LONG-ACTING RECOMBINANT FOLLICLE-STIMULATING HORMONE AND USE THEREOF

Abstract

Disclosed in the present invention is a long-acting recombinant human follicle-stimulating hormone-Fc fusion protein (referred to as hFSH-Fc for short) and a preparation method thereof, wherein the hFSH-Fc protein is a dimerized fusion protein and the amino acid sequence thereof successively comprises an hFSH subunit, CTP, an hFSH subunit, a flexible peptide linker and human IgG2 Fc variant from N-terminal to C-terminal. Also disclosed in the present invention is the use of the recombinant hFSH-Fc fusion protein composition in preparing drugs in the animal breeding field.

Claims

1. A recombinant hFSH-Fc fusion protein, wherein the fusion protein is a dimerized fusion protein, and the amino acid sequence of the fusion protein sequentially comprises an hFSH subunit, CTP, an hFSH subunit, a peptide linker and human IgG2 Fc variant from N-terminal to C-terminal.

2. The recombinant hFSH-Fc fusion protein of claim 1, wherein the amino acid sequence of the hFSH subunit is an amino acid sequence shown in hFSH SEQ ID NO: 5 in which amino acid residues 1-18 in conventional hFSH subunit are deleted; wherein the amino acid sequence of the CTP is 28-34 amino acid residues from carboxy-terminal of HCG chain, preferably, the CTP is the sequence of 33 amino acid residues from carboxy-terminal of HCG chain, as shown in SEQ ID NO: 4; wherein the sequence of the amino acid residues of the is hFSH subunit is an amino acid sequence shown in hFSH SEQ ID NO: 3 in which amino acid residues 1-24 in conventional hFSH subunit are deleted; wherein the peptide linker comprises 2 to 20 amino acids, the peptide linker is present between the hFSH subunit and the human IgG2 Fc variant, and the peptide linker contains two or more amino acids selected from glycine, serine, alanine, and threonine, the preferred sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.

3. The recombinant hFSH-Fc fusion protein of claim 1, wherein the human IgG2 Fc variant contains a human IgG2 hinge region with Pro331Ser mutation, a CH2 domain, and a CH3 domain.

4. The recombinant hFSH-Fc fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 2.

5. A nucleotide sequence encoding the recombinant hFSH-Fc fusion protein of claim 1, wherein the sequence thereof is shown in SEQ ID NO: 1.

6. A method of preparing the recombinant hFSH-Fc fusion protein of claim 1, comprising the following steps: i) constructing a gene expression vector encoding the recombinant hFSH-Fc fusion protein: a gene encoding the hFSH-Fc fusion protein is obtained by a DNA synthetic method, and is inserted into a mammalian cell expression vector, and thus an expression plasmid containing the gene encoding the hFSH-Fc fusion protein is obtained; ii) stably expressing the recombinant hFSH-Fc fusion protein in mammalian host cells: the expression vector containing the gene encoding the hFSH-Fc fusion protein is transfected into the mammalian host cells, and a cell strain with stable expression of hFSH-Fc fusion protein is screened; iii) culturing high-density cells for the production of the recombinant hFSH-Fc fusion protein: the above-mentioned screened stable cell strain is transferred into a shake flask or a bioreactor to scale up cell production; when the cell density reaches 110.sup.7/mL, the temperature is reduced from 37 C. to 33 C., and cultivation is performed at this temperature till the expression yield no longer increases; iv) purifying and preparing the recombinant hFSH-Fc fusion protein: a) Protein A affinity chromatography: performing centrifugation, collecting the supernatant, and performing chromatography using Protein A affinity column according to the characteristics of a protein-coupled Fc fragment of the present disclosure; b) purification by hydrophobic chromatographic column: performing hydrophobic chromatographic purification to further remove the impurities in target protein after Protein A purification; the hydrophobic column includes Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow, Butyl-S Sepharose 6 Fast Flow, Butyl Sepharose 4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B; preferably, Phenyl Sepharose 6 Fast Flow.

