Recombinant chorionic gonadotropin, preparation process, pharmaceutical composition and use thereof

Abstract

The present invention relates to a recombinant single-chain chorionic gonadotropin polypeptide having an amino acid sequence encoding the beta chain and the alpha chain of equine chorionic gonadotropin, the amino acid sequence being linked to one or more sequences including a glycosylation site, which will result in excellent in vitro and in vivo activity. The present invention also teaches a DNA molecule encoding the recombinant polypeptide, and the present invention also discloses an expression vector comprising these DNA molecules, a composition comprising the recombinant protein, and a method for preparing the same.

Description

Background Art

[0001] Equine chorionic gonadotropin (eCG) is a hormone produced by the endometrial cup of pregnant mares (Christakos and Bahl, 1979). Formerly known as pregnant mare serum gonadotropin (PMSG), this hormone is commonly used to induce ovulation prior to artificial insemination in livestock operations. It is a heavily glycosylated glycoprotein composed of two heterodimeric chains, α and β. The α subunit is common to all glycoprotein hormones (LH, FSH, TSH, CG), while the β subunit is unique to each hormone and is responsible for receptor binding specificity, although in the case of CG it binds to the same receptor as LH. In equines (horses, donkeys, zebras), placental CG and pituitary LH are expressed from the same gene and therefore share the same protein sequence, differing only in their glycosyl side chains, particularly in the β subunit, leading to the consensus term eLH / CG (Sherman et al., 1992).

[0002] eLH and eCG are encoded by the same gene as described above and have an O-glycosylated C-terminal extension and N-glycosylation in their β subunits, and two N-glycosylation sites in their α subunits (Murphy and Martinuk, 1991), but when expressed and secreted in different tissues, they differ greatly in the N and O of their glycosyl side chains (Smith et al. 1993, Matsui et al. 1994, Bousfield and Butnev 2001), which gives them different biological capabilities in vivo, as eCG has a longer half-life than eLH (Klett et al. 2003) and different thermal stabilities (Galet et al. 2004).

[0003] eCG is currently produced by bleeding pregnant mares between 40 and 130 days of gestation (Allen and Moor, 1972). Once purified, formulated, and controlled, it is marketed for artificially inducing estrus in female goats, cattle, and pigs, among others. eCG has two key characteristics that distinguish it from its use in animal husbandry. Unlike horses, where it possesses only luteinizing activity, in other species it possesses both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activity. Furthermore, due to its quaternary structure and multiple glycosylation sites, it has an elevated half-life in the bloodstream (Stewart et al., 1976; Combarnous et al., 1981).

[0004] Current production processes have all the problems associated with the production and purification of extracted hormones, particularly those related to the maintenance and health costs of the animals, as well as the difficulty of maximizing the purity of the final preparation. Therefore, it is very important to be able to develop an alternative production method.

[0005] It is known that the differences in bioactivity between pituitary-secreted eLH and trophoblast-secreted eCG are fundamentally attributed to differences in glycan length and sialylation. Furthermore, it is clear that the lack of adequate glycosylation in recombinant protein production systems has thus far prevented recombinant eLH / CG from possessing a plasma half-life sufficient to demonstrate bioactivity in vivo.

[0006] Galet et al., "Expression of a single BETA ALPHA chain protein of equine LH / CG in milk of transgenic rabbits and its biological activity," published in Molecular and Cellular Endocrinology 174 (2000) 31-40, teach the construction of a β / α chain fusion protein without the addition of a linker. The construct was expressed in the milk of transgenic rabbits. While the resulting protein exhibited in vitro activity similar to that of the native protein, it was inactive in vivo, and the half-life of the recombinant hormone produced was very short.

[0007] On the other hand, in "Biological Activities of Tethered Equine Chorionic Gonadotropin (eCG) and Its Deglycosylated Mutants," published by MIN Kwan-Sik et al. in Journal of Reproduction and Development, Vol. 50, No. 3, 2004, they demonstrated the fusion of β and α chains in CHO-K1 cells, with or without mutations that reduce their glycosylation, without using any linker to fuse the α and β chains. These researchers only evaluated in vitro activities (cell culture) for LH and FSH activities, and these activities were lower in the non-glycosylated mutants. Although they obtained active hormones, they only obtained active hormones in vitro.

