CN114736848B - Application of long-beard nereis polypeptide in chicken egg vacuole granular cell proliferation and progesterone secretion - Google Patents
Application of long-beard nereis polypeptide in chicken egg vacuole granular cell proliferation and progesterone secretion Download PDFInfo
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Abstract
The invention provides application of long-beard clam worm polypeptide in chicken egg vacuole granular cell proliferation and progesterone secretion, and belongs to the technical field of animal husbandry feeding. Experiments show that the long-beard clam worm polypeptide can effectively promote the proliferation of chicken egg-like granular cells and can effectively promote the protein expression of proliferation-related proteins Cyclin-D1 and CDK-1. Meanwhile, the long-beard clam worm polypeptide can also effectively promote chicken follicle granular cells to secrete progesterone.
Description
Technical Field
The invention belongs to the technical field of animal husbandry and breeding, and particularly relates to application of long beard nereis polypeptide in chicken follicle granular cell proliferation and progesterone secretion.
Background
With the gradual improvement of the living standard of people, the demand of China for poultry meat and eggs is more and more large. Eggs are a low-price and high-efficiency animal protein source, and are deeply popular with consumers due to the advantages of rich nutrition, amino acid proportion suitable for the needs of human bodies and easy absorption. Meanwhile, eggs are eggs laid by chickens biologically and are important carriers for breeding the next generation. Therefore, the egg-laying capacity of hens has an important influence on both laying and broiler production.
The egg-laying performance of chickens is influenced by a variety of properties, wherein the development of follicles in the chicken ovaries plays a decisive role in the high and low laying rate of the chickens. Thus promoting the normal development of the follicles will contribute to increasing the egg-laying capacity of the chickens. Meanwhile, steroid synthesis also has important influence on the development of follicles, and steroids can effectively promote ovarian development.
The Nereid gruffs is one of Annelida and Nereidae, and is mainly distributed in Bohai sea, yellow sea and east sea of China. At present, the research on the long-hair nereis polypeptide is not available, and the application of the long-hair nereis polypeptide in chicken follicle development and progesterone secretion is unknown, so that the preparation method disclosed by the invention is used for researching the application of the long-hair nereis polypeptide in promoting chicken follicle granular cell proliferation and progesterone secretion.
Disclosure of Invention
The invention aims to provide application of long-hair nereis polypeptide in promoting chicken egg vacuole granular cell proliferation and progesterone secretion.
In order to realize the purpose, the invention provides the application of the long beard clam worm polypeptide in preparing the biological preparation for promoting the proliferation of the granular cells of the chicken egg.
Secondly, the invention provides the application of the long-beard clam worm polypeptide in preparing a biological preparation for promoting the secretion of progesterone from chicken egg granular cells.
Preferably, the preparation method of the long beard clam worm polypeptide comprises the following steps:
(1) Cleaning dried Nereis longibracteata, grinding into powder, adding 5 times of water, and stirring to obtain homogenate;
(2) Adding 0.05 times of trypsin into the clamworm homogenate, adjusting the pH value to be 8, and carrying out enzymolysis at 37 ℃ for 4 hours to obtain clamworm enzymolysis liquid;
(3) Adjusting the temperature of the Nereis longipalpis enzymatic hydrolysate to 100 ℃, and heating at constant temperature for 20min for inactivation to obtain the Nereis longipalpis enzymatic hydrolysate;
(4) Putting the clamworm enzymatic hydrolysate into a centrifuge, and centrifuging at 12000rpm for 15min to obtain clamworm supernatant;
(5) Filtering the clamworm supernatant by using a nanofiltration membrane of 2nm to obtain a clamworm polypeptide extract;
(6) And (3) freeze-drying the nereis longipalpis polypeptide extracting solution to obtain the nereis longipalpis polypeptide.
The invention has the beneficial effects that:
1. the invention prepares the long-hair nereis polypeptide and finds that the long-hair nereis polypeptide can effectively promote the proliferation of follicular granulosa cells and can effectively promote the protein level expression of proliferation-related proteins Cyclin-D1 and CDK-1;
2. the invention discovers that the long-hair nereis polypeptide prepared by the invention can effectively promote the follicular granular cells to secrete progesterone and can effectively promote the progesterone to secrete mRNA level expression of related genes STAR, CYP19A1 and CYP11A 1.