7. The method of preparing the recombinant hFSH-Fc fusion protein of claim 6, wherein the gene expression vector in step i) is pCDNA3, pCMV/ZEO, ORES, pDR, pBK, pSPORT or pCMV-DHFR, preferably, pCDNA3; wherein cell transfection method in step ii) includes electroporation transfection method, calcium phosphate transfection, liposome transfection and protoplast fusion, preferably, electroporation transfection method; the mammalian host cell includes CHO, HEK293, BHK, NSO and Sp2/0 cells, preferably, CHO cells, more preferably, suspended DHFR-deficient CHO cells (CHO DHFR-).

8. The method of preparing the recombinant hFSH-Fc fusion protein of claim 6, wherein step iii) also includes: adding additives to culture medium, preferably, adding 100 M Cu.sup.2+ to basal medium, adding 2 mM ManNAc (N-acetyl-D-amino mannose) to feeding medium.

9. A pharmaceutical composition, wherein it comprises a pharmaceutically acceptable carrier or excipient or diluent, and an effective amount of the recombinant hFSH-Fc fusion protein of any one of claims 1-8.

10. An application of the recombinant hFSH-Fc fusion protein of claim 1 in preparing drugs in the field of animal breeding.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows the comparison of the amino acid sequences of the hinge region of human IgG2 and its variants and CH2 domain. These three parts of the amino acid sequence are compared: amino acid domains 228, 234-237, and 330-331. The amino acid mutation of the variants is indicated in bold italics. The number of the amino acid residues is determined according to the EU numbering system.

[0052] FIG. 2 shows the schematic diagrams of the single-chain hFSH-Fc and the dimerized structure. a) single-chain hFSH-Fc; b) dimerized hFSH-Fc.

[0053] FIG. 3 shows the nucleotide sequence and the deduced amino acid sequence of hFSH-Fc of the HindIII-EcoRI fragment in the pCDNA3 expression vector. The nucleotide sequence of hFSH-Fc comprises the nucleotide sequence encoding leader peptide (1-18), hFSH chain, CTP, mature hFSH chain, peptide linker, and IgG2Fc variant (vIgG2Fc). The mature recombinant hFSH-Fc fusion protein contains mature hFSH chain (19-129), CTP (130-162), mature chain (163-254), peptide linker (255-270) and IgG2Fc variant (vIgG2Fc) (271-493).

[0054] FIG. 4 shows a plasmid map of the constructed eukaryotic expression plasmid encoding the hFSH-Fc fusion protein. The full length of this expression plasmid is 9063 bp, comprising 10 major gene fragments, including: 1. CMV promoter, 2. target gene hFSH-Fc, 3. IRES, 4. Zeocin resistance screening gene, 5. BGH terminator, 6. SV40 promoter, 7. DHFR amplification gene, 8. SV40 terminator, 9. Ampicillin resistance gene (Ampicillin), 10. ColE1 replication origin (Ori).

[0055] FIG. 5 shows the graph of the cumulative trend of the hFSH-Fc protein expressed and secreted by the recombinant hFSH-Fc cell strain in a 7L bioreactor.

[0056] FIG. 6 shows successful expression of the recombinant hFSH-Fc fusion protein in CHO cells by the result of Western blotting analysis. Non-reduced gel, Lane 1: porcine pituitary FSH (about 43 kDa); Lane 2: the recombinant hFSH-Fc fusion protein of the present disclosure (about 140 kDa).

[0057] FIG. 7 shows a 10% SDS-PAGE electrophoretogram of the single-chain hFSH-Fc and the dimerized hFSH-Fc under reduced condition and non-reduced condition. a) reduced gel, single-chain hFSH-Fc (about 70 kDa); b) non-reduced gel, dimerized hFSH-Fc (about 140 kDa).

[0058] FIG. 8 shows the metabolic profiles of the purified recombinant hFSH-Fc fusion protein and the porcine pituitary FSH in rats.