[0008] Legardinier et al., Journal of Molecular Endocrinology (2005) 34, 47-60, "Biological activities of recombinant equine luteinizing hormone / chorionicgonadotropin (eLH / CG) expressed in Sf9 and Mimic insect cell lines." This study demonstrates that the β chain and α chain were expressed in SF9 and Mimic insect cells (five glycosyltransferases were added to SF9), respectively, but attempts to obtain the recombinant hormones reported in this article were unsuccessful. Both eLH / CG produced in Sf9 and Mimic cells were active in in vitro bioassays for LH and FSH, with potencies similar to those of eCG. However, they exhibited no significant in vivo biological activity, nor did they exhibit activity for follicle-stimulating hormone (FSH), nor in eCG-specific assays. Although recombinant eLH / CG produced in mock cells were more highly glycosylated than those produced in Sf9 cells, there was no difference in their in vivo activities because the terminal sialylation of their glycosyl chains was insufficient, which led to their rapid clearance from the blood.

[0009] Another group reported the fusion of β and α chains without any linker in "Expression and bioactivity of a single chain recombinantequine luteinizing hormone (reLH).", Jablonka-Shariff et al., Theriogenology 67 (2007) 311-320. Expression was achieved in CHO-K1 cells. They described LH activity in vitro (testosterone production by Leydig cells) and in vivo, and also described it by the peak of testosterone production in horses. However, they did not show the presence of FSH activity. The researchers knew that the degree of glycosylation of LH produced in the pituitary gland is lower than that of GC produced in the trophoblast. The recombinant product they obtained (reLH) was more like eLH than eCG, so they believed that CHO cells lacked the appropriate glycosyltransferase activity required for eCG synthesis.

[0010] Finally, Sogayar et al., in patent application WO 2017 / 112987 A1, reported on the transformation of CHO dg44 cells (a CHO K1 line with the dhfr gene knocked out by fusion as described in Jablonka-Shariff), screening them and expanding them to select clones with high production of recombinant glycosylated eCG. They assessed that their CHO cells expressed all the enzymes involved in glycosylation and evaluated why their eCG was active both in vitro and in vivo (rat and cow). However, the proposed process requires the selection of certain clones with accidental glycosylation and similar characteristics to trophoblasts, so the stability of the clones makes the strategy relatively useful for large-scale production.

[0011] This is why we decided to modify the eLH / CG structure to increase the chances of more efficient glycosylation after translation in a recombinant expression system without changing its affinity for its receptor, increasing its hydrodynamic volume and, therefore, its bioactivity in vivo. SUMMARY OF THE INVENTION

[0013] The present invention relates to a recombinant single-chain chorionic gonadotropin polypeptide, characterized in that the polypeptide has an amino acid sequence encoding the β chain and α chain of equine chorionic gonadotropin, and the amino acid sequence is connected to one or more sequences including glycosylation sites.

[0014] In a preferred embodiment, the recombinant single-chain chorionic gonadotropin polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10.

[0015] A DNA molecule encoding a recombinant polypeptide is also a related embodiment of the present application. Specifically, the DNA molecule has a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7.

[0016] Expression vectors comprising these DNA molecules are also an object of the present application.

[0017] In one embodiment, these polypeptides are part of a pharmaceutical composition.

[0018] These recombinant proteins and compositions can be used to prepare drugs for treating conditions related to mammalian reproduction or ovulation, such as hyperovulation, ovulation blockage, ovarian subfunction, postpartum estrus induction, or testicular hypofunction in mammals such as cattle, pigs, sheep, dogs, rabbits, deer, goats, and experimental animals.

[0019] Another embodiment relates to a process for producing a recombinant single-chain chorionic gonadotropin polypeptide by transfecting a vector, expressing it in CHO cells, and optionally purifying it by immunoaffinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Expression vectors are shown that allow variants of the β / α fusion protein to be obtained, linked by different linkers.

[0021] Figure 2 Shown are measurements of in vitro gonadotropin bioactivity (FSH), and typical dose-response curves for eCG.

