Recombinant bovine interferon coated sustained-release microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a recombinant bovine interferon coated sustained release microsphere and a preparation method and application thereof, an ultrasonic re-emulsification method is used for replacing bare-handed re-emulsification, 2.5% of trehalose is added into an internal water phase W1, and the protein concentration of the internal water phase W1 is reduced by 30 mg / ml to 25 mg / ml. According to the method provided by the invention, the encapsulation efficiency reaches 90% or above, the recovery rate reaches 95%, the drug loading rate is close to 5%, the balling rate exceeds 90%, and the in-vitro effective slow release time reaches about 10 days; through clinical supplement of the coated slow-release rbIFNT, the pregnancy rate of the receptor cattle in the initial test and the re-test is obviously improved by about 10%, and the pregnancy rate of the repeat breeding infertility young cattle in the initial test is obviously improved by about 15%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a recombinant bovine interferon coated sustained-release microsphere and a preparation method and application thereof. BACKGROUND

[0002] Improving the reproductive rate of dairy cows and accelerating the breeding speed of good breeds are the key to improving the core competitiveness of the dairy industry and the transformation and upgrading of the industry. The bovine fertilization rate can reach more than 90%, but severe embryo implantation failure is the main reason for the low pregnancy rate of many ranches below 50%. The key is that the reproductive tract and embryo development are out of sync due to insufficient uterine receptivity. Therefore, focusing on the products related to uterine receptivity at the implantation stage is an urgent need to promote the efficient application of modern breeding technology and improve the reproductive rate of cattle. The specific pregnancy recognition signal interferon tau (IFNT) secreted by the conceptus of ruminants plays a core role in mediating signal transduction between mother and fetus, prolonging the life of corpus luteum, regulating embryo development, and inducing uterine receptivity formation to ensure embryo implantation and subsequent placental development, thereby establishing pregnancy. In cattle, the expression and secretion of IFNT have a unique regularity, which starts at the morula or blastocyst stage, and peaks at 15-24 days as the conceptus extends, and the secretion decreases rapidly after 28 days. The time coincides with the maternal pregnancy recognition period, so supplementing IFNT is of great significance to improve the uterine receptivity of cows and increase the pregnancy rate.

[0003] In the prior art, large-scale preparation of high-activity soluble rbIFNT has been achieved (ZL202110622916.0, method for constructing soluble bIFNT mature peptide gene cloning strain, expression strain and induced expression of product), but based on the secretion regularity of natural IFNT, rbIFNT has a short half-life, which requires continuous administration for a long time in actual application. Not only is the operation cumbersome, but also early embryo death may occur due to improper administration, which greatly limits its promotion in clinical practice. The encapsulation efficiency, drug loading rate and sphere formation rate in the preparation process of traditional recombinant bovine interferon are low, which cannot meet the demand. SUMMARY

[0004] In order to improve the encapsulation efficiency, drug loading rate and sphere formation rate in the preparation process of recombinant bovine interferon, the present application provides a recombinant bovine interferon (rbIFNT) coated sustained-release microsphere and a preparation method and application thereof. The method provided by the present application has an encapsulation efficiency of more than 90%, a recovery rate of 95%, a drug loading rate of nearly 5%, a sphere formation rate of more than 90%, and an in vitro effective sustained-release time of about 10 days. Clinical supplementation of coated sustained-release rbIFNT significantly improves the pregnancy rate of recipients by about 10% in the initial and re-examination of embryo transfer, and significantly improves the initial pregnancy rate of repeat breeding young cattle by about 15%.

[0005] The application provides a preparation method of recombinant bovine interferon coated sustained-release microspheres, which comprises the following steps: mixing rbIFNT solution with 20-25% trehalose solution in mass fraction to obtain rbIFNT freeze-dried powder; adding the rbIFNT freeze-dried powder into 2.0-2.5% trehalose solution in mass fraction to obtain an inner water phase, with the final concentration of rbIFNT being 20-25 mg / mL; dissolving PLGA in dichloromethane to obtain a PLGA dichloromethane solution, which is used as an oil phase, with the final concentration being 40-50 mg / mL; using 1.0-1.2% polyethylene solution containing 0.8-1% NaCl in mass fraction as an outer water phase; mixing the inner water phase and the oil phase according to a volume ratio of 1:8-10 to obtain a primary emulsion through ultrasonic emulsification; mixing the primary emulsion and the outer water phase according to a volume ratio of 1:8-10 to obtain a multiple emulsion through ultrasonic emulsification; adding the multiple emulsion into the 1.0-1.2% polyethylene solution containing 0.8-1% NaCl in mass fraction drop by drop at a constant speed to perform solidification, so as to obtain a solidified microsphere emulsion; centrifuging the microsphere emulsion to collect the precipitate, resuspending the precipitate in sterile distilled water, and then vacuum freeze-drying to obtain microsphere dry powder, namely, the recombinant bovine interferon coated sustained-release microspheres.

[0006] Further, the concentration of the rbIFNT solution is 2-4 mg / mL.

[0007] Further, the freeze-drying is vacuum freeze-drying at a temperature of-80 DEG C and a pressure of 9999 mbar for 48 h.

[0008] Further, the ultrasonic emulsification condition of the mixture of the inner water phase and the oil phase is ultrasonic emulsification at a power of 325 W for 20 s, with a working time of 3 s and an intermittent time of 2 s.

[0009] Further, the ultrasonic emulsification condition of the mixture of the primary emulsion and the outer water phase is ultrasonic emulsification at a power of 390 W for 30 s, with a working time of 3 s and an intermittent time of 2 s.