DETAILED DESCRIPTION

[0059] The present disclosure will be further elaborated with specific examples hereinafter. It should be understood that these examples are merely used to illustrate the present disclosure and not to limit the scope of this disclosure. In the following examples, the experimental methods which specific conditions are not stated can be operated according to the conventional conditions such as the conditions mentioned in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

EXAMPLE 1

Construction of the Gene Expression Vector Encoding the Recombinant hFSH-Fc Fusion Protein

[0060] The design of gene sequence was optimized on the basis of the preferred codons of CHO cells. The optimized fusion gene which contained gene encoding the signal peptide of the chain of hFSH protein and its mature peptide fragment, CTP and the mature peptide fragment of hFSH chain was synthesized by an artificial synthetic method. The synthesized DNA fragment of 756 bp in length was inserted into a transfer vector such as between the EcoRV restriction enzyme sites in pUC57 to give the hFSH plasmid (phFSH). The correctness of the inserted sequence was confirmed by DNA sequencing.

[0061] The fusion genes L-vIgG2Fc encoding a flexible peptide linker (Linker, detection L) and an Fc variant (vIgG2Fc) fragment containing the restriction enzyme sites of BamHI (5-end) and EcoRI (3-end) were artificially synthesized respectively. The resulting fusion gene fragments were inserted into a transfer vector such as between the BamHI and EcoRI sites in PUC19 respectively to give a pL-vIgG2Fc plasmid which contained the gene encoding the Fc variant. The gene sequence of L-vIgG2Fc was confirmed by DNA sequencing. To prepare the hFSH-L-Fc fusion gene, the phFSH plasmid was double digested by the restriction enzymes Spel and BamHI. The fusion gene fragments encoding the signal peptide of the chain of hFSH protein and its mature peptide fragment, CTP and the mature peptide fragment of hFSH chain were recycled after the gel electrophoresis. The purified above-mentioned gene fragments were then inserted to the 5-end of the peptide linker in the pL-vIgG2Fc plasmid, linked by T4 ligase to construct a phFSH-L-vIgG2Fc plasmid. The constructed fusion gene comprised the gene encoding hFSH, CTP, hFSH, peptide linker, and Fc variant. Its single-stranded structure was shown in FIG. 2a, the dimerized structure was shown in FIG. 2b.

[0062] The restriction enzymes Spel/EcoRI were used to double digest the phFSH-L-vIgG2Fc plasmid, and the hFSH-L-vIgG2Fc fragment was obtained by DNA gel purification. The purified hFSH-L-Fc fragment was inserted between the corresponding restriction enzyme sites of the mammalian cell expression plasmid, such as pCDNA3 (Invitrogen), to finally obtain the expression plasmid pCDNA3-hFSH-L-vIgG2Fc (simply referred to as pCDNA3-hFSH-Fc plasmid) comprising the fusion gene, as shown in FIG. 4. This plasmid comprised the promoter CMV which was necessary for the mammalian cells to express the heterologous proteins with high efficiency; this plasmid also comprised two kinds of selective marker gene, leading to ampicillin resistance in bacteria and zeocin resistance in mammalian cells. In addition, when the host cells were deficient in DHFR gene expression, the dihydrofolate reductase (DHFR) gene of mice contained in PCDNA3 expression vector enabled the co-amplification of the pFSH-L-Fc fusion gene and the DHFR gene in the presence of methotrexate (MTX).

[0063] Connecting the chain and the chain of hFSH with the CTP peptide fragment was convenient for the right folding of the two chains. Coupling of hFSH and the Fc fragment by peptide linkers (preferably flexible linkers) might increase the bioactivity of the protein. For the present disclosure, a peptide linker of about 20 or fewer (but not less than 2) amino acids in length was preferred. As a matter of course, a peptide linker comprised of a single amino acid was within the protective scope of the present disclosure, it was preferred to use a peptide linker comprising or being comprised of two or more amino acids selected from the following amino acids: glycine, serine, alanine, and threonine. The peptide linker of an example of the present disclosure contained a Gly-Ser peptide member, and the amino acid sequence thereof was GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.