Drawings
FIG. 1 effect of Nereis longissima polypeptides at different concentrations on proliferation of chicken oocytes;
FIG. 2 shows the results of the effect of the Nereis longissimus polypeptides on the proliferation of chicken oocyst granulosa cells detected by Edu;
FIG. 3 is a graph showing the effect of a Nereis longissimus polypeptide on the expression of the proliferation-associated proteins Cyclin-D1 and CDK-1 in chicken oocyst granulosa cells;
FIG. 4 Effect of Nereis longipalpis polypeptide on progesterone secretion from chicken oocyst granulosa cells;
FIG. 5 Effect of Nereis longipalpis polypeptide on mRNA expression of the chicken oocyst progesterone secretion-associated genes STAR, CYP19A1, CYP11A 1.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
Preparation of Nereis longibracteata polypeptide
(1) Cleaning dried Nereid longipalpis, grinding into powder, adding 5 times of water, and stirring to obtain Nereid longipalpis homogenate;
(2) Adding 0.05 times of trypsin into the clamworm homogenate, adjusting the pH value to 8, and carrying out enzymolysis at 37 ℃ for 4h to obtain clamworm enzymolysis liquid;
(3) Adjusting the temperature of the Nereis longipalpis enzymatic hydrolysate to 100 ℃, and heating at constant temperature for 20min for inactivation to obtain the Nereis longipalpis enzymatic hydrolysate;
(4) Putting the clamworm enzymatic hydrolysate into a centrifuge, and centrifuging at 12000rpm for 15min to obtain clamworm supernatant;
(5) Filtering the clamworm supernatant by using a nanofiltration membrane of 2nm to obtain a clamworm polypeptide extract;
(6) And (3) freeze-drying the nereis longipalpis polypeptide extracting solution to obtain the nereis longipalpis polypeptide.
Example 2
(1) After the hens in the egg laying peak period die, taking out the whole ovary and placing the whole ovary in precooled sterilized PBS;
(2) Taking out the yellow follicle from the ovary, washing the yellow follicle by using PBS, cutting the yellow follicle by using a sterile scalpel, releasing yolk, and rinsing by using PBS again;
(3) After the granular cell layer is carefully separated, transferring the granular cell layer into a 50mL centrifuge tube, standing for 5min, and then removing the supernatant;
(4) Adding 5mL of preheated 0.2% collagenase II, digesting at 37 ℃ for 10min, adding 5mL of 4 ℃ precooled M199 culture medium to stop the digestion reaction after the tissue blocks are digested, and obtaining cell suspension;
(5) Filtering the cell suspension by using a 70-micron cell filter screen, and centrifuging for 8min at 1000 r/min;
(6) Discarding the supernatant, adding M199 culture medium, blowing and beating the precipitate uniformly, and centrifuging in a centrifuge again at 1000r/min for 8min;
(7) Adding another 5mL of M199 culture medium to suspend and precipitate, inoculating the cells into a cell culture dish, and placing the cell culture dish in 5% CO 2 Culturing at 37 deg.C in incubator;
(8) After 24h of culture, the culture medium is changed for the first time, and then the culture medium is changed every 2-3d by observation every day.
Example 3
Effect of Long beard Nereid Polypeptides on proliferation of chick oocyst granulosa cells
(1) Inoculating chicken egg granular cells into a 96-well cell culture plate, wherein each well contains 3000 cells and 100 mu L;
(2) The 96-well cell culture plate is placed in a cell culture box, and after 24 hours, the plate is replaced by M199 culture medium containing 0. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL, 120. Mu.g/mL Nereis brevicauna polypeptide, 37 ℃ and 5% CO 2 Culturing for 48h under the condition;
(3) After the culture was completed, 10. Mu.L of CCK-8 was added to each well, and after further culturing for 2 hours, the OD value at a wavelength of 450nm was measured on a microplate reader, and 5 replicates for each treatment were set.
Example 4
Edu experiment for further detecting the influence of the long-hair clamworm polypeptide on the proliferation of chicken egg granular cells
(1) Inoculating chicken egg granular cells into a 96-well cell culture plate, wherein 3000 cells are per well, and the volume is 100 mu L;
(2) Placing 96-well cell culture plate in cell culture box, 24 hr later, replacing with M199 culture medium containing 0 μ g/mL,80 μ g/mL long beard clam worm polypeptide, 37 deg.C, 5% CO 2 Culturing for 48h under the condition;
(3) After the culture is finished, adding 100 mu L of Edu to incubate the cells for 2h, removing the cells after the incubation is finished, and washing the cells for 2 times by using PBS;
(4) Adding 4% paraformaldehyde to fix cells for 30min, removing, adding 100 μ L0.2% glycine, incubating for 5min, and washing cells with PBS for 2 times;
(5) Cells were treated with 100. Mu.L of 0.5% Triton-100 for 10min and washed with PBS
(6) Adding 100 μ L Apollo to treat cells for 10min, washing the cells with PBS, and adding 100 μ L0.5% Triton-100 to treat the cells for 10min;
(7) After the treatment was completed, 100 μ L of methanol was added to wash 2 times, and then the cells were washed 2 times with PBS;
(8) 100 μ L of DAPI stain was added for 5min, washed with PBS, and photographed under a fluorescent microscope.