[0022] Figure 3 Shown are measurements of in vitro luteinizing hormone (LH) bioactivity and dose-response curves for hCG.

[0023] Figure 4 In vitro bioactivity measurements of constructs expressed in HEK293T cells are shown.

[0024] Figure 5 In vitro FSH bioactivity measurements of constructs expressed in HEK293T or CHOK1 cells are shown.

[0025] Figure 6 Analysis of supernatants from expression products in HEK293T or CHOK1 cells is shown. A 10% native acrylamide gel is shown. Each lane was seeded with 17 μg of total protein. The H series was synthesized in HEK and C cells in CHO K1. BSA (5 μg) and PMSG (0.25 IU) were used. The units corresponding to the 17 μg mass are: 2H (pCG 2) 4 IU; 5H (pCG 5) 7.2 IU; 8H (pCGβ) 4.5 IU; 10H (pCG 10) 1.4 IU; 5C (pCG5) 0.55 IU; and 8C (pCG 8) 0.22 IU.

[0026] Figure 7 Shown are measurements of in vitro FSH bioactivity of stable protein-producing clones.

[0027] Figure 8 Analysis of purified reCG protein by immunoaffinity is shown. The eCG samples (patterns) seeded were 10 and 1 ug, where for Coomassie and protein staining, 3.5 and 0.5 ug were loaded into constructs #5 and #8 under the same conditions.

[0028] Figure 9 Shown are measurements of in vitro FSH and LH bioactivity of stable clones 5G and 8F.

[0029] Figure 10Shown are measurements of follicle number during treatment with a progesterone-containing device in a field trial in dairy cows. Follicle number (mean) according to follicle size and treatment day in cows treated with eCG (naive; n=13) or reCG (n=14) before device removal on P4 (P>0.1).

[0030] Figure 11 Figure 2 shows measurements of follicle number after removal of a progesterone-containing device in a field trial in dairy cows. Follicle number (mean) according to follicle size and treatment day in cows treated with eCG (naive; n=13) or reCG (n=14) after device removal on P4 (P>0.1).

[0031] Figure 12 Shown are measurements of follicle number after device removal at P4 level in both treatment groups. DETAILED DESCRIPTION

[0032] Obtaining recombinant variants of eCGα-β fusion protein

[0033] In this paper, we describe the generation of a single recombinant α / β chain (reCG) of eCG that increases its plasma half-life by adding glycosylation sites. The construction of a single chain allows the introduction of sequences with multiple glycosylation sites as linkers and avoids the problem of subunit association / dissociation.

[0034] To do this, we modified the gene sequence by adding two sequences with multiple O-glycosylation or N-glycosylation sites. The O-glycosylation sequence is unique to the β chain and is located in the carboxyl-terminal peptide (CTP). The N-glycosylation sequence was designed based on the consensus sequence of mammalian N-glycosylation. We call it NTP instead of CTP because it is used for the amino (N)-terminal peptide.

[0035] To evaluate the functionality of different linkers, expression vectors containing various constructs were prepared, e.g. Figure 1 The nucleotide and amino acid sequences of each of these variants are presented in the accompanying sequence listing:

[0036] SEQ ID NO: 1 corresponds to the polynucleotide sequence of the construct encoding NTP-βCTP-NTP-α (Construct #2), and SEQ ID NO: 2 corresponds to its amino acid sequence. SEQ ID NO: 3 corresponds to the polynucleotide sequence of the construct encoding NTP-βCTP-CTP-α (Construct #5), and SEQ ID NO: 4 corresponds to its amino acid sequence. SEQ ID NO: 5 corresponds to the polynucleotide sequence of the construct encoding NTP-βCTP-α (Construct #8), and SEQ ID NO: 6 corresponds to its amino acid sequence. SEQ ID NO: 7 corresponds to the polynucleotide sequence of the construct encoding NTP-β-NTP-α (Construct #10), and SEQ ID NO: 8 corresponds to its amino acid sequence. SEQ ID NO: 9 corresponds to the polynucleotide sequence of the construct encoding CTP-βCTP-CTP-α (Construct #6), and SEQ ID NO: 10 corresponds to its amino acid sequence. SEQ ID NO: 11 corresponds to the polynucleotide sequence of the construct encoding βCTP-CTP-α (construct #4), and SEQ ID NO: 12 corresponds to its amino acid sequence.