[0010] Further, the centrifugation condition of the microsphere emulsion is centrifugation at 4 DEG C and 10000 rpm for 10 min.

[0011] The application further provides the recombinant bovine interferon coated sustained-release microspheres prepared by the preparation method of the recombinant bovine interferon coated sustained-release microspheres.

[0012] The application further provides application of the recombinant bovine interferon coated sustained-release microspheres in preparation of drug-loaded reagents.

[0013] The application further provides application of the recombinant bovine interferon coated sustained-release microspheres in preparation of pregnancy-promoting drugs.

[0014] Further, the drug functions are improving the pregnancy rate of infertile young cattle in initial examination and prolonging the life of non-pregnant cattle corpus luteum.

[0015] Compared with the prior art, the present application has the beneficial effects that: The present application provides a scalable, high encapsulation, high balling and high recovery rate rbIFNT coated sustained-release microsphere preparation method, which can stabilize and ensure the biological function activity release of rbIFNT for about 10 days (10±1 days). It can improve the initial detection pregnancy rate of different types of dairy cows, prolong the life of corpus luteum, increase the blood progesterone level and corpus luteum blood flow. The present application provides a low-cost freeze-drying method for effectively protecting the activity of rbIFNT raw materials and the recovery rate, so that the raw material recovery rate is more than 90%, and the activity after freeze-drying is maintained at about 80%.

[0016] The present application provides a stable, good encapsulation and high balling rbIFNT microsphere preparation method, which can improve the microsphere encapsulation, drug loading rate and balling rate by 36, 1.83 and 9 percentage points respectively. The microsphere encapsulation rate can be more than 90%, the recovery rate can be 95%, the drug loading rate can be nearly 5%, the cost is relatively low, the operation is relatively simple, the process variables are controllable, the finished product is stable, and it is suitable for large-scale production.

[0017] The rbIFNT coated microspheres prepared by the method of the present application can ensure stable long-acting sustained release (up to about 10 days) under the premise of biological activity, and the sustained release characteristics meet the secretion rules of physiological IFNT, which is beneficial to the use as a biological agent in clinical addition.

[0018] The coated sustained-release rbIFNT and its clinical application method provided by the present application can significantly improve the pregnancy rate of embryo transfer in recipient cows, high-yield high-litter and repeated infertility young artificial insemination, and improve the efficiency of cow reproduction. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 The present application provides a stable, good encapsulation and high balling rbIFNT microsphere preparation method, which can improve the microsphere encapsulation, drug loading rate and balling rate by 36, 1.83 and 9 percentage points respectively. The microsphere encapsulation rate can be more than 90%, the recovery rate can be 95%, the drug loading rate can be nearly 5%, the cost is relatively low, the operation is relatively simple, the process variables are controllable, the finished product is stable, and it is suitable for large-scale production.

[0021] Figure 2 The present application provides a stable, good encapsulation and high balling rbIFNT microsphere preparation method, which can improve the microsphere encapsulation, drug loading rate and balling rate by 36, 1.83 and 9 percentage points respectively. The microsphere encapsulation rate can be more than 90%, the recovery rate can be 95%, the drug loading rate can be nearly 5%, the cost is relatively low, the operation is relatively simple, the process variables are controllable, the finished product is stable, and it is suitable for large-scale production.

[0022] Figure 3Changes of endometrial receptivity key genes ISG15 (A), MX1 (B), MX2 (C), IFI6 (D) and OAS2 (E) caused by the rbIFNT lyophilized redissolution and non-lyophilized of the application in bEECs.

[0023] Figure 4 Preparation flowchart of the coated sustained-release rbIFNT microspheres of the application.

[0024] Figure 5 Finished product diagram of the coated sustained-release rbIFNT microspheres of the application; wherein, the left diagram is the finished product diagram of the coated sustained-release rbIFNT microspheres prepared by the method of Comparative Example 1, and the right diagram is the finished product diagram of the coated sustained-release rbIFNT microspheres (rbIFNT coated sustained-release microspheres) prepared by the method of Example 1 of the application.

[0025] Figure 6 Average particle size distribution diagram of the coated sustained-release rbIFNT microspheres of the application.

[0026] Figure 7 Scanning electron microscope diagram of the coated sustained-release rbIFNT microspheres of the application; wherein, the left diagram is the scanning electron microscope diagram of the coated sustained-release rbIFNT microspheres prepared by the method of Comparative Example 1, and the right diagram is the scanning electron microscope diagram of the coated sustained-release rbIFNT microspheres prepared by the method of Example 1 of the application.

[0027] Figure 8 Cumulative drug release diagram of the coated sustained-release rbIFNT microspheres of the application in bovine simulated uterine fluid in vitro.

[0028] Figure 9 Changes of endometrial receptivity key genes ISG15 (A), MX1 (B), MX2 (C), IFI6 (D) and OAS2 (E) caused by the coated sustained-release rbIFNT microspheres of the application in bovine simulated uterine fluid for 1 day, 4 days and 10 days.

[0029] Figure 10 Schematic diagram of the coated sustained-release rbIFNT ET and embryo "five-stage method" vascularization of the application.

[0030] Figure 11 Disposable uterine perfusion device diagram of the coated sustained-release rbIFNT microspheres of the application for artificial insemination of dairy cows; wherein, the left diagram is respectively an embryo transfer gun, a sterile vinyl catheter and a sterile 5 mL syringe, and the right diagram is a disposable uterine perfusion device prepared from the devices of the left diagram.