EXAMPLE 2

Stable Expression of the Recombinant hFSH-Fc Fusion Protein in Mammalian Cells

[0064] The expression plasmid pCDNA3-hFSH-L-Fc constructed in example 1 was transfected into DHFR-deficient CHO host cells (CHO-DHFR.sup.). FIG. 2b showed the schematic diagram of the recombinant dimerized hFSH-Fc fusion protein. The transfection was performed by electroporation method. A Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) with a capacitance of 960 Fd was used and its electric field was set at 250V. 10 g of plasmid DNA linearized with Pvul was added to 2510.sup.7 cells in a cuvette. Two days following the transfection, the culture medium was changed to a growth medium containing 100 g/mL of Zeocin resistance marker gene to obtain a transfectant that underwent the primary resistance screening. The expression of hFSH-Fc was examined by Western blotting method using anti-hFSH antibody, as shown in FIG. 6. DHFR amplifiable selectable marker gene was used to increase the expression level of the recombinant dimerized protein. For this purpose, the transfected recombinant dimerized protein gene was co-amplified using the DHFR gene in a growth medium containing increasing concentrations of MTX. Transfectants that could grow in a medium containing up to 10 MTX were subcloned using the limiting dilution method. The secretion rate of the subcloned cell lines was further analyzed. Cell strains with secretion level of more than about 10 (preferably about 20) g/10.sup.6 (i.e., million) cells/24 hours were screened to obtain a cell line with stable and high expression of the recombinant hFSH-Fc fusion protein.

EXAMPLE 3

Production and Purification of the Recombinant hFSH-Fc Fusion Protein

[0065] The high-yield cell line obtained from Example 2 was first subjected to serum-free acclimation cultivation in a culture dish, and then transferred to a shake flask for suspension acclimation cultivation. During the acclimation process, medium screening was carried out at the same time. Different ingredients were added to observe the growth status and the growth trend of cells, as well as the biochemical indexes such as the activity of the expression products, sialic acid, etc. The following cell culture condition was preferred: basal medium comprising 100 M Cu.sup.2+, feeding medium comprising 2 mM ManNAc (N-acetyl-D-amino mannose). This method could increase the glycosylation extent of the recombinant hFSH-Fc fusion protein, and increase the content of sialic acid by about 20%. After successful acclimation, the cells were amplified to sufficient quantity. The cells were monitored and cultured in a 7 L bioreactor. When the cell density exceeded 110.sup.7/mL, the culture temperature was reduced to 33 C., and the growth cycle for one batch was 20 days. The expression amount of the recombinant hFSH-Fc fusion protein was measured by affinity chromatography using a 1 ml Protein A column. The results showed that the cumulative yield expressed by the recombinant hFSH-L-vIgG2Fc cell line was 1.87 g/L (FIG. 5).

[0066] The purification of the recombinant hFSH-Fc fusion protein included the following steps: 1) Protein A affinity chromatography: performing centrifugation, collecting the supernatant, and according to the characteristics of the protein-coupled Fc fragment of the present disclosure, the supernatant was loaded onto a Protein A column equilibrated with phosphate buffer saline (PBS) by using affinity chromatography; after the binding of the recombinant fusion protein to Protein A, the column was washed with PBS until the OD 280 value was below 0.01. The bound recombinant FSH-Fc fusion protein was eluted with 20 mM sodium acetate buffer (pH 4.0), and lastly the active collected liquid was neutralized with 1 M Tris-HCl buffer (pH 10.0). The purity of the purified hFSH-Fc protein could reach 95% or more.

[0067] 2) Hydrophobic column chromatography: the above-mentioned active collected liquid from the Protein A column was changed to 20 mM Tris-HCl-1.5 M NaCl (pH8.0) buffer by ultrafiltration method, and this sample was loaded onto a phenyl-6 Fast Flow column equilibrated with 20 mM Tris-HCl-1.5 M NaCl (pH8.0). The column was first washed with the same equilibration buffer, and then washed with 20mM Tris-HCl-1.35 M NaCl (pH 8.0) before its elution with 20 mM Tris-HCl-0.5 M NaCl (pH 8.0) buffer.

[0068] The result of Western blotting indicated the successful expression of the recombinant hFSH-Fc fusion protein in CHO cells. As shown in FIG. 6, in SDS-PAGE gel electrophoretogram under non-reduced condition, the porcine pituitary FSH (commercial product) and the recombinant hFSH-Fc fusion protein of the present disclosure showed the corresponding hybrid bands of the target protein at 43 kDa and 140 kDa respectively, confirming that the recombinant hFSH fusion protein obtained in the present disclosure contained FSH protein. FIG. 7 was the SDS-PAGE gel electrophoretogram of the purified hFSH-Fc fusion protein under reduced and non-reduced conditions. The result demonstrated that the purity of the purified hFSH-Fc protein could reach 98% or more. The molecular weight of the hFSH-Fc under reduced condition was half of that under non-reduced condition.

