited the angiogenesis of chick chorioallantoic membrane (CAM) in a dose-dependent manner (Table 1).

Table 1 Inhibitory effect of red ray cartilage polypeptide angiogenesis inhibitor on the angiogenesis of chick embryo chorioallantoic membrane (CAM)

The obtained red ray cartilage polypeptide angiogenesis inhibitor GYICPQW was used to determine the expression of the pro-angiogenesis factor. The measurement method was referred to "Experimental study on the inhibition of Lewis lung cancer angiogenesis by scorpion venom polypeptide extract combined with chemotherapy" by Sun Xiaojia, Zhang Yueying, Jia Qing, etc. (Recorded in "Chinese Journal of Traditional Chinese Medicine", 2011 No. 12 (Vol. 36): pp. 1644-1649). The red stingray angiogenesis inhibitor GYICPQW (20.0 mg/kg·d×14) was intraperitoneally injected into Lewis lung cancer-bearing mice, and the lung cancer tissues were taken for immunohistochemical examination. The expression of three pro-angiogenic factors including cell growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was significantly inhibited (Table 2).

Table 2 Inhibitory effects of red ray cartilage polypeptide angiogenesis inhibitory factors on the expression of pro-angiogenic factors

Although the present invention has been described in conjunction with preferred embodiments, it is not intended to limit the present invention, and any person skilled in the art, without departing from the spirit and scope of the present invention, can implement various aspects of the subject matter set forth herein. Therefore, the protection scope of the present invention should be determined by the scope defined by the appended claims.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> A red ray cartilage polypeptide angiogenesis inhibitor

<130> zjou-2015-wb0501

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Synthetic

<400> 1

Gly Tyr Ile Cys Pro Gln Trp

1 5

Claims (1)

1. The dasyatis akajei cartilage polypeptide angiogenesis inhibiting factor is characterized in that the amino acid sequence of the dasyatis akajei cartilage polypeptide angiogenesis inhibiting factor is Gly-Tyr-Ile-Cys-Pro-Gln-Trp, and the molecular weight of ESI-MS detection is 865.98 Da.

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