[0031] Figure 12 Changes of serum progesterone levels of the bovine on the 7th day, 16th day and 21st day with and without the coated sustained-release rbIFNT microspheres prepared by the application.

[0032] Figure 13 Figure A is the corpus luteum blood flow of a cow on the 16th day after application of the rbIFNT coated sustained-release microspheres prepared by the present application; Figure A is the corpus luteum blood flow of a cow on the 16th day after application of the rbIFNT coated sustained-release microspheres prepared by the present application; Figure B is the corpus luteum blood flow of a cow on the 16th day after application of the rbIFNT coated sustained-release microspheres prepared by the present application. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0034] The present application is directed to the situation that the pregnancy rate is less than 50% due to embryo implantation failure in early pregnancy of dairy cows and the half-life of recombinant bovine INF-t is short. A recombinant bovine interferon coated sustained-release microsphere is developed. Compared with the technology of Comparative Example 1, the ultrasonic re-emulsification method is replaced by manual re-emulsification, 2.5% trehalose is added to the inner water phase W1, and the protein concentration of the inner water phase W1 is reduced from 30 mg / ml to 25 mg / ml. The percentage points of the microsphere encapsulation rate, drug loading rate, and sphere formation rate of the present application are increased by 36, 1.83, and 9, respectively, compared with the method of Comparative Example 1. The encapsulation rate is more than 90%, the recovery rate is 95%, the drug loading rate is nearly 5%, the sphere formation rate is more than 90%, the in vitro effective sustained-release time is about 10 days, the clinical supplement of coated sustained-release rbIFNT significantly improves the pregnancy rate of the recipient cow embryo transplantation by about 10% in the initial examination and re-examination, significantly improves the initial pregnancy rate of the young cow with repeated failure by about 15%, significantly prolongs the corpus luteum life of non-pregnant cows, and improves the progesterone level on the 16th and 21st days after estrus and the corpus luteum blood flow on the 16th day. A low-cost freeze-drying method of raw materials of recombinant bovine interferon (rbIFNT) is provided, the recovery rate of raw materials is more than 90%, and the activity after freeze-drying is maintained at about 80%.

[0035] Example 1: A preparation method of recombinant bovine interferon (rbIFNT) coated sustained-release microspheres.

[0036] I. Experimental methods 1. Freeze-drying of recombinant bovine interferon (rbIFNT) (1) Obtaining of soluble rbIFNT Referring to the purified soluble rbIFNT in ZL202110622916.0, the steps are mainly as follows: (1) construction of soluble rbIFNT gene cloning strain; (2) expression and purification of soluble rbIFNT, for details, see ZL202110622916.0. A total of 200 mg of soluble rbIFNT was prepared in each batch, wherein 200 mg of soluble rbIFNT was dissolved in 400 ml of PBS to obtain a rbIFNT solution with a concentration of 2 mg / ml.

[0037] (2) Freeze-drying of rbIFNT solution Trehalose as a freeze-drying protective agent for soluble rbIFNT, sterile trehalose solution was prepared: 3 g of trehalose was accurately weighed into 12 mL of sterile distilled water, and microwaved for 1 min to dissolve into a 25% trehalose solution by mass fraction, denoted as 25% trehalose solution, and filtered under sterile conditions using a sterilized 0.45 μm filter membrane for standby use.

[0038] A 50 mL centrifuge tube was weighed in triplicate, and the average weight was taken. 24 mL of rbIFNT solution with a concentration of 2 mg / ml was added, and 1 mL of 25% trehalose solution was added to prepare a 25 mL mixture containing 1% trehalose, denoted as rbIFNT+trehalose mixed solution.

[0039] The rbIFNT+trehalose mixed solution was placed in a freeze-dried bottle with a weight taken in advance, and was pre-cooled in a -80°C refrigerator for 8 h. Before freezing, a sealing film was wound around the bottle mouth and a sterile 0.3 mm needle was used to pierce a dense and uniform small hole. The pre-cooled vacuum freeze-drier was used for 1 h, and dry ice was used to transport the pre-cooled rbIFNT+trehalose mixed solution. The bottle mouth was opened and placed on the freeze-drying tray under the conditions of a temperature of -80°C and a pressure of 9999 mbar. Vacuum freeze-drying was carried out for 48 h to obtain rbIFNT freeze-dried powder.

[0040] (3) The obtained rbIFNT freeze-dried powder was observed for reconstitution, biological activity determination and function identification of pregnancy recognition signal factor. The specific methods and results are as follows:

[0041] Reconstitution observation: After freeze-drying, the freeze-dried bottle was taken out, and the 50 mL centrifuge tube containing the rbIFNT freeze-dried powder was weighed three times, and the average weight was taken. The empty tube weight was subtracted from the sum of the weights of the raw material drug and trehalose to calculate the recovery rate, which was more than 90% after freeze-drying. A certain amount of rbIFNT freeze-dried powder was placed in a 60 mm dish, and its appearance was observed. The rbIFNT freeze-dried powder was light yellow, flocculent crystalline, and had a light texture without clumping. Another 25 mg of rbIFNT freeze-dried powder was taken and 1 mL of sterile distilled water was added to observe its solubility. The rbIFNT freeze-dried powder was quickly dissolved without precipitation, and the color was clear and transparent. The results are shown in Table 1 andFigure 1 as shown.

