Cow recombinant follicle-stimulating hormone as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal breeding, and particularly discloses a cow recombinant follicle-stimulating hormone as well as a preparation method and application thereof, and the preparation method of the cow recombinant follicle-stimulating hormone comprises the following steps: constructing a recombinant plasmid pcDNA3.1-rFSH; the recombinant plasmid is used as a template, an alpha and beta-CTP target fragment is recovered through PCR amplification, the alpha and beta-CTP target fragment is subjected to homologous recombination and transformation to obtain a recombinant FSH lentiviral vector pCD513B-rFSH, then CHO-K1 cells are transfected, a monoclonal cell strain is obtained through screening, the monoclonal cell strain is cultured, cell supernatant is collected, and the recombinant FSH lentiviral vector pCD513B-rFSH is obtained. And separating and purifying to obtain the milk recombinant bovine follicle-stimulating hormone. The cow recombinant follicle-stimulating hormone prepared by the method provided by the invention has stable biological activity, and can significantly promote the proliferation of granular cells.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and particularly relates to a recombinant follicle-stimulating hormone for dairy cows, a preparation method thereof, and an application thereof. Background Art

[0002] The reproductive efficiency of excellent breeding livestock is an important factor restricting the high-quality development of ranch breeding. The popularization of technologies such as artificial insemination (AI), multiple ovulation and embryo transfer technology (MOET), ovum pick-up (OPU) and in vitro production (IVP) of embryos for ranch animals mainly including cattle and sheep has provided technical support for improving the overall level of breeding and establishing an excellent breeding livestock reproduction system in China. Along with the development and application of improved breeding technologies, the application of animal reproductive hormones has become increasingly widespread, and the commercial market has been expanding day by day.

[0003] Ovarian granulosa cells are a type of cells in the ovarian cortex. They surround the eggs and form gap junctions through the matrix between cells to "communicate" with the eggs, transport nutrients, and promote the growth, maturation, and ovulation of the eggs. In the ovary, follicle-stimulating hormone mainly acts on granulosa cells in the follicle to promote their proliferation and differentiation. Follicle-stimulating hormone promotes the development and maturation of follicles by stimulating the proliferation and differentiation of ovarian granulosa cells.

[0004] Follicle-stimulating hormone (FSH) is an essential gonadotropin in reproductive physiological activities such as follicle growth and development, granulosa cell proliferation, steroid synthesis, and sperm production, and plays a very important role in human and animal assisted reproduction. The source of natural follicle-stimulating hormone is very limited, and its promoting effect on ovarian granulosa cells is also limited. To maintain the follicle cycle of mammals, frequent administration of drugs is required, but frequent drug administration is likely to cause stress to animals. Therefore, it is necessary to prepare recombinant follicle-stimulating hormone to replace natural follicle-stimulating hormone. However, in the current preparation of recombinant follicle-stimulating hormone, generally, different subunits of follicle-stimulating hormone are artificially connected in series into linear fragments, which results in low stability and affects its biological activity. Therefore, it is necessary to provide a preparation method for mammalian recombinant follicle-stimulating hormone with good biological activity. Summary of the Invention

[0005] To develop a method for preparing recombinant mammalian follicle-stimulating hormone, the present invention provides a recombinant bovine follicle-stimulating hormone, its preparation method and application. By means of genetic engineering, the α and β-CTP target fragments are recovered; using pCD513B as a vector, the recombinant FSH lentiviral vector pCD513B-rFSH is obtained by homologous recombination and transformation of the α and β-CTP target fragments, and then by transfecting CHO-K1 cells and screening to obtain monoclonal cell lines, and then by culturing, separating and purifying to obtain recombinant bovine follicle-stimulating hormone (rFSH). The prepared recombinant bovine follicle-stimulating hormone has stable biological activity, significantly promotes the proliferation of granulosa cells, and improves the effect of superovulation.

[0006] The present invention provides a method for preparing recombinant bovine follicle-stimulating hormone. By recombining, transforming and extracting the gene fragment containing the CMV promoter and the FSHα subunit with linearized pcDNA3.1-FSHβ, the recombinant plasmid pcDNA3.1-rFSH is obtained; using the recombinant plasmid pcDNA3.1-rFSH as a template, PCR amplification is carried out with the primers pCD513B-rFSH F shown in SEQ ID NO.9 and pCD513B-rFSH R shown in SEQ ID NO.10, the α and β-CTP target fragments are recovered, using pCD513B as a vector, the recombinant FSH lentiviral vector pCD513B-rFSH is obtained by homologous recombination and transformation of the α and β-CTP target fragments, and then by transfecting CHO-K1 cells and screening to obtain monoclonal cell lines, and by culturing the monoclonal cell lines and collecting the cell supernatant, recombinant bovine follicle-stimulating hormone is separated and purified.