EXAMPLE 4

In vivo and in vitro Activity Assay of the Recombinant hFSH-Fc Fusion Protein

[0069] The in vitro activity (immunogenic activity) of the recombinant hFSH-Fc fusion protein of the present disclosure was assayed by the quantitative FSH enzyme immunoassay kit produced by BIOCHECK (USA) Company. Experimental method was conducted referring to the specification of the kit. The in vivo activity was assayed by the ovarian weight gain method in the 2010 edition of the British Pharmacopoeia. The measurement of the protein content was determined by LOWRY quantitative method. The HCG preparation was taken, and phosphate buffer (pH 7.20.2) solution containing 0.1% albumin was added to prepare a diluent of the test sample containing 70 IU/ml HCG Based on the labeled amount of the standard, the estimated potency of the porcine pituitary FSH and the recombinant hFSH-Fc fusion protein, the standard, the porcine pituitary FSH and the recombinant hFSH-Fc fusion protein were formulated with the diluent of the test sample (pH 7.20.2) into working solutions containing 3.33 IU/ml, 1.67 IU/ml and 0.83 IU/ml FSH (high, medium and low dose) respectively. Female

[0070] Wistar rats of 19-28 days old were selected, however, the age difference should be no more than 3 days and the weight difference should be no more than 10 grams. The standard group, the porcine pituitary FSH group and the hFSH-Fc group were all divided into high-, medium- and low-dose group, and each group had 6 animals. The rats were injected subcutaneously into the back of the neck twice a day, 0.2 ml each time for 3 consecutive days, and the rats were dosed at the same time each day. 24 hours after the last injection, animals were killed in accordance with the sequence of administration by cervical dislocation. Ovaries were taken out and weighed after the surface moisture was blotted dry, and the weights of the organs were recorded. The activities of the porcine pituitary FSH and hFSH-Fc were calculated by the parallel line assay method based on quantitative response according to the ovarian weight gain of the standard group. The measured in vitro activities of the recombinant hFSH-Fc fusion protein and the porcine pituitary FSH were 10105 and 8321 IU/ml, respectively, and the in vivo activities thereof were 10230 and 7523 IU/ml, respectively. These results indicated that the recombinant hFSH-Fc fusion protein of the present disclosure had biological activity both in vitro and in vivo.

EXAMPLE 5

Pharmacokinetic Assay of the Recombinant hFSH-Fc Fusion Protein

[0071] The administration groups were divided into the recombinant hFSH-vIgG2Fc fusion protein of the present disclosure group and the porcine pituitary FSH group. In each group, five male Wistar rats weighing 200-250 grams per group were injected intramuscularly at 15 IU/kg respectively. Blood samples were collected at 1, 2, 3, 4, 6, 8, 12, 36 and 60h after administration for the porcine pituitary FSH group and at 1, 2, 4, 8, 12, 24, 56, 120, 176, 200, 264 and 340h after administration for the recombinant hFSH-Fc fusion protein group. The above samples were centrifuged at 3000 rpm for 5 min and the serum was taken and stored at 20 C. The immunological activity of FSH in plasma at each time point was tested by ELISA kit (BIOCHECK, USA). The main pharmacokinetic parameters of each group were calculated by statistical moment method using kinetica 4.4 software. The pharmacokinetic curves of each group were shown in FIG. 8. The results of half-life were shown in Table 1. The results indicated that the elimination half-life of the porcine pituitary FSH in rats was about 3.05 h. However, the elimination half-life of the equal dose of the recombinant hFSH-vIgG2Fc fusion protein was approximately 47.24 h, which was 10 times or more of that of the porcine pituitary FSH.