[0042] Biological activity determination of rbIFNT freeze-dried powder: According to the second method (reporter gene method) of "3523. Interferon biological activity determination" in the 2015 Chinese Pharmacopoeia, the purchased commercial HEK293 cells containing ISRE-firefly luciferase and mCherry protein were resuscitated and cultured in DMEM high-sugar medium containing 2 μg / ml puromycin at 37°C, 5% CO2 for 30 h for passage. After the passage cells were completely adherent, they were trypsinized for 2 min, and the digestion was terminated with the above-mentioned medium and centrifuged at 1000 rpm for 5 min. The precipitate was resuspended in DMEM high-sugar medium to prepare a cell suspension of 4.5 x 10 5 6 IU / mg, the activity of rbIFNT freeze-dried and redissolved was 2.87 x 10 6 IU / mg, and there was no significant difference between the two, indicating that freeze-drying had no significant effect on the activity of rbIFNT.

[0043] After treatment, the supernatant was discarded, 100 μL of purchased commercial firefly luciferase reagent cell lysis solution was added, and it was blown and mixed uniformly. Then the above-mentioned cell lysis solution was transferred to a 96-well white plate, 80 μL per well, and placed on the sample plate of the enzyme marker. The fluorescence intensity detection conditions were set as excitation wavelength 587 nm and emission wavelength 610 nm to detect the fluorescence intensity of mCherry protein. The uniformity of the fluorescence intensity of the cells in each well was investigated, and the results are shown in Table 3. It can be seen that the fluorescence intensity of mCherry protein of each sample is close, indicating that the cells are uniformly laid.

[0044] The above measured samples were added with 100 μL of commercial firefly luciferase substrate reagent, blown and mixed uniformly for 2 s, and the multifunctional enzyme marker was set to luminescence detection mode. Each well was detected for 2 s with an interval of 1 s, and the chemiluminescence intensity of each well was detected. According to the standard concentration, the interferon activity-fluorescence intensity curve is shown in Figure 2 , and the activity of non-freeze-dried rbIFNT was calculated to be 3.10 x 10 6 IU / mg, and the activity of rbIFNT freeze-dried and redissolved was 2.87 x 10 6 IU / mg, and there was no significant difference between the two, indicating that freeze-drying had no significant effect on the activity of rbIFNT.

[0045] Pregnancy recognition signal factor function identification of rbIFNT freeze-dried powder: ① Preparation and treatment of bEECs The immortalized bEECs were routinely thawed and recovered, and when the confluence of the adherent cells reached 80% under an inverted microscope, the cells were washed and trypsin was added for digestion. When the cells on the dish bottom were spherical or short spindle-shaped, the digestion was terminated by adding DME / F-12 culture medium containing 10% fetal bovine serum, and a single-cell suspension was formed by gentle blowing. After counting, it was used for subsequent experiments. After reseeding and culturing, when the confluence of the adherent cells reached 80%, the DME / F-12 culture medium containing 10% fetal bovine serum was replaced, 20 ng / mL of lyophilized and resolubilized rbIFNT, non-lyophilized rbIFNT, and commercial eukaryotic expression recombinant sheep IFNT (Shengong Bioengineering Co., Ltd., item number C600063) were added to the culture medium, respectively, and a control group was set without adding any rbIFNT. After 24 h of culture at 37°C in a cell culture incubator containing 5% CO2, the experiment was repeated three times.

[0046] ② Extraction of total RNA from bEECs The total RNA of the treated bEECs was extracted using the Trizol method. Specifically, the supernatant of the treated cells was discarded, 1 mL of Trizol was added to each well of the culture dish, and the cells were repeatedly blown and collected into a 1.5 mL RNase-free centrifuge tube. 200 μL of chloroform was added to the centrifuge tube, which was quickly shaken up and down for 15 s, and then placed at room temperature for 10 min. The centrifuge was placed in a pre-cooled 4°C centrifuge, and centrifuged at 12000 rpm for 15 min. After centrifugation, the water supernatant was carefully transferred to an empty RNase-free centrifuge tube, and an equal volume of isopropanol was added. After rotating and mixing, it was placed at room temperature for 10 min, and then centrifuged at 4°C and 12000 rpm for 15 min. The supernatant was discarded, 1 mL of freshly prepared 75% ethanol was added to each tube, and the tube was vortexed to suspend the RNA precipitate. The tube was centrifuged at 4°C and 7500 rpm for 10 min, and the supernatant was discarded. The tube was air-dried at room temperature for 5 min, and when the precipitate was observed to be translucent, 30 μL of DEPC water was added according to the amount of precipitate, and the RNA was fully dissolved by repeatedly blowing and mixing with a pipette.

[0047] ③ Reverse transcription of RNA The concentration of the extracted RNA was measured using a spectrophotometer, and a commercial kit (Evolvence Bioengineering Co., Ltd. EVO M-MLV Reverse Transcription Kit II, item number AG11711) was used for reverse transcription to cDNA. RNA can also be reverse transcribed by other methods or products. The reverse transcription reaction system and reaction conditions are shown in Table 4.

[0048] ④ Design of fluorescent quantitative PCR primers According to the gene sequences of bovine ISG15, IFI6, MX1, MX2 and OAS2 and the internal reference gene GAPDH registered in GeneBank, the fluorescence quantitative PCR amplification primers were designed as shown in Table 5 below.