[0007] By means of genetic engineering, the α and β-CTP target fragments are recovered; using pCD513B as a vector, the recombinant FSH lentiviral vector pCD513B-rFSH is obtained by homologous recombination and transformation of the α and β-CTP target fragments, and then by transfecting CHO-K1 cells and screening to obtain monoclonal cell lines, and then by culturing, separating and purifying to obtain recombinant bovine follicle-stimulating hormone (rFSH). The prepared recombinant bovine follicle-stimulating hormone has stable biological activity, significantly promotes the proliferation of granulosa cells, and improves the effect of superovulation.

[0008] Furthermore, the preparation steps of linearized pcDNA3.1-FSHβ are as follows: using the FSHβ-CTP-α sequence shown in SEQ ID NO.1 as a template, and using pcDNA3.1-FSHαF / R and pcDNA3.1-FSHβF / R as primers respectively, amplify the bovine FSHα and FSHβ-CTP target gene fragments; the FSHβ-CTP target gene fragment is recombinantly ligated with the linearized pcDNA3.1 vector to obtain the recombinant plasmid pcDNA3.1-FSHβ, and linearized by enzymatic digestion to obtain linearized pcDNA3.1-FSHβ.

[0009] Furthermore, the linearized pcDNA3.1 vector is obtained by linearizing the empty vector pcDNA3.1 with the restriction endonucleases BamHⅠ and HindⅢ.

[0010] Furthermore, the reaction system for the recombinant ligation of the bovine FSHα and FSHβ-CTP target gene fragments with the linearized pcDNA3.1 vector is 20 μL: 0.87 μL - 0.89 μL of the linearized vector, 5.6 μL - 5.7 μL of the target gene fragment FSHβ-CTP, 6 μL - 6.1 μL of FSHα, 4 μL - 4.1 μL of 5×CEⅡ Buffer, 2 μL - 2.1 μL of ExnaseⅡ, and the balance is ddH 2 O.

[0011] The present invention also provides a recombinant bovine follicle-stimulating hormone, which is obtained by the above preparation method. The recombinant bovine follicle-stimulating hormone includes a β subunit with an amino acid sequence as shown in SEQ ID NO.11 and an α subunit as shown in SEQ ID NO.12;

[0012] The amino acid sequence of the β subunit encoded by FSHβ-CTP is as shown in SEQ ID NO.11:

[0013] MKSVQFCFLFCCWRAICCRSCELTNITITVEKEECGFCISINTTWCAGYCYTRDLVYRDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCSKCDSDSTDCTVRGLGPSYCSFREIKEDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQHHHHHH*. The "*" represents a stop codon.

[0014] The amino acid sequence of the α subunit encoded by FSHα is as shown in SEQ ID NO.12:

[0015] MDYYRKYAAVILAILSLFLQILHSFPDGEFTMQGCPECKLKENKYFSKPD APIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNV RVENHTECHCSTCYYHKS*. "*" represents the stop codon.

[0016] The present invention also provides a recombinant FSH lentiviral vector, which is obtained by the preparation steps of the above-mentioned recombinant FSH lentiviral vector.

[0017] The present invention also provides a monoclonal cell line, which is obtained by transfecting CHO-K1 cells with the recombinant FSH lentiviral vector.

[0018] The present invention also provides a method for constructing a monoclonal cell line, which includes the following steps: transfecting HEK293T cells with the recombinant FSH lentiviral vector, collecting virus particles and then transfecting CHO-K1 cells, screening positive cells with puromycin drug and green fluorescent protein, so as to obtain the monoclonal cell line.

[0019] Further, the process of transfecting HEK293T cells with the recombinant FSH lentiviral vector is as follows: mixing the recombinant FSH lentiviral vector pCD513B-rFSH with the helper plasmids pGag / Pol, pRev and pVSV-G according to the mass ratio of 3 - 3.2:1:1:1, adding them into the MEM medium containing transfection reagent, changing to the complete culture medium after 15h - 16h, collecting the cell supernatant after 70h - 72h, centrifuging to aspirate the supernatant, and filtering to collect virus particles.

[0020] Further, after the density of CHO-K1 cells reaches 60% - 70%, they are used for lentiviral transfection.

[0021] The present invention also provides the application of the recombinant bovine follicle-stimulating hormone, recombinant FSH lentiviral vector or monoclonal cell line as described above in the drug for promoting the proliferation of ovarian granulosa cells.