TABLE-US-00001 TABLE 1 Half-life of the recombinant hFSH-Fc fusion protein and the porcine pituitary FSH Group Half-life T.sub.1/2 (h) Recombinant hFSH-vIgG2Fc 47.24 13.92 Porcine pituitary FSH 3.05 1.12

EXAMPLE 6

Effect of the Recombinant hFSH-Fc Fusion Protein on Promoting the Early Estrus of Young Gilts

[0072] Replacement gilts (6 months old, 90-100 kg) before puberty were selected and randomly divided into three groups: a recombinant hFSH-vIgG2Fc administration group (200 IU/head), a porcine pituitary FSH control group (200 IU/head), and a negative control group (physiological saline). In the above administration groups, 400 IU/head of HCG was used in combination, which might synergistically promote follicular maturation. The gilts were injected intramuscularly by group respectively, their estrus status were observed and recorded, and the estrus rate and synchronous estrus status were counted. The results were shown in Table 2. The data indicated that both the recombinant hFSH-Fc fusion protein and the porcine pituitary FSH could promote the early estrus of young gilts. However, the synchronous estrus rate within 3-4 days reached 80% or more in the recombinant hFSH-vIgG2Fc fusion protein group. The effect of the recombinant hFSH-vIgG2Fc fusion protein on promoting the early estrus of young gilts was better than that of the porcine pituitary FSH.

TABLE-US-00002 TABLE 2 Effect of the recombinant hFSH-Fc fusion protein on the early estrus of young gilts Number of the tested Synchronous estrus Group gilts (head) Estrus rate rate within 3-4 days Recombinant hFSH- 30 93.3%**.sup. 83.3%**.sup. vIgG2Fc Porcine pituitary FSH 30 53.3%* 40%** Negative control 30 0 0 Note: .sup.2 test, compared to the negative control group, **p < 0.01, *p < 0.05; compared to the porcine pituitary FSH control group, .sup.p < 0.05.

EXAMPLE 7

Therapeutic Effect of the Recombinant hFSH-Fc Fusion Protein on Replacement is Sows in Anestrus

[0073] Replacement sows in anestrus older than 10 months and weighing 140 kg or more were selected and injected with 1 ml of cloprostenol injection to eliminate the non-estrus cases caused by the generation of permanent corpus luteum, and were then randomly divided into three groups: a recombinant hFSH-vIgG2Fc fusion protein administration group (200 IU/head), a porcine pituitary FSH control group (200 IU/head), and a negative control group (physiological saline). In the above administration groups, 400 IU/head of HCG was used in combination, which might synergistically promote follicular maturation. The sows were intramuscularly injected by group respectively, and their estrus and conception status were observed and recorded. The results were shown in Table 3. The data indicated that the recombinant hFSH-vIgG2Fc fusion protein could significantly increase the estrus rate of the replacement sows in anestrus, and showed significant difference (P<0.01) relative to the negative control group. The recombinant hFSH-vIgG2Fc fusion protein group also achieved higher results as compared to the porcine pituitary FSH control group (P<0.05). In addition, the recombinant hFSH-vIgG2Fc fusion protein group also had a conception rate in estrus that was significantly higher than that of the negative control group and the porcine pituitary FSH control group. Besides, the difference among the above groups was statistically significant (P<0.05).

TABLE-US-00003 TABLE 3 Therapeutic effect of the recombinant hFSH-Fc fusion protein on replacement sows in anestrus Number of the Estrus Conception Group tested sows rate rate in estrus Recombinant hFSH-vIgG2Fc 60 40%**.sup. 75%*.sup. Porcine pituitary FSH 60 28.3%* 47.1% Negative control 60 6.7% 25% Note: .sup.2 test, compared to the negative control group, **p < 0.01, *p < 0.05; compared to the porcine pituitary FSH group, .sup.p < 0.05.