[0037] Development of a method for determining eCG via competitive ELLSA

[0038] The capture antibody (Ab) was a rabbit anti-eCG polyclonal antibody provided by Syntex. Specific immunoglobulins were purified using an eCG affinity column covalently bound to CL agarose. The tracer was biotinylated with sialic acid residues using alkoxyamine-PEG12-biotin.

[0039] Alkaline phosphatase-streptavidin (Strp-FA) was used to quantify the non-displaced Biot-eCG present in samples or standard curves.

[0040] The assay was performed in 96-well ELISA plates, and a standard curve was constructed using Standard 1eCG Syntex standards. Alkaline phosphatase concentrations were determined using 4-nitrophenyl phosphate as a substrate in 100 mM Tris pH 9.2, 1 mM MgCl₂. The reading was at 405 nm. The dynamic range of the assay was 20–0.16 ng / well.

[0041] In vitro gonadotropin (FSH) bioactivity assay

[0042] HEK 293T cells were transfected with a plasmid carrying the bovine FSH hormone receptor transgene and the selective antibiotic neomycin. After selection for antibiotic resistance and expansion of resistant clones, expression of the bFSH receptor was verified by binding to 125I-FSH in the presence or absence of 1 IU of a standard provided by INAME as a competitor to confirm specific binding.

[0043] Under the CRE promoter (pGL4Promega), the clone of the reaction of the FSH receptor and the synthesis of the second messenger cAMP was expressed with high affinity and ligand-bound FSH with luciferase reporter construct transfection. In the presence of cAMP, the cell with this construct can produce a luciferase proportional to the quantity of the second messenger. In the presence of ATP+O2, the enzyme luciferase converts luciferin into metabolites and light. When the whole system was assembled, this luminescence was proportional to the analyte concentration with follicle stimulating activity. The dynamic range of the assay was 12.5-0.4mU / tube.

[0044] Typical dose-response curves for in vitro biological activity are shown in Figure 2. Figure 2 shown.

[0045] In vitro luteinizing bioactivity assay

[0046] The mouse Leydig cell line constitutively expresses the LH receptor and responds to gonadotropin stimulation with cAMP production and progesterone secretion prior to gonadotropin stimulation. Due to partial loss of LH / hCG responsiveness, the current cell line was recloned and the clone with the highest hCG response was screened and transfected with the CRE-Luc reporter gene, as were the HEK cells used for the FSH activity assay.

[0047] The assay was performed on collagen-coated P96 plates, seeding 25,000 cells per well. After a 48-hour preincubation, the hGC standard curve or samples were added and incubated for an additional 3 hours. After the stimulation period, the medium was removed, and the same reagents were added as in the previous assay to determine luciferase activity. The dynamic range of the assay was 50–0.8 mlU / well.

[0048] Typical dose-response curves for hCG are as follows Figure 3 shown.

[0049] In vivo bioactivity assay

[0050] The samples were analyzed using the European Pharmacopoeia, 9th edition (Eh.Ph.9) (Equine Chorionic Gonadotropin Monograph for veterinary use) as the analytical standard.

[0051] The presence of equine chorionic gonadotropin (eCG), either natural or recombinant, and its attendant biological activity (FSH), resulted in an increase in ovarian mass in immature female rats injected with the product. This increase was determined by comparing the average weight of a pair of ovaries obtained after treatment with the weight of an untreated pair of ovaries (baseline weight). The baseline weight of an ovary pair from the strain used in the study (Sprague Dawley (sd)) was less than 25 mg.

[0052] Methodology: 0.2 mL of sample was injected subcutaneously into selected rats.

[0053] The injections were repeated 18, 21, 24, 42, and 48 hours after the first injection (the solution was kept in a refrigerator). The animals were sacrificed no less than 40 hours and no more than 72 hours after the last injection. Subsequently, the ovaries were removed from each animal, and the fat, adhesions, and oviducts were separated. Finally, both ovaries of each treated rat were weighed.