[0022] The present invention also provides the application of the recombinant bovine follicle-stimulating hormone as described above in the preparation of drugs for improving superovulation.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] In the present invention, the cDNA sequences of the FSHα and β subunits of dairy cows were cloned, combined with chorionic gonadotropin (CG) and the carboxyl-terminal peptide (CTP) of CGβ. Eukaryotic expression vectors containing restriction enzyme sites and target genes (pcDNA3.1, pEGFP-N1) were constructed and expressed in Escherichia coli (E. coli DH5α), and monoclonal cell lines (4204 strains) of dairy cow rFSH with stable expression and biological activity in cells were screened. Using the 4204 strains, recombinant follicle-stimulating hormone (rFSH) of dairy cows with relatively high purity can be obtained. The recombinant follicle-stimulating hormone of dairy cows prepared in the present invention is close to the natural follicle-stimulating hormone in structure, has stable biological activity, significantly promotes the proliferation of granulosa cells, and improves the effect of superovulation. It accelerates the process of the application of commercial rFSH for animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It is the technical route for the construction of the recombinant FSH vector.

[0027] Figure 2 It is the double enzyme digestion agarose gel electrophoresis identification of the α-CMV-β-CTP-6×His fragment of dairy cow recombinant follicle-stimulating hormone and the constructed pCD513B-rFSH vector; in the figure, M: DL 5000 Marker; 1: the double enzyme digestion result of the MluⅠ and HindⅢ of the α-CMV-β-CTP-6×His fragment with homologous arms added; 2: the double enzyme digestion result of the BamHⅠ and HindⅢ of pCD513B-rFSH.

[0028] Figure 3 It is the screening of monoclonal cell lines of dairy cow rFSH;

[0029] In the figure, A is the white light image of the polyclonal cells screened by puromycin after lentiviral transfection;

[0030] B is the fluorescence image of the polyclonal cells screened by puromycin after lentiviral transfection;

[0031] C is the white light image of the monoclonal cells screened by puromycin after lentiviral transfection;

[0032] D is the fluorescence image of the monoclonal cells screened by puromycin after lentiviral transfection.

[0033] Figure 4 WB detection of bovine rFSH protein secreted into the cell supernatant

[0034] In the figure, A: 1. Control of the supernatant of transfected cells, 2. Supernatant of the monoclonal cell line of bovine rFSH

[0035] B: 1, 2, 3, 4, 5 are the treatments of boiling the supernatant of the monoclonal cell line of bovine rFSH for 1, 2, 3, 4, 5 minutes respectively

[0036] Figure 5 Effect of commercially available FSH at different concentrations on the proliferation of bovine granulosa cells

[0037] Figure 6 Effect of bovine rFSH prepared in Example 1 at different concentrations on the proliferation of bovine granulosa cells

[0038] Figure 7 Difference in the effect of commercially available FSH and bovine rFSH prepared in Example 1 on the proliferation of bovine granulosa cells Detailed implementation mode

[0039] The following is a detailed description of the specific implementation mode of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation mode. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified

[0040] Example 1: A bovine recombinant follicle-stimulating hormone and its preparation method

[0041] 1. Experimental method

[0042] 1. Primer design and synthesis

[0043] The CDS region of the bovine FSHα gene (NM_173901.3) and the CDS region of the bovine FSHβ gene (NM_174060.1) were obtained from the NCBI database. The CTP gene sequence was selected with reference to Homo-HCGβ3 (NM_000737.5)

[0044] Xi'an Qingke Biotechnology Co., Ltd. synthesized the bovine FSHβ-CTP-α sequence according to the above genes. The bovine FSHβ-CTP-α sequence is shown in SEQ ID NO.1

[0045] SEQ ID NO.1

[0046] ATGAAGTCTGTCCAGTTCTGTTTCCTTTTCTGTTGCTGGAGAGCAATCTGCTGCAGAAGCTGCGAGCTGACCAACATCACCATCACGGTGGAGAAAGAGGAATGTGGCTTCTGCATAAGCATCAACACCACGTGGTGTGCAGGCTACTGCTACACCCGGGACTTGGTATACAGGGACCCAGCAAGGCCCAATATCCAGAAAACGTGTACCTTCAAGGAGCTGGTCTACGAGACGGTGAAAGTGCCTGGCTGTGCTCACCATGCAGACTCCCTGTACACGTACCCAGTAGCCACTGAATGTCACTGCAGCAAGTGCGACAGCGACAGCACTGACTGCACCGTGAGAGGCCTGGGGCCCAGCTACTGCTCCTTCAGGGAAATCAAAGAAGATGACCCCCGCTTCCAGGACTCCTCTTCCTCAAAGGCCCCTCCCCCCAGCCTTCCAAGCCCATCCCGACTCCCGGGGCCCTCGGACACCCCGATCCTCCCACAAGATTACTACAGAAAATATGCAGCTGTCATTCTGGCCATTTTGTCTCTGTTTCTGCAAATTCTCCATTCCTTTCCTGATGGAGAGTTTACAATGCAGGGCTGTCCTGAATGCAAGCTAAAAGAAAACAAATACTTCTCCAAGCCAGATGCTCCAATCTATCAGTGCATGGGGTGCTGCTTCTCCAGGGCATACCCCACTCCAGCGAGGTCTAAGAAGACAATGTTGGTCCCCAAGAACATCACCTCGGAAGCTACATGCTGTGTGGCCAAAGCATTTACCAAGGCCACAGTGATGGGAAATGTCAGAGTGGAGAACCACACCGAGTGCCACTGCAGCACTTGTTATTATCACAAATCCTAA。