[0049] ⑤Fluorescence quantitative PCR reaction The concentration of the cDNA obtained by reverse transcription was detected using NanoDrop 2000, and the concentration of each sample was adjusted to a uniform concentration for subsequent experiments. The amplification reaction was performed using a commercial kit (Nanjing Novozyme Biological Company SYBR q PCR MasterMix kit, item number Q311-02). The reaction conditions and reaction system are shown in Table 4. GAPDH was used as an internal reference, and the 2-ΔΔt method was used for calculation. The statistical analysis of the expression changes of ISG15, IFI6, MX1, MX2 and OAS2 genes showed that the freeze-dried rbIFNT could cause significant up-regulation of the above genes, and there was no significant difference in the up-regulation fold compared with the non-freeze-dried rbIFNT (such as Figure 3 ).

[0050] 2. Preparation of recombinant bovine interferon (rbIFNT) coated sustained-release microspheres (1) Preparation of recombinant bovine interferon (rbIFNT) coated sustained-release microspheres in Comparative Example 1 ①Configuration of internal water phase W1: weigh the freeze-dried powder containing 30 mg of rbIFNT, dissolve in 1 ml of sterile distilled water, and blow uniformly to prepare a 30 mg / mL rbIFNT solution as the internal water phase W1.

[0051] Configuration of oil phase O liquid: take 5 mL of dichloromethane into a 10 mL beaker, accurately weigh 150 mg of PLGA, and put it into an ultrasonic water bath at 4°C for 30 min until it is completely dissolved. Filter with a sterilized 0.45 μm filter membrane to prepare a 30 mg / ml PLGA dichloromethane solution as the oil phase O liquid.

[0052] Configuration of polyvinyl alcohol aqueous solution: accurately weigh 6 g of emulsifier polyvinyl alcohol powder and 3 g of NaCl into 300 mL of sterile distilled water, heat at 90°C for 15 min until completely dissolved, and put into a 4°C refrigerator until completely cooled. Prepare a 2% polyvinyl alcohol solution containing 1% NaCl. Take 18 mL of 2% polyvinyl alcohol solution containing 1% NaCl into a 50 mL beaker as the external water phase W2, and take 200 mL of 2% polyvinyl alcohol solution containing 1% NaCl into a 400 mL beaker as the solidification.

[0053] ②Preparation of W1O primary emulsion: Take the freeze-dried rbIFNT and configure it as a 30mg / mL rbIFNT aqueous solution. Take 0.5mL of the 30mg / mL rbIFNT aqueous solution into 5mL of a dichloromethane solution containing 30mg / mL of PLGA. Under the condition of ice water bath, ultrasonic at a power of 50% (325W) for 20s (work for 3s, intermittent for 2s) to prepare W1O primary emulsion.

[0054] ③Preparation of W1O W2 multiple emulsion: manually add 5.5mL of W1O primary emulsion into 18ml of 2% polyvinyl alcohol aqueous solution containing 1% NaCl to prepare W1O W2 multiple emulsion.

[0055] ④Microsphere solidification: 23.5mL of W1OW2 multiple emulsion is extracted with a 20mL disposable syringe and added dropwise at a uniform speed (3mL / min) into 200mL of 2% polyvinyl alcohol aqueous solution. The dropping position is kept 5cm away from the liquid level and the falling point is located at the edge of the central vortex. Solidify for 2h under the condition of 500rpm / min magnetic stirring and volatilize the remaining dichloromethane to obtain solidified microsphere emulsion.

[0056] ⑤Microsphere collection: The solidified microsphere emulsion is divided into 50mL centrifuge tubes and centrifuged at 4℃ 10000rpm for 10min. The precipitate is resuspended with 10mL of sterile distilled water. Repeat the operation for 3 times to remove the residual organic solvent and polyvinyl alcohol to obtain resuspended emulsion. Then the resuspended emulsion is vacuum freeze-dried at-80℃, 9999mbar for 48h to obtain microsphere dry powder. The freeze-dried product is shown in Figure 5 .

[0057] (2) Preparation of recombinant bovine interferon (rbIFNT) coated sustained-release microspheres ①Configuration of internal water phase W1 (add 2.5% trehalose): First, take 25mg of trehalose and place it in 1mL of sterile distilled water. Heat to dissolve into a 2.5% trehalose solution. Then take 25mg of freeze-dried rbIFNT and dissolve it in sterile distilled water containing 2.5% trehalose. Blow and mix uniformly to prepare a 25mg / mL rbIFNT solution containing 2.5% trehalose as the internal water phase W1.

[0058] The present application adjusts the viscosity of the internal water phase W1 by adding trehalose to increase the encapsulation efficiency and drug loading rate based on the low encapsulation efficiency and drug loading rate.

[0059] Preparation of oil phase O liquid: 5 mL of dichloromethane was taken in a 10 mL beaker, 250 mg of polylactic acid-glycolic acid copolymer (PLGA) was precisely weighed and put in, and was completely dissolved in an ultrasonic water bath at 4 DEG C for 30 min, and was filtered with a sterilized 0.45 mu m filter membrane to prepare a 50 mg / mL PLGA dichloromethane solution as the oil phase O liquid.

[0060] The polylactic acid-glycolic acid copolymer (PLGA) was purchased from Jilin Province Zhongke Kang Technology Co., Ltd., and the item number was 20241123.

[0061] Preparation of polyvinyl alcohol aqueous solution: 3.6 g of emulsifier polyvinyl alcohol powder and 3 g of NaCl were precisely weighed and put in 300 mL of sterile distilled water, and were completely dissolved at 90 DEG C for 15 min, and were put in a 4 DEG C refrigerator until completely cooled, to prepare a 1.2% polyvinyl alcohol solution containing 1% NaCl, 50 mL of the 1.2% polyvinyl alcohol solution containing 1% NaCl was taken in a 100 mL beaker as the external water phase W2, and 200 mL of the 1.2% polyvinyl alcohol solution containing 1% NaCl was taken in a 400 mL beaker as the solidification phase.