[0054] All animal handling, injections, slaughter, extraction, cleaning, and ovary weighing were performed by personnel specialized in these methods and animal handling.

[0055] Female rats of the breed Sprague Dawley (sd) aged 21 to 28 days (within three days of age difference) and weighing approximately 50-55 g (range ± 10 g) were used in the selected groups.

[0056] Determination of construct functionality

[0057] A-Evaluation in HEK293T cells

[0058] To test the gonadotropin activity of various reCG constructs, the HEK293T cell line was selected as a platform for recombinant protein synthesis. This cell line harbors the T antigen, which means that if they harbor the SV40 gene, plasmids can be propagated. This means that recombinant proteins cloned in suitable plasmids can be aberrantly expressed via transient transfection. However, it also suffers from the disadvantage of being a glycosylated sample, meaning that recombinant proteins synthesized using this cell line have a very short half-life once exposed to complex systems. Activity is generally unaffected in systems lacking clearance, so these proteins were used to test activity in in vitro systems.

[0059] The in vitro bioactivity of constructs 2, 5, 8, and 10 expressed in HEK293T cells was determined as follows: Figure 4 The results of HEK cells in vitro ( Figure 4 is a typical result) leading us to conclude that the designed fusion protein also possesses eGC activity in vitro.

[0060] On the other hand, the in vivo bioactivity of these same reCG constructs (recombinants) synthesized in the HEK293T cell line was investigated. Based on the results obtained from the in vitro assays, the bioactivity of the samples was estimated to be 500 IU / mL.

[0061] In all cases, the results obtained showed a mean ovary weight below 25 mg, ie similar to the baseline weight of the studied species.

[0062] Therefore, it can be concluded that the construct synthesized in HEK cells, although biologically active in vitro, lacks biological activity in vivo.

[0063] B-Evaluation in CHOK1 cells

[0064] Due to the results obtained in HEK293T cells, the development of reCG continued, as glycoproteins produced using CHOK1 cells as a platform generally have longer plasma half-lives than HEK and SF9 cells. Constructs #5 and #8 were selected because they expressed in greater amounts in in vitro assays.

[0065] Comparison of in vitro FSH bioactivity of proteins synthesized by 10×106 HEK or CHO cells Figure 5 shown.

[0066] On the other hand, the proteins present in the supernatant were analyzed in two native polyacrylamide gels, one of which was used to detect total proteins using Coomassie G250 staining, and then transferred to another nitrocellulose membrane to analyze it using an antibody raised against native eCG in rabbit. Figure 6 shown.

[0067] The apparent size of the recombinant protein synthesized in HEK cells was similar to that of the extracted eGC samples, suggesting that the lack of observed in vivo activity was not due to the hydrodynamic volume or amount of glycosylation of the polypeptide synthesized in HEK cells, but rather to its mass.

[0068] Therefore, recombinant protein production in CHOK1 cells continued as well with constructs #5 and #8 being expressed in greater amounts.

[0069] In vivo bioactivity assays showed an increase in ovarian weight within the minimum stimulation value of the standard curve. Control conditioned medium did not produce values ​​other than the control.

[0070] Given that the recombinant hormone was recognized in Western blots using antibodies raised against native eCG, supernatants from transiently transfected CHO cells were purified using an affinity column containing the eCG#5 construct. 13 μg of protein was obtained, with a total in vitro FSH activity of 61 IU. In vivo activity was determined in three animals, demonstrating ovarian weights of 203.9 mg, 119.8 mg, and 172.9 mg, respectively, with stimulation values ​​similar to the maximum value of the standard curve.

[0071] Generation of stable protein-producing clones

[0072] CHO K1 cells were transfected with eCG#5 and #8 constructs and selected with G418 antibiotic to obtain cells that stably harbored the transgene. Once confluence was reached, hormone expression was determined by ELISA, with a setting of detecting 0.6 ng activity in a 50 ul aliquot of supernatant.

[0073] The criteria for screening clones were good growth and a yield greater than 1.5 IU / ml, of which 4 clones of construct #5 and 3 clones of construct #8 remained as candidates. Figure 7 shown.