[0047] The CDS region of the FSHα gene is shown in SEQ ID NO.2:

[0048] ATGGATTACTACAGAAAATATGCAGCTGTCATTCTGGCCATTTTGTCTCTGTTTCTGCAAATTCTCCATTCCTTTCCTGATGGAGAGTTTACAATGCAGGGCTGTCCTGAATGCAAGCTAAAAGAAAACAAATACTTCTCCAAGCCAGATGCTCCAATCTATCAGTGCATGGGGTGCTGCTTCTCCAGGGCATACCCCACTCCAGCGAGGTCTAAGAAGACAATGTTGGTCCCCAAGAACATCACCTCGGAAGCTACATGCTGTGTGGCCAAAGCATTTACCAAGGCCACAGTGATGGGAAATGTCAGAGTGGAGAACCACACCGAGTGCCACTGCAGCACTTGTTATTATCACAAATCCTAA。

[0049] The CDS region of the bovine FSHβ gene is shown in SEQ ID NO.3:

[0050] ATGAAGTCTGTCCAGTTCTGTTTCCTTTTCTGTTGCTGGAGAGCAATCTGCTGCAGAAGCTGCGAGCTGACCAACATCACCATCACGGTGGAGAAAGAGGAATGTGGCTTCTGCATAAGCATCAACACCACGTGGTGTGCAGGCTACTGCTACACCCGGGACTTGGTATACAGGGACCCAGCAAGGCCCAATATCCAGAAAACGTGTACCTTCAAGGAGCTGGTCTACGAGACGGTGAAAGTGCCTGGCTGTGCTCACCATGCAGACTCCCTGTACACGTACCCAGTAGCCACTGAATGTCACTGCAGCAAGTGCGACAGCGACAGCACTGACTGCACCGTGAGAGGCCTGGGGCCCAGCTACTGCTCCTTCAGGGAAATCAAAGAATAA。

[0051] The CTP gene sequence is shown in SEQ ID NO.4:

[0052] GATGACCCCCGCTTCCAGGACTCCTCTTCCTCAAAGGCCCCTCCCCC CAGCCTTCCAAGCCCATCCCGACTCCCGGGGCCCTCGGACACCCCGATCC TCCCACAA。

[0053] 2. Acquisition of Cow FSH Gene Fragment and Construction of Recombinant Plasmid

[0054] (1) Cloning of Gene CDS Region

[0055] Using the FSHβ-CTP-α sequence synthesized in the above steps as a template, and pcDNA3.1-FSHαF / R and pcDNA3.1-FSHβF / R in Table 1 as primers respectively, amplify the target gene fragments: the CDS region fragments of cow FSHα and FSHβ-CTP genes with homologous arms.

[0056] Use SnapGene 4.0 software to design the corresponding PCR primers for vector plasmids pcDNA3.1 and pCD513B, as shown in Table 1.

[0057] Table 1 Construction Vector Names and Primer Sequences

[0058]

[0059]

[0060] The PCR reaction system is 50 μL: 4 μL of cDNA (50 ng / μL), 1.5 μL each of upstream and downstream primers (10 μM), 25 μL of 2×PrimeSTAR Max Premix, ddH 2 O 18 μL. The PCR reaction program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 35 s, 35 cycles (step 2-4); total extension at 72°C for 5 min; 16°C ∞.

[0061] After the PCR reaction, obtain the PCR products: FSHβ-CTP gene fragment and FSHα gene fragment. Identify the PCR products by agarose gel electrophoresis, and purify and recover the target gene DNA fragments according to the instructions of the universal DNA purification and recovery kit.