EXAMPLE 8

Therapeutic Effect of the Recombinant hFSH-Fc Fusion Protein on Delayed Estrus of Multiparous Sows

[0074] Multiparous sows that did not enter estrous two weeks after weaning were selected and injected with 1 ml of cloprostenol injection to eliminate the non-estrus cases caused by the generation of permanent corpus luteum, and were then randomly divided into three groups: a recombinant hFSH-vIgG2Fc fusion protein administration group (200 IU/head), a porcine pituitary FSH control group (200 IU/head) and a negative control group (physiological saline). is In the above administration groups, 400 IU/head of HCG was used in combination, which might synergistically promote follicular maturation. The sows were intramuscularly injected by group, and their estrus and conception status were observed and recorded. The results were shown in Table 4. The data indicated that both the recombinant hFSH-Fc fusion protein and the porcine pituitary FSH could increase the estrus rate of the multiparous sows which did not enter estrus 2 weeks after weaning. However, as compared to the negative control group and the porcine pituitary FSH group, the recombinant hFSH-vIgG2Fc fusion protein group had better therapeutic effect and had significant difference relative to the negative control group (P<0.01). The recombinant hFSH-vIgG2Fc fusion protein group also had higher conception rate in estrus than that of the porcine pituitary FSH control group and the negative control group. The difference among the above groups was statistically significant (P<0.05).

TABLE-US-00004 TABLE 4 Therapeutic effect of the recombinant hFSH-Fc fusion protein on delayed estrus of multiparous sows Number of the Estrus Conception Group tested sows rate rate in estrus Recombinant hFSH-vIgG2Fc 38 63.1%**.sup. 87%*.sup. Porcine pituitary FSH 37 27.3%* 60% Negative control 38 7.9% 33.3% Note: .sup.2 test, compared to the negative control group, **p < 0.01, *p < 0.05; compared to the porcine pituitary FSH group, .sup.p < 0.05.

EXAMPLE 9

Synchronous Estrus Effect of the Recombinant hFSH-Fc Fusion Protein on Dairy Goats in the Central Shaanxi Plain During the Breeding Season

[0075] During the breeding season (from September to November) of dairy goats in the central

[0076] Shaanxi plain, healthy ewes aged 1 to 3 years old and weighing 50 to 75 kg, with moderate or better body condition and no reproductive disease, were selected to conduct the test. The test was conducted in three groups: a recombinant hFSH-vIgG2Fc administration group, a pituitary FSH control group, and a blank negative control group. Each group of ewes was treated with progesterone vaginal suppository sponge (CIDR) for 12 days. In the recombinant hFSH-vIgG2Fc group, 40 units of the corresponding drug were intramuscularly injected 24 hours before the removal of the suppository. The ewes of the pituitary FSH control group were intramuscularly injected with 25 units of pituitary FSH at 24h and 12h before the removal of the suppository, respectively. The ewes of the blank negative control group were intramuscularly injected with the same volume of physiological saline at 24h and 12h before the removal of the suppository. All three groups were injected with 0.1 mg cloprostenol at the removal of the suppository. From 12h after the removal of the suppository, rams were used every 12 hours to test the estrus. It was deemed as estrus when the ewes approached the rams, fluttered tails, allowed the mounting of rams or ewes. The synchronous estrus treatment was deemed effective for ewes entering estrus within 96 h, and the estrus rate was calculated. 5 days after the completion of estrus, ovulation and the development of corpus luteum in ovaries of the ewes in estrus were observed with laparoscope. The number of the ewes with normally developed ovarian follicle, ovulation and the formation of normally functioned corpus luteum in ovary were recorded, and the rate of ovulating and forming functional corpus luteum was calculated.

[0077] The results (Table 5) indicated that, as compared to the negative control group, both the recombinant hFSH-vIgG2Fc and the pituitary FSH could significantly increase the estrus rate (P<0.01), which meant that both drugs had significant effect on promoting the estrus of goats. However, the recombinant hFSH-Fc fusion protein of the present disclosure demanded smaller dose and fewer administrations as compared to the pituitary FSH.

TABLE-US-00005 TABLE 5 Synchronous estrus effect of the recombinant hFSH-Fc fusion protein on dairy goats in central Shaanxi plain during the breeding season within 96 h after the removal of the suppository Number of the Rate of ovulating and dairy goats forming functional Group under treatment Estrus rate corpus luteum Blank negative 45 22% 20% control group Pituitary FSH 45 91.1%* 68.3%* control group Recombinant 45 93.3%* 71.4%* hFSH-vIgG2Fc administration group .sup.2 test: compared to the blank negative control group, *P < 0.01.