[0074] Clones 5D, 5G, 5J and 8F were amplified to determine in vivo biological activity, and based on the results, clones 5G and 8F were selected to proceed with the isolation of recombinant proteins by affinity chromatography of both constructs for their further characterization.

[0075] ReCG purification by immunoaffinity

[0076] Syntex sheep-raised anti-eCG immune serum was used. Specific IgG was affinity-isolated using an eCG agarose column, yielding 3 mg of anti-eCG IgG per ml of serum. An aliquot of anti-eCG was dialyzed against a coupling buffer (bicarbonate-NaCl) to yield a total of 90 mg of IgG conjugated to 9 ml of agarose-NHS.

[0077] 0.22 μg of filtered conditioned medium was chromatographed at 1 ml / cm2 / min, the bed was washed with PBS until it returned to the baseline and eluted with 3 ml of glycine pH 3, collecting the absorbance peak at 280 nm. The glycine eluate pH 3 was neutralized, diafiltered with PBS and concentrated.

[0078] 850 μg of construct #5 and 360 μg of construct #8 were obtained. They were analyzed by PAGE without reducing agent, and the total protein bands were visualized by Coomassie staining and the specific bands were visualized by Western blotting using Syntex antibodies raised against natural rabbit hormone as the primary antibody. Figure 8 shown.

[0079] It should be noted that in the case of the recombinant hormone, the only band revealed by the antibody is identical to the band stained with Coomassie. The extremely high-weight band is likely a dimer of the fusion protein, while the low-weight band observed with the native protein is due to partial dissociation of the heterodimer under running conditions in the presence of SDS but without reducing agents.

[0080] The in vitro gonadotropic and luteinizing activities of both the protein and the standard 1eCG Syntex were determined. The results of the dose-response activity in terms of mass and luciferase activity in terms of relative luminescence units (RLU) were shown in Figure 2. Figure 9 The differences in specific activity between recombinant and natural hormones are evident and are even more pronounced in the case of luteinizing hormone, where the natural hormone does not saturate the activity even at higher doses.

[0081] Study the biological activity of stably generated protein-producing clones

[0082] CHOK1 cells were transfected with constructs #5 and #8 and screened to obtain cells stably harboring the transgene.

[0083] The clones with good growth and yield were screened, and clones 5D, 5G, 5J, and 8F were expanded and their biological activities were determined. The results are shown below:

[0084] Table 3. In vivo biological activities of stable clones of reCG.

[0085]

[0086] Due to the positive eCG identification and the high ovarian growth of the treated animals, it was decided to make a first approximation of the estimated values ​​for the samples compared to a reference weight of 25 mg.

[0087] To this end, samples of different clones were analyzed by analytical technology based on standard three-point eCG curves at different concentrations using Syntex secondary eCG standards, which were generated in Syntex and evaluated according to the standard: "International Standard NIBSC Code 62 / 001 (NIBSC)".

[0088] Due to the high response in the previous test, the sample was estimated at a theoretical potency of 30 IU / mL and the results are shown in the form used to process the obtained raw data:

[0089] Bioactivity determination standard at a certain point in PMSG: Secondary SYNTEX concentration information from injection of std:8.1667

[0090]

[0091] Table of ovarian weight (mg) depending on dose

[0092]

[0093]

[0094]

[0095] It was concluded that the clones analyzed produced recombinant protein with positive biological activity, accompanied by a significant increase in ovary weight.

[0096] All treated animals (8 per analyzed sample) responded to the treatment without exception.

[0097] Furthermore, the responses demonstrated with the recombinant were identical to those known and obtained in animals treated with the natural hormone, and no abnormal signals or atypical behaviors were observed in the treated animals.

[0098] Based on the data obtained, the cloning performance is as follows:

[0099] 5D: 26 IU / ml

[0100] 5G: 33 IU / ml

[0101] 5J: 20 IU / ml

[0102] 8F: 17 IU / ml

[0103] Purification process research:

[0104] The goal is to assess whether purification has degraded the molecule or resulted in loss of the molecule as a result of the process.