[0062] (2) Linearization of pcDNA3.1 Vector

[0063] The empty vector pcDNA3.1 was linearized with the restriction enzymes BamHⅠ and HindⅢ, and the enzyme digestion products were detected by agarose gel electrophoresis experiment. The reaction system was 50 μL: 5 μL of 10×QuickCut Green Buffer, 5 μL each of the restriction enzymes BamHⅠ and HindⅢ, 10 μL (5 μg) of the pcDNA3.1 vector, ddH 2 O 25 μL, reacted at 37 °C for 30 min. Subsequently, the target band was recovered from the gel using a universal DNA purification and recovery kit to obtain the linearized vector for standby.

[0064] (3) Recombination reaction

[0065] The target gene fragments FSHβ-CTP and FSHα were recombinantly ligated with the linearized pcDNA3.1 vector. The reaction system was 20 μL: 0.87 μL of the linearized vector, 5.6 μL of the target gene fragment FSHβ-CTP, 6 μL of FSHα, 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, and then add ddH 2 O to 20 μL, reacted at 37 °C for 30 min, cooled to 4 °C or immediately placed on ice to obtain the recombinant plasmid product.

[0066] (4) Transformation, extraction and identification of the recombinant plasmid

[0067] According to the operation instructions of the transformation reaction, the recombinant plasmid product was transferred into DH5α Escherichia coli competent cells and spread on a solid medium containing ampicillin (1 μL / mL). It was cultured in a 37 °C incubator for 16 h. Then, a single colony was picked and cultured in 30 mL of LB culture medium containing 30 μL of carbenicillin on a 37 °C shaker for 12 h to obtain the bacterial solution. Using the bacterial solution DNA as a template for PCR amplification to check whether positive clones were contained in the bacterial solution. The reaction system: 7.5 μL of 2×Quick Taq HSDyeMix, 0.5 μL each of the pcDNA3.1-FSHαF / R primers (10 μM) in Table 1 or 0.5 μL each of the pcDNA3.1-FSHβF / R primers (10 μM), 2 μL of the bacterial solution, ddH 2 O was added to 15 μL. The reaction program: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 68 °C for 35 s, 35 cycles (step 2-4); total extension at 68 °C for 5 min; 16 °C ∞. The positive plasmid was extracted according to the instructions of the endotoxin-free plasmid miniprep midiprep kit and its concentration was detected. Subsequently, it was singly digested and identified with the corresponding restriction enzyme. The plasmids that met the expectations were retained and named pcDNA3.1-FSHα and pcDNA3.1-FSHβ respectively for subsequent experiments.

[0068] 3. Construction of Recombinant Plasmid of Bovine FSH

[0069] (1) Referring to the linearization method of the above pcDNA3.1 vector, use QuickCut TM Mlu I and BamHⅠ to linearize pcDNA3.1-FSHα according to the enzyme digestion reaction system in the linearization step of the pcDNA3.1 vector, and recover the gene fragment containing the CMV promoter and the FSHα subunit. At the same time, use QuickCut TM Mlu I to linearize pcDNA3.1-FSHβ for recovery and reserve. Referring to the aforementioned recombination reaction, transformation, extraction and identification methods of recombinant plasmids, recombine, transform and extract the gene fragment containing the CMV promoter and the FSHα subunit with the linearized pcDNA3.1-FSHβ, and use QuickCut TM Mlu I, BamHⅠ and HindⅢ for enzyme digestion identification, and name the plasmid pcDNA3.1-rFSH recombinant plasmid.

[0070] 4. Construction of Recombinant FSH Lentiviral Vector

[0071] Using the primers pCD513B-rFSH F and pCD513B-rFSH R in Table 1, referring to the PCR step in the linearization of the above pcDNA3.1 vector, use the pcDNA3.1-rFSH recombinant plasmid as a template in the reaction system, extend for 2 min, and after the reaction ends, perform agarose gel electrophoresis identification on the PCR product, and purify and recover the target fragments containing α and β-CTP.

[0072] Referring to the recombination reaction method, use pCD513B as a vector to perform homologous recombination, transformation and extraction on the target fragments containing α and β-CTP to obtain a recombinant FSH lentiviral vector, identify and reserve it, and name it pCD513B-rFSH.

[0073] 5. Cell Culture and Transfection

[0074] In a 60 mm culture dish, add the revived HEK293T cells to 3 mL of DMEM high-glucose medium containing 10% fetal bovine serum. When the density of 2 dishes of HEK293T cells in good condition reaches 50%, use them for transfection. During transfection, change the medium to serum-free and antibiotic-free medium. During the transfection process, refer to the operation procedure of Lipo8000 TM Transfection Reagent (Shanghai Beyotime Biotechnology Co., Ltd.). Add the recombinant FSH lentiviral vector pCD513B-rFSH and the helper plasmids pGag / Pol, pRev and pVSV-G in a mass ratio of 3:1:1:1 to 4 μL of Lipo8000 TM125 μL of Opti MEM medium was then slowly mixed well and added drop by drop into the HEK293T cell culture dish. After 16 h, it was replaced with complete culture medium. The cell supernatants were collected at 48 h and 72 h respectively. The supernatant was aspirated after centrifugation at 2000 r / min for 5 min, filtered through a 0.22-μm filter, aliquoted, and stored at -80 °C for later use to obtain virus particles. The experimental group was transfected with the pCD513B-rFSH recombinant plasmid, and the empty plasmid pCD513B was transfected as the control group.