Example 5
Influence of long-hair nereis polypeptide on expression of chicken egg vacuole granular cell proliferation related protein Cyclin-D1 and CDK-1 protein
(1) Inoculating chicken egg granular cells into 6-well culture plate, and changing to M199 culture medium containing 0 μ g/mL,80 μ g/mL long beard clam worm polypeptide when cell density reaches 70%, 37 deg.C, and 5% CO 2 Culturing for 48h under the condition;
(2) After the culture is finished, washing the cells by using PBS, adding 100 mu L of RIPA cell lysate into each hole, scraping the cells by using a cell scraper, and transferring the cell lysate into a centrifuge tube by using a pipette gun;
(3) After the centrifugal tube is placed on ice for cracking for 30min, the centrifugal tube is placed in a centrifugal machine, the centrifugal tube is centrifuged for 10min at the temperature of 4 ℃ and the rpm of 12000, and after centrifugation, the supernatant is transferred to a new centrifugal tube;
(4) Sucking 2 mu L of supernatant, performing protein quantitative detection by using a BCA method, adding 5 Xupstream buffer solution, and boiling for 5min to obtain a protein sample;
(5) Preparing 5% of upper layer glue and 12% of lower layer glue, installing an electrophoresis tank, adding 10 mu L of protein sample and Marker into each hole, and adding electrophoresis liquid to start electrophoresis;
(6) After electrophoresis is finished, assembling an electric transfer clamp, transferring a 250mA film for 1.5h, after the electric transfer is finished, transferring the PVDF film into 5% of skimmed milk powder, and sealing the PVDF film for 1h by a shaking table at room temperature;
(7) After the end of blocking, membranes were washed with TBST, primary antibodies to Cyclin-D1 (1, 2000), CDK-1 (1, 2000), β -actin (1, 10000) were incubated, blocked for 1h at 4 ℃;
(8) After washing the membrane with TBST, incubating corresponding secondary antibody, and incubating on a shaking table for 1h at room temperature;
(9) In the dark, the development exposure was performed with reference to the kit instructions.
Example 6
Effect of Long beard Nereis polypeptide treatment on Progesterone secretion from Chicken oocyst granulosa cells
(1) Inoculating chicken egg granular cells into 6-well culture plate, and changing to M199 culture medium containing 0 μ g/mL,80 μ g/mL long beard clam worm polypeptide when cell density reaches 70%, 37 deg.C, and 5% CO 2 Culturing for 48h under the condition;
(2) After the culture is finished, collecting the culture medium, centrifuging for 5min at 1000r/min, and storing the culture medium at-20 ℃;
(3) And (3) detecting the content of the progesterone by referring to the specification of the progesterone ELISA detection kit.
Example 7
Effect of Long beard Nereis polypeptide treatment on the Synthesis of genes involved in Progesterone in granular cells of egg yolk
(1) Inoculating chicken egg granular cells into 6-well culture plate, and changing to M199 culture medium containing 0 μ g/mL,80 μ g/mL long beard clam worm polypeptide when cell density reaches 70%, 37 deg.C, and 5% CO 2 Culturing for 48h under the condition;
(2) After the culture is finished, adding TRIZOL to extract RNA;
(3) Reverse transcribing the extracted RNA into cDNA with reference to the TaKaRa reverse transcription kit instructions;
(4) The relative expression quantity of STAR, CYP19A1, CYP11A1, beta-actin is detected by referring to a TaKaRa fluorescent quantitative PCR kit, and the sequences of primers are as follows:
| gene | Primer sequences | Size of product |
| STAR | An upstream primer: GAGCTGGTGGACAACATGGA; SEQ ID NO.1, downstream primer: CGTGGGTGATCAGAGTGTCC; SEQ ID NO.2; | 100bp |
| CYP19A1 | an upstream primer: CGATTTGGGAGCAAGCTTGG; SEQ ID NO.3, downstream primer: TGGCTATCATGCGCACAAGA; SEQ ID No.4; | 145bp |
| CYP11A1 | an upstream primer: GCACTTCAAGGGACTGAGCT; SEQ ID NO.5, downstream primer: ACTTGGTCCCAACTTCCACC; SEQ ID No.6; | 146bp |
| β-actin | an upstream primer: GAAGGAGATCACAGCCCTGG; SEQ ID NO.7, downstream primer: ACTCCTGCTTGCTGATCCAC; SEQ ID No.8; | 143 bp |
(5) According to 2 -∆∆Ct The relative expression amounts of STAR, CYP19A1, CYP11A1 genes were calculated.