[0062] (2) Preparation of W1O initial emulsion: 0.5 mL of the 25 mg / mL rbIFNT internal water phase W1 solution containing 2.5% trehalose was taken into 5 mL of the 50 mg / mL PLGA oil phase O liquid, and was ultrasonically treated at 50% power (325 W) for 20 s (working for 3 s and intermittent for 2 s) in an ice water bath to prepare the W1O initial emulsion.

[0063] (3) Preparation of W1O W2 multiple emulsion: 5.5 mL of the W1O initial emulsion was added to 50 mL of the 1.2% polyvinyl alcohol solution containing 1% NaCl, and was ultrasonically treated at 60% power (390 W) for 30 s (working for 3 s and intermittent for 2 s) in an ice water bath to prepare the W1O W2 multiple emulsion.

[0064] The present application is based on low encapsulation rate and short sustained release time, and ultrasonic method is used for multiple emulsification, and the particle size, encapsulation rate, drug loading rate, sustained release time and the like of the microspheres are adjusted. Figure 6 , Figure 7 and Figure 8 .

[0065] (4)Microspheres solidification: 55.5 mL of W1O W2 double emulsion was extracted by 20 mL disposable syringe, and was added dropwise into 200 mL of 1.2% polyvinyl alcohol solution containing 1% NaCl at a uniform speed (3 mL / min) under the condition of 500 rpm magnetic stirring. The dropping position was kept 5 cm away from the liquid level, and the dropping point was located at the edge of the central vortex. The remaining dichloromethane was volatilized after 2 h of solidification, and the solidified microsphere emulsion was obtained. The preparation process of the above-mentioned recombinant bovine interferon (rbIFNT) coated sustained-release microspheres is shown in Figure 4 .

[0066] (5) Microspheres collection: The solidified microsphere emulsion was divided into 50 mL centrifuge tubes and centrifuged at 10000 rpm for 10 min at 4°C. The precipitate was resuspended with 10 mL of sterile distilled water, and the operation was repeated 3 times to remove the residual organic solvent and polyvinyl alcohol. The resuspended emulsion was then vacuum freeze-dried at -80°C and 9999 mbar for 48 h to obtain microsphere dry powder. The freeze-dried product is shown in Figure 5 .

[0067] The present application is adjusted according to the actual situation. The optimized preparation scheme is shown in Example 2, and the main changes are as follows: first, 2.5% trehalose is added to the inner water phase W1, and the protein concentration of the inner water phase W1 is reduced from 30 mg / ml to 25 mg / ml. The inner water phase W1 is adjusted to the appropriate viscosity to reduce the drug leakage, encapsulation efficiency and drug loading rate caused by the fluctuation of the viscosity of the inner water phase W1. The results are shown in Table 6; secondly, the ultrasonic double emulsification method (power 60%, time 30 s) is replaced by manual double emulsification to improve the microsphere encapsulation efficiency, drug loading rate and sustained-release condition. The results are shown in Table 6 and Figure 8 ; the PLGA concentration is increased from 30 mg / ml to 50 mg / ml to improve the encapsulation efficiency. The results are shown in Table 6; the volume ratio of W1O primary emulsion to outer water phase W2 is adjusted from 3:10 to 1:4, and the volume ratio of W1O W2 double emulsion to solidification phase is adjusted from 1:8.5 to 1:4 to reduce the width of particle size distribution. The results are shown in Figure 6 ; finally, the polyvinyl alcohol concentration is reduced from 2% to 1.2% to avoid the adhesion of microspheres caused by the high viscosity of the outer water phase W2. The results are shown in Figure 6 and Figure 7 .

[0068] 3. Parameter determination of recombinant bovine interferon (rbIFNT) coated sustained-release microspheres First, accurately weigh 50 mg each of the lyophilized microspheres prepared by the method of Comparative Example 1 and the lyophilized microspheres of this invention. Then, add 3 mL of sterile distilled water and 2 mL of dichloromethane to the microsphere powders respectively, vortex for 10 min, centrifuge at 10000 rpm for 10 min, collect 2 mL of supernatant, add 3 mL of distilled water, and repeat the operation 3 times. Finally, mix the extracted supernatants, determine the protein concentration of the supernatant using the BCA method, and calculate the microsphere encapsulation efficiency. The actual drug loading rate is calculated by weighing the lyophilized microsphere powder based on the theoretical drug loading rate and encapsulation efficiency. The sphere formation rate is calculated using the online software ImageJ. Based on the scanning electron microscope images of the microspheres, more than 200 particles are randomly selected. Particles without deformation, breakage, obvious adhesion, or relatively smooth are considered as spheres. The percentage of spheres formed is the sphere formation rate. The recovery rate is the percentage of the final weighed product compared to the sum of the added active pharmaceutical ingredient and wall material. The results are shown in Table 6 below.

[0069] The WOW double emulsification method of this invention achieves an encapsulation rate, sphericity, and recovery rate of rbIFNT microspheres exceeding 95%, with a drug loading rate approaching 5%. The percentage points of encapsulation rate, drug loading rate, and sphericity of the microspheres in this invention are increased by 36, 1.83, and 9, respectively, which is a significant improvement compared to the method of Comparative Example 1.