[0075] Resuscitate CHO-K1 cells in advance. When the cell density reaches 60%, it is used for lentivirus transfection. Add Polybrene (6 μg / mL) and 3 mL of 10 7 TU / mL of virus particles to the CHO-K1 cells. After thorough mixing, an equal volume of fresh medium was added and cultured for 2 days. During this period, passage or medium change can be carried out according to the cell growth conditions.

[0076] Add 1 μg / mL puromycin for drug screening and positive cells screened by green fluorescent protein. After the cells grow stably, through the pressure screening method: puromycin at 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, change the cell culture medium every 48 h until the cells stably passaged and grew, and use the limiting dilution method to select monoclonal cell lines.

[0077] 6. Detection of the expression of bovine rFSH by Western blotting (WB)

[0078] Collect the monoclonal cell culture supernatant into a centrifuge tube, centrifuge at 8000 rpm for 10 min, collect the supernatant, add 5×Loading Buffer, mix well, and place it in a 100 °C water bath for 1 min, 2 min, 3 min, 4 min, and 5 min respectively, and then perform SDS-PAGE gel electrophoresis. After the electrophoresis is completed, transfer the protein to a PVDF membrane; prepare 10% skim milk powder and block it on a shaker for 2 h. After blocking, wash the membrane 3 times with TBST solution, 10 min each time. Place the successfully transferred PVDF membrane in the rabbit anti-His-tag primary antibody working solution (diluted 1:1000), and incubate it slowly on a shaker at room temperature for 2 h and then overnight at 4 °C; after the primary antibody incubation, wash the membrane 3 times with TBST, 10 min each time. Then place the PVDF membrane in the HRP-labeled goat anti-rabbit secondary antibody working solution (diluted 1:5000), and incubate it on a shaker at room temperature for 2 h; after completion, wash the membrane 3 times with TBST, 10 min each time. After slightly drying, place the PVDF membrane in 2 mL of ECL luminescent solution for 2 min, expose it in a darkroom, and save the result map of the target protein.

[0079] II. Experimental Results

[0080] 1. Synthesis of target gene fragment and construction of lentiviral vector

[0081] Using the FSHβ-CTP-α sequence as a template, plasmid pcDNA3.1-rFSH was obtained according to the recombinant technical roadmap shown in Figure 1 , and was identified by digestion with QuickCut TM Mlu I, BamHⅠ and HindⅢ.

[0082] The results are shown in Figure 2 . There are three fragments in lane 1, namely: FSHα subunit (363bp); FSHβ subunit and CTP-6×His (510bp); CMV promoter (676bp), which are consistent with the expected results.

[0083] The obtained pCD513B-rFSH plasmid was identified by digestion with QuickCut TM Mlu I, BamHⅠ and HindⅢ.

[0084] The results are shown in Figure 2 . Lane 2 shows the FSHα subunit (363bp); FSHβ subunit and CTP-6×His (510bp); CMV promoter (676bp); meanwhile, there is a band above 5000bp that is the same as the band of the linear vector pCD513B, which is consistent with the vector construction results and structure.

[0085] The results of agarose gel electrophoresis showed that the amplified target fragment was single, bright and without non-specific amplification. Thus, it can be proved that the double subunit fragment of bovine rFSH was successfully cloned, and the lentiviral vector pCD513B-rFSH of bovine recombinant follicle-stimulating hormone was successfully obtained according to the recombinant technical roadmap.

[0086] 2. Screening of bovine rFSH monoclonal

[0087] The lentiviral vector pCD513B-rFSH containing the target gene was co-transfected with the helper plasmids pMDLg / RRE, pRSV-Rev and pMD2.G into the CHO-K1 cell line at a mass ratio of 3:1:1:1. After puromycin selection, positive bovine rFSH monoclonal cell lines were obtained by the 96-well limited dilution method and GFP protein labeling, and were named strain 4204 (see Figure 3 ).