[0105] Samples of the purified 5G construct were injected into three animals. The responses obtained were as follows:

[0106] 203.9mg-172.9mg-119.8mg

[0107] Since the growth values ​​were consistent with those obtained with the unpurified clone, it was concluded that eCG was not lost or degraded during purification.

[0108] In the case of construct 8F, subsequent attempts to obtain sufficient mass for in vivo bioactivity assays were unsuccessful. Therefore, only construct #5 continued with the project until a production method was determined.

[0109] Luteinizing activity studies (LH):

[0110] The biological responses of male Sprague Dawley (sd) rats aged 21 to 28 days after treatment with sample #5 were studied.

[0111] Methodology:

[0112] A group of 21 to 28 day old Sprague Dawley male rats (sd) (no more than three days between them) and selected groups weighing approximately 60-65 g (with an interval of ±10 g) were prepared.

[0113] The samples were injected subcutaneously for 4 consecutive days at a volume of 0.5 mL every 24 hours.

[0114] Rats were sacrificed 24 hours after the last injection, and the seminal vesicles were removed, washed, dried with filter paper, and weighed.

[0115] All animal handling, injections, slaughter, extraction, cleaning, and spermatocyst weighing tasks were performed by dedicated personnel at the animal care facility.

[0116] The sample solution was prepared by calculating the mass or volume based on its potency and then adjusting the concentration to 5.264 IU / mL.

[0117] Results: Four animals were treated, and the weights of the seminal vesicles after treatment were as follows:

[0118] 44.0mg-58.0mg-48.0mg-52.0mg

[0119] Knowing that for the rat strain used, the baseline weight of vesicles (untreated) is below 12 mg and that the reaction begins to saturate within 50 mg, we can conclude that the presence of luteinizing activity is confirmed.

[0120] This study confirmed the dual activity of recombinant eCG: follicle-stimulating activity and luteinizing activity.

[0121] Determination of follicle stimulation and luteinizing activity of the final 5G sample

[0122] The luteinizing and follicle-stimulating activities were analyzed according to the European Pharmacopoeia, 9th edition.

[0123] Groups of at least 8 animals were analyzed using the national INAME standard for hCG and the Syntex secondary standard for eCG.

[0124] result:

[0125]

[0126] Based on the above, it was concluded that the recombinant eCG produced from construct #5 possessed two prominent activities, follicle stimulating hormone (FSH) and luteinizing hormone (LH), similar to the natural hormones.

[0127] Determining the advantages of amino-terminal peptide modifications relative to in vivo activity

[0128] Fusion proteins using CTP of the β chain of eCG as a linker lacked in vivo bioactivity in eCG synthesized in SF9 and mock cells (Legardinier, J Mol Endo (2005) 34), CHO K1 (Min, Endocrine Journal (1996), 43 585-593), or in eCG produced in the mammary glands of transgenic rabbits (Galet et al., Mol Cell Endocrinol 174 (2000) 31), suggesting that not only the quantity of glycosylation is important, but also the quality of glycosylation. Therefore, we tested two alternative constructs to #5: one, designated #4, which is a fusion of the α and β chains of eCG, like construct #5, but without modification at the amino terminus, and another, construct #6, which is similar to #5 in having a modified amino terminus, but in this case with a CTP peptide sequence. This modification evaluated the importance of amino-terminal modification for the same linker sequence.

[0129] CHO K1 cells were transfected with plasmids encoding constructs #4 and #6 and screened for neomycin resistance. Resistant cells were cloned by dilution and those expressing the highest amounts of protein as determined by ELLSA were selected (clones 4C and 6A). These cells were cultured in DMEN-F12 5% SFB medium, and the conditioned medium was collected. The protein was precipitated with ammonium sulfate, suspended in PBS, and dialyzed against PBS for 36-48 hours with three changes of buffer. The recombinant protein concentration was determined by ELISA, and in vitro biological activity was determined by stimulation with luciferase activity.