Experimental results for example 3:
as shown in FIG. 1, OD values of 0. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 100. Mu.g/mL and 120. Mu.g/mL are 0.398. + -. 0.028,0.455. + -. 0.034,0.547. + -. 0.068,0.670. + -. 0.060,0.686. + -. 0.045,0.690. + -. 0.050, respectively; from the above results, it can be seen that when the concentration of the Nereis longissimus polypeptide is greater than 20 μ g/mL, the growth of chicken egg granular cells can be promoted, and the difference has statistical significance. Meanwhile, it can be seen that, when the concentration reaches 80. Mu.g/mL, the promotion effect reaches the highest level basically, and the promotion effect cannot be increased continuously even if the concentration is increased subsequently, so the concentration is taken as the optimum concentration in the invention.
Experimental results of example 4
As shown in fig. 2, it can be seen from the results of EDU staining that the number of stained cells in the cells treated with the long silk nereis polypeptide is significantly higher than that of the cells not treated with the polypeptide, and the results further verify that the long silk nereis polypeptide can effectively promote the proliferation of chicken egg granule cells.
Experimental results of example 5
As shown in FIG. 3, it can be seen from the results of Western Blot that the gray values of Cyclin-D1 and CDK-1 in the cells treated with the Long silk worm polypeptide are significantly higher than those in the cells not treated with the polypeptide, indicating that the Long silk worm polypeptide can effectively promote the protein expression of proliferation-related proteins Cylin-D1 and CDK-1 in chicken egg granule cells.
Experimental results of example 6
As shown in fig. 4, it can be seen from the results that the amount of progesterone secreted by the cells treated with the long beard nereis polypeptide is significantly higher than that secreted by the cells not treated with the polypeptide, and the difference has statistical significance, which indicates that the long beard nereis polypeptide can effectively promote the chicken follicle granule cells to secrete progesterone.
Experimental results of example 7
As shown in FIG. 5, in the 80. Mu.g/mL long-hair Nereid polypeptide-treated group, the relative expression amount of the STAR gene was 1.618. + -. 0.280, the relative expression amount of the CYP19A1 gene was 1.528. + -. 0.281, and the relative expression amount of the CYP111A1 gene was 2.331. + -. 0.288. From the results, the long-hair nereis polypeptide can effectively promote the mRNA expression of the chicken follicle granular cell progesterone secretion related gene STAR, CYP19A1 and CYP11A 1.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
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Claims (2)
1. The application of the long-beard clam worm polypeptide in preparing the biological preparation for promoting the proliferation of the granular cells of the chicken egg vacuoles is characterized in that the preparation method of the long-beard clam worm polypeptide comprises the following steps:
(1) Cleaning dried Nereid longipalpis, grinding into powder, adding 5 times of water, and stirring to obtain Nereid longipalpis homogenate;
(2) Adding 0.05 times of trypsin into the clamworm homogenate, adjusting the pH value to 8, and carrying out enzymolysis at 37 ℃ for 4h to obtain clamworm enzymolysis liquid;
(3) Adjusting the temperature of the Nereis longipalpis enzymatic hydrolysate to 100 ℃, and heating at constant temperature for 20min for inactivation to obtain the Nereis longipalpis enzymatic hydrolysate;
(4) Putting the clamworm enzymolysis solution into a centrifuge, and centrifuging at 12000rpm for 15min to obtain clamworm supernatant;
(5) Filtering the clamworm supernatant by using a nanofiltration membrane of 2nm to obtain a clamworm polypeptide extract;
(6) Freeze-drying the clam worm polypeptide extract to obtain the clam worm polypeptide;
the long-hair nereis polypeptide promotes the protein expression of proliferation-related proteins Cyclin-D1 and CDK-1.
2. The application of the long-beard clam worm polypeptide in preparing the biological preparation for promoting the secretion of the progesterone from the chicken follicle granular cells is characterized in that the preparation method of the long-beard clam worm polypeptide comprises the following steps:
(1) Cleaning dried Nereid longipalpis, grinding into powder, adding 5 times of water, and stirring to obtain Nereid longipalpis homogenate;
(2) Adding 0.05 times of trypsin into the clamworm homogenate, adjusting the pH value to be 8, and carrying out enzymolysis at 37 ℃ for 4 hours to obtain clamworm enzymolysis liquid;
(3) Adjusting the temperature of the Nereis longibracteata enzymolysis solution to 100 ℃, and heating at constant temperature for 20min for inactivation to obtain the Nereis longibracteata enzymolysis solution;
(4) Putting the clamworm enzymolysis solution into a centrifuge, and centrifuging at 12000rpm for 15min to obtain clamworm supernatant;
(5) Filtering the clamworm supernatant by using a nanofiltration membrane of 2nm to obtain a clamworm polypeptide extract;
(6) Freeze-drying the nereis longissima polypeptide extracting solution to obtain nereis longissima polypeptide;
the long-beard nereis polypeptide promotes mRNA level expression of progesterone secretion related genes STAR, CYP19A1 and CYP11A 1.
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