[0088] 3. Obtaining of bovine recombinant follicle-stimulating hormone (rFSH)

[0089] Cell expansion culture and supernatant collection: Inoculate the monoclonal cell line (strain 4204) into a T125 cell culture flask, add 50 mL of medium every 2 days. On the 7th day, collect all the cell culture medium, centrifuge at 8000 g at 4 °C for 10 min, and collect the culture supernatant. Use an AKTA flux 6 (manufactured by Cytiva, USA) membrane separation and concentrator to ultrafilter with a membrane package with a molecular weight cut-off of 10 kDa, and collect the ultrafiltered and concentrated solution. Then, use 10 mL PE tubes, aliquot 3 mL into each tube, and use a Freezon e (manufactured by LABCONCO, USA) freeze dryer to freeze-dry overnight. Store for later use.

[0090] 4. WB detection of the expression of bovine rFSH protein in cell supernatant

[0091] Culture of monoclonal cell line 4204: Extract the proteins from the cell supernatants of the experimental group (strain 4204) and the control group (CHO-K1 cells transfected with the lentiviral vector pCD513B). After WB detection, the results are as Figure 4 shown. A distinct and single band appears around 35 kDa in the bovine rFSH monoclonal cell line group, which is consistent with the expected target protein, while there is no band at this position in the empty transfection control group. The above results indicate that the lentiviral vector pCD513B-rFSH was successfully transfected into CHO-K1 cells, and bovine rFSH protein that can be secreted into the cell supernatant was obtained after screening.

[0092] It can be detected that the recombinant follicle-stimulating hormone for cows prepared in the present invention comprises a β subunit linked to CTP with an amino acid sequence as shown in SEQ ID NO.11 and an α subunit as shown in SEQ ID NO.12.

[0093] The amino acid sequence of the protein encoded by FSHβ-CTP is as shown in SEQ ID NO.11:

[0094] MKSVQFCFLFCCWRAICCRSCELTNITITVEKEECGFCISINTTWCAGYCYTRDLVYRDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCSKCDSDSTDCTVRGLGPSYCSFREIKEDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQHHHHHH*. "*" represents the stop codon.

[0095] The amino acid sequence of the protein encoded by FSHα is as shown in SEQ ID NO.12:

[0096] MDYYRKYAAVILAILSLFLQILHSFPDGEFTMQGCPECKLKENKYFSKPDAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNVRVENHTECHCSTCYYHKS*。

[0097] Example 2: Application of the recombinant follicle-stimulating hormone for dairy cows prepared in Example 1.

[0098] I. Experimental methods

[0099] 1. Culture of granulosa cells

[0100] Collect dairy cow ovaries from a slaughterhouse in Yangling District, Shaanxi Province, and place them in physiological saline at 37°C containing 1% penicillin-streptomycin solution, and then transport them back to the laboratory. Use sterilized forceps and scissors to remove excess tissue and mesentery around the ovaries. First, quickly wash with 75% alcohol 3 times to remove blood and bacteria. Finally, wash the tissue 3 times with physiological saline. Pierce the follicles with a 5 mL disposable syringe to collect granulosa cells (GCs), transfer them to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, add DF12 medium to resuspend the cell pellet, and repeat washing the cell pellet 3 times. Then, add DF12 medium + 10% fetal bovine serum (FBS) to the cell pellet, inoculate in a cell culture dish and culture. Observe the cell growth under a microscope 24 h later to remove non-adherent cells and prevent cell contamination. Replace the fresh medium with fresh culture solution every other day.

[0101] 2. Effects of different follicle-stimulating hormones on the proliferation of granulosa cells

[0102] Use CCK-8 to measure cell proliferation.

[0103] The experimental design is as follows: Dilute the commercially available follicle-stimulating hormone (FSH, batch number: B2203291) from the Second Hormone Factory in Ningbo City with complete medium, and then filter and sterilize it with a 0.22 μm filter to obtain a medium containing FSH. Then, use complete medium to configure FSH into the following concentrations: 0 ng / mL, 12 ng / mL, 120 ng / mL, 1.2 μg / mL, 12 μg / mL, and 120 μg / mL).

[0104] Configure the membrane-separated and concentrated dairy cow rFSH obtained in Example 1 into the following concentrations with complete medium in the same way: 0 ng / mL, 2 ng / mL, 20 ng / mL, 200 ng / mL, 2 μg / mL, and 20 μg / mL.

[0105] Dilute the cultured granulosa cells to a density of 2×10 4 cells / mL with the above-configured culture medium, and evenly inoculate 100 μL per well into a 96-well plate and culture at 37°C. Add CCK-8 reagent (10 μL / well) at different culture times (1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d), place it in a 37°C incubator and incubate in the dark for 2 h, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of the solution at 450 nm and statistically analyze the cell proliferation coefficient. Determine the optimal concentrations of FSH and bovine rFSH.