[0070] Characterization of rbIFNT-coated microspheres: The microsphere dry powder was reconstituted with water and then placed in a laser particle size analyzer to determine its particle size and distribution. Figure 6 The microsphere powder was subjected to electron microscopy for observation and characterization, and the sphericity was calculated. Figure 7 ).from Figure 6 As we can see, the method in Comparative Example 1 exhibits a significant multi-peak phenomenon, indicating a high degree of microsphere adhesion. Figure 7 As can be seen from the above, the present invention significantly reduces the multi-peak phenomenon and the degree of adhesion by using ultrasonic re-emulsification and adjusting the amount of polyvinyl alcohol.

[0071] 4. Evaluation of the in vitro sustained-release effect of rbIFNT-coated microspheres (1) Preparation of bovine simulated uterine fluid To more closely resemble the physiological uterine environment of cattle, a slow-release experiment was conducted using simulated uterine fluid. The simulated uterine fluid formula (NaCl, 4.970g; KCl, 0.224g; CaCl, 0.250g; NaH2PO4·2H2O, 0.072g; glucose, 0.5g) was accurately weighed and dissolved in 1000ml of sterile distilled water. The mixture was stirred thoroughly, and the pH value was measured. The pH was then adjusted to 6.8 using 1mol / L HCl.

[0072] (2) Evaluation of the sustained-release effect of rbIFNT-coated microspheres in bovine uterine simulated fluid Take 50 mg rbIFNT coated microspheres, add 1 mL of simulated uterine fluid in a 2 mL centrifuge tube, a total of 7 tubes, placed in a 38℃, 200 rpm constant temperature shaker, respectively at 12h, 1 day, 2 days, 4 days, 6 days, 8 days and 10 days sampling, centrifugation to take supernatant and add 1ml of fresh bovine simulated uterine fluid, and take the precipitate at each time point, resuspend and freeze-dry, extract rbIFNT in microspheres using the method described above, and finally the supernatant and extract are subjected to BCA protein quantification, and the cumulative release curve is drawn, the results are shown in Figure 8 , from Figure 8 It can be seen that the method of Comparative Example 1 has obvious burst release in simulated uterine fluid, about 80% of IFNT is released in 2 days, and the release time (about 4 days) is much shorter than the target release time (10 days), while the present application only releases about 50% of the total drug in 2 days, and the cumulative release time can reach about 10 days, which meets the target release time (10 days).

[0073] 5. Function identification of slow-release rbIFNT pregnancy recognition signal factor The stimulation effect of coated microsphere rbIFNT on bEECs in bovine simulated uterine fluid for 1 day, 4 days and 10 days is detected by the method described above (see Figure 9 ). It can be seen that although the stimulation up-regulation multiple of coated microsphere rbIFNT on bEECs in bovine simulated uterine fluid for 1 day, 4 days and 10 days has a decreasing trend, but until the 10th day, it can still cause significant up-regulation of such genes.

[0074] 6. Clinical application method of coated slow-release rbIFNT (recombinant bovine interferon coated slow-release microspheres) (1) Preparation of adding coated slow-release rbIFNT in bovine embryo transfer If coated slow-release rbIFNT is used in the breeding method of bovine embryo transfer, the embryo to be transferred and the coated slow-release rbIFNT are loaded into the same vessel using the "five-section method", as shown in Figure 10 , only the third section of liquid is used as embryo transfer preservation solution, and 1 embryo is sucked into it, and the other four sections of liquid are all coated slow-release rbIFNT solution with a concentration of 20 mg / mL, the concentration is calculated based on the rbIFNT raw material, the solvent is PBS, the total volume of the four sections of liquid is about 150 μL, and the total amount of rbIFNT raw material is about 2.5 mg.

[0075] (2) Preparation of adding coated slow-release rbIFNT in bovine artificial insemination If coated slow-release rbIFNT is used in the breeding method of bovine artificial insemination, first, a disposable uterine perfusion device is prepared using an embryo transfer gun, a sterile vinyl catheter and a sterile 5 mL syringe (see Figure 11) and the coated sustained-release rbIFNT was configured as a 1.25 mg / mL solution, the concentration was calculated based on the raw material of rbIFNT, and the solvent was PBS.

[0076] (3) Administration of coated sustained-release rbIFNT in bovine embryo transfer The coated sustained-release rbIFNT can be simultaneously transferred into the uterus with the embryo by using the conventional bovine embryo transfer method.

[0077] (4) Administration of coated sustained-release rbIFNT in bovine artificial insemination On the seventh day after bovine artificial insemination, the coated sustained-release rbIFNT was perfused into the luteal-side uterine horn using a disposable uterine perfusion device. The method was as follows: after the cow was fixed by a neck clamp or a six-column bar, epidural anesthesia was performed using 2% lidocaine hydrochloride, and then the accumulated feces was cleaned, the perineum and vulva were wiped and disinfected; the disposable embryo transfer gun sleeve and the embryo transfer gun were assembled and covered with a plastic protective film; one hand of the operator was inserted into the rectum to hold the cervix and uterine horn, and the other hand held the embryo transfer gun; the embryo transfer gun was inserted into the vagina, extended to the vicinity of the cervix, and the plastic protective film was pierced with the gun head; the cervix was held, and the other hand helped to pass the embryo transfer gun through the cervix and into the uterus, and the stability of the embryo transfer gun was maintained as much as possible; the cervix was pulled back, and the luteal-side uterine horn was held to guide the embryo transfer gun to be gently and slowly inserted into the uterine horn; when the gun head was located at the front 1 / 2 of the uterine horn, the relative stability of the uterine horn and the embryo transfer gun was maintained as much as possible; the embryo transfer gun was removed outside the vulva by the assistant, and only the disposable embryo transfer gun sleeve remained in the uterus; a sterile vinyl catheter was connected to the outer end of the disposable embryo transfer gun sleeve, the other end of the sterile vinyl catheter was connected to a 5 mL sterile syringe, 2 mL of coated sustained-release rbIFNT solution was pre-aspirated in the sterile 5 mL syringe, and all were stored in an ice box before uterine perfusion; the sterile syringe was used to slowly inject each liquid into the uterine horn, and the piston of the sterile syringe was pushed to the top end, but at this time there was still some liquid remaining in the vinyl catheter and the disposable embryo transfer gun sleeve; the sterile syringe was removed to aspirate air (about 2 mL), and the sterile vinyl catheter was connected again to slowly inject air to inject all the liquid into the uterine horn; the disposable embryo transfer gun sleeve was slowly removed from the uterus, and the uterus should not be massaged; the end of the disposable embryo transfer gun was checked for bleeding, shedding, bending, and liquid residue, and if no abnormalities were found, the uterine perfusion was qualified.