[0106] The results are as Figures 5 to 7 shown. The commercially available FSH at 1.2 μg / mL had a highly significant effect on the proliferation of bovine granulosa cells starting from the 3rd day. Bovine rFSH at 20 ng / mL and 0.2 μg / mL had a highly significant effect on the proliferation of bovine granulosa cells starting from the 3rd day, and the proliferation of granulosa cells was significantly higher than that of the commercially available FSH.

[0107] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts.

[0108] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and deformations.

Claims

1. A method for preparing recombinant bovine follicle-stimulating hormone, characterized in that: The gene fragment containing CMV promoter and FSH alpha subunit is recombined, transformed and extracted with linearized pcDNA3.1-FSH beta to obtain recombinant plasmid pcDNA3.1-rFSH; the recombinant plasmid pcDNA3.1-rFSH is used as a template, pCD513B-rFSH F shown in SEQ ID NO.9 and pCD513B-rFSH R shown in SEQ ID NO.10 are used as primers to perform PCR amplification, and the target fragments of alpha and beta-CTP are recovered; pCD513B is used as a vector, and the target fragments containing alpha and beta-CTP are homologously recombined and transformed to obtain the recombinant FSH lentiviral vector pCD513B-rFSH; then, CHO-K1 cells are transfected and screened to obtain a monoclonal cell line; the monoclonal cell line is cultured and the cell supernatant is collected to separate and purify the recombinant bovine follicle-stimulating hormone.

2. The method for preparing recombinant follicle-stimulating hormone for dairy cows according to claim 1, characterized in that: The preparation steps of linearized pcDNA3.1-FSHβ are as follows: using the FSHβ-CTP-α sequence shown in SEQ ID NO.1 as a template, using pcDNA3.1-FSHαF / R and pcDNA3.1-FSHβF / R as primers, respectively, to amplify the target gene fragments of dairy cow FSHα and FSHβ-CTP; recombining the FSHβ-CTP target gene fragment with the linearized pcDNA3.1 vector to obtain the recombinant plasmid pcDNA3.1-FSHβ, and linearizing by enzyme digestion to obtain the linearized pcDNA3.1-FSHβ.

3. The method for preparing recombinant follicle-stimulating hormone for dairy cows according to claim 2, characterized in that: The linearized pcDNA3.1 vector is obtained by linearizing the empty vector pcDNA3.1 with restriction endonucleases BamHI and HindⅢ.

4. The method for preparing recombinant follicle-stimulating hormone for dairy cows according to claim 2, characterized in that: The recombination ligation reaction system of bovine FSHα and FSHβ-CTP target gene fragments and linearized pcDNA3.1 vector is 20μL: linearized vector 0.87μL~0.89μL, target gene fragment FSHβ-CTP 5.6μL~5.7μL, FSHα 6μL~6.1μL, 5×CEⅡBuffer 4μL4.1μL, ExnaseⅡ2μL~2.1μL, and the balance is ddH2O.

5. A recombinant bovine follicle-stimulating hormone, characterized in that: The recombinant bovine follicle-stimulating hormone is prepared by the method according to any one of claims 1 to 4, and comprises the amino acid sequence shown in SEQ ID NO.11 to SEQ ID NO.

12.

6. A recombinant FSH lentiviral vector, characterized in that: Obtained by the construction steps of the recombinant FSH lentiviral vector pCD513B-rFSH in claim 2.

7. A monoclonal cell line, characterized in that The recombinant FSH lentiviral vector of claim 6 is used to transfect CHO-K1 cells.

8. A method for constructing a monoclonal cell line, characterized in that: The recombinant FSH lentiviral vector was transfected into HEK293T cells, and the viral particles were collected and transfected into CHO-K1 cells. Positive cells were screened by puromycin drug screening and green fluorescent protein screening to obtain the monoclonal cell line described in claim 7.

9. The method for constructing a monoclonal cell line according to claim 8, characterized in that: The process of transfecting HEK293T cells with the recombinant FSH lentiviral vector is as follows: the recombinant FSH lentiviral vector pCD513B-rFSH is mixed with the auxiliary plasmids pGag / Pol, pRev and pVSV-G in a mass ratio of 3 to 3.2:1:1:1 and added to the MEM culture medium containing the transfection reagent, replaced with complete culture medium after 15h to 16h, and the cell supernatant is collected after 70h to 72h, the supernatant is aspirated by centrifugation, and the virus particles are collected by filtration.

10. Use of the recombinant bovine follicle-stimulating hormone according to claim 5, the recombinant FSH lentiviral vector according to claim 6 or the monoclonal cell line according to claim 7 in the preparation of a drug for promoting the proliferation of ovarian granulosa cells.

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