[0078] 7. Evaluation of the clinical application effect of coated sustained-release rbIFNT The differences in pregnancy rate, serum progesterone level, and luteal blood flow were evaluated by setting a control group to compare the application of coated sustained-release rbIFNT with the non-application of coated sustained-release rbIFNT in bovine embryo transfer, high-yield high-litter dairy cows, and artificial insemination of young cows with repeated pregnancy.

[0079] As shown in Table 7, supplementation with coated sustained-release rbIFNT significantly increased the initial and follow-up pregnancy rates of recipient cows after embryo transfer by approximately 10%; significantly increased the initial pregnancy rate of repeatedly infertile heifers by approximately 15%; and significantly prolonged the lifespan of the corpus luteum in non-pregnant cows. Although no significant differences were observed, it increased the initial and follow-up pregnancy rates of high-yielding, high-parity dairy cows by more than 10%. Overall, the application of coated sustained-release rbIFNT effectively improved the reproductive efficiency of cows undergoing embryo transfer and artificial insemination. Furthermore, it increased progesterone levels on days 16 and 21 postestrus. Figure 12 ) and luteal blood flow on day 16 ( Figure 13 This confirms that it effectively enhances luteal function.

[0080] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0081] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a recombinant bovine interferon coated sustained release microspheres, characterized by, The method comprises the following steps: mixing rbIFNT solution with 20%-25% trehalose solution by mass fraction to obtain rbIFNT freeze-dried powder; adding the rbIFNT freeze-dried powder into 2.0%-2.5% trehalose solution by mass fraction to obtain an inner water phase, wherein the final concentration of rbIFNT is 20 mg / mL-25 mg / mL; dissolving PLGA in dichloromethane to obtain a PLGA dichloromethane solution, which is used as an oil phase, and the final concentration of the PLGA is 40 mg / mL-50 mg / mL; using 1.0%-1.2% polyethylene glycol solution containing 0.8%-1% NaCl by mass fraction as an outer water phase; mixing the inner water phase and the oil phase according to a volume ratio of 1:8-10 to obtain a primary emulsion by ultrasonic emulsification; mixing the primary emulsion and the outer water phase according to a volume ratio of 1:8-10 to obtain a multiple emulsion by ultrasonic emulsification; adding the multiple emulsion into the 1.0%-1.2% polyethylene glycol solution containing 0.8%-1% NaCl by mass fraction drop by drop at a uniform speed to obtain a solidified microsphere emulsion; centrifuging the microsphere emulsion to collect the precipitate; and resuspending the precipitate in sterile distilled water and vacuum freeze-drying to obtain a microsphere dry powder, i.e., the recombinant bovine interferon coated sustained-release microsphere.

2. The method for preparing recombinant bovine interferon-coated sustained-release microspheres according to claim 1, characterized in that, The concentration of the rbIFNT solution is 2 mg / mL-4 mg / mL.

3. The method for preparing recombinant bovine interferon-coated sustained-release microspheres according to claim 1, characterized in that, The freeze-drying is vacuum freeze-drying for 48 h under the condition of a temperature of-80 ℃ and a pressure of 9999 mbar.

4. The method for preparing recombinant bovine interferon-coated sustained-release microspheres according to claim 1, characterized in that, The ultrasonic emulsification condition of the mixture of the inner water phase and the oil phase is ultrasonic emulsification for 20 s, working for 3 s and intermittently for 2 s under a power of 325 W.

5. The method for preparing recombinant bovine interferon-coated sustained-release microspheres according to claim 1, characterized in that, The ultrasonic emulsification condition of the mixture of the primary emulsion and the outer water phase is ultrasonic emulsification for 30 s, working for 3 s and intermittently for 2 s under a power of 390 W.

6. The method for preparing recombinant bovine interferon-coated sustained-release microspheres according to claim 1, characterized in that, The centrifugation condition of the microsphere emulsion is centrifugation for 10 min under the condition of 4 ℃ and 10,000 rpm.

7. The recombinant bovine interferon coated sustained-release microsphere prepared by the method of any one of claims 1-6.

8. The use of the recombinant bovine interferon coated sustained-release microsphere of claim 7 in the preparation of a drug carrier.

9. The use of the recombinant bovine interferon coated sustained-release microsphere of claim 7 in the preparation of a pregnancy promoting drug.

10. Use according to claim 9, characterized in that, The function of the drug is to improve the pregnancy rate of infertility young cattle in the initial examination and prolong the life of non-pregnant cattle corpus luteum.

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