[0130] eCG construct 4C 13341 IU / mg

[0131] eCG construct 5G 4013 IU / mg

[0132] eCG construct 6A 4768 IU / mg

[0133] The higher in vitro bioactivity of the protein synthesized by the 4C construct is similar to that found with β-α chain fusions (Min, Endocrine Journal (1996), 43 585-593). It is also expected that in vivo systems, if this protein exhibits activity, it will be lower than that of other recombinant proteins (reCG-5G and reGC-6C).

[0134] When these proteins were tested in vivo, the protein produced with the 5G construct having an NTP peptide at the amino terminus showed an activity of 5477 IU / mg, whereas the construct with a CTP peptide at the amino terminus and the construct with no modification at the amino terminus (6A and 4C, respectively) did not produce an increase in ovarian weight in treated animals.

[0135] These data clearly demonstrate that modification of the amino terminus with the NTP peptide of a fusion protein of the α and β chains of equine chorionic gonadotropin confers an increase in half-life and, therefore, activity in vivo.

[0136] Comparative field tests

[0137] To evaluate the field behavior of recombinant hormones, we tested whether intramuscular injection of an estimated dose of 2000 IU of recombinant eCG protein derived from construct #5 (reCG#5) could produce the same superstimulatory and superovulatory responses as injection of a 2000 IU dose of native eCG (Novormon 5000, Syntex).

[0138] Cows treated with either native eCG or reCG#5 at a dose of 2000 IU had similar progesterone (P4) levels on days 3 and 7 after ovulation.

[0139] The trials took place at the Santa Julia Zootechnics Station of the Catholic University of Córdoba (UCC) during May, June and July 2019.

[0140] Fourteen open-bred Angus / Hereford cows aged 3 to 5 years were used (two replicates, 7 cows per group per replicate); all cows had corpora lutea (cyclical) at the start of treatment. Body condition (BCS) averaged 2.5 (scale 1-5; image 1).

[0141] Animals were fed ad libitum high-quality alfalfa rolls and 15 kg per cow per day of crushed corn silo, fed twice daily in feeders and delivered by a tractor equipped with a ration mixer.

[0142] Treatment group:

[0143] A total of 18 cows were treated with cloprostenol (Cyclase, Syntex) at a dose of 500 μg on day -10 of treatment and were tested for fever after PGF administration. Fourteen cows that developed fever after PGF were selected for inclusion in the trial.

[0144] All cows (14) were treated on day 0 of treatment with an intravaginal device (DIB, 1 g P4, Syntex) supplemented with 2 mg estradiol benzoate (Gonadiol, Syntex) to synchronize the emergence of a new follicle wave around day 4 of treatment (Bo et al., 1994).

[0145] On day 4, the cows were divided into two groups to receive either 2000 IU of natural eCG (eCG group, n=7) or a 350 μg dose of a product produced by Syntex (reCG group #5, n=7), which is estimated to be equivalent to 2000 IU of natural eCG. All injections were made in a volume of 10 ml and were deep intramuscular injections.

[0146] On day 6.5 of treatment, a dose of 500 μg of PGF (Cyclase, Syntex) was administered, and a second dose was administered on day 7, when the device was removed using P4 (DIB 1 g, Syntex Argentina). One day later (day 8), a dose of 100 μg of GnRH (Gonasyn, Syntex) was administered.

[0147] Twenty days after the end of replication 1 (day 17) (day -10 of replication 2), cows were treated with the same protocol, but in this case, cows in the naive eCG group were treated with reCG#5 and vice versa. In this way, all cows were treated with both groups.

[0148] Table 4. Replication 1

[0149]

[0150]

[0151] Table 5. Replication 2

[0152]

[0153]

[0154] *Dose per cow = 10ml-im

[0155] Ovarian ultrasound scans were performed on the days indicated above in Tables 4 and 5. Ovarian structures were plotted and recorded on a spreadsheet designed for this purpose.

[0156] Animals were examined by transrectal ultrasonography (Chison 500; 7.5 MHz, Doppler). All follicles with a diameter greater than 3 mm were identified, measured, and their position in the ovary was charted to assess changes in each of them individually. The subsequent technique used was that described in the work of (Knopf et al., 1989) and adapted by Bo (Bo et al., 1994). The data to be evaluated were: the diameter of the dominant follicle and the main secondary follicle, the number of follicles >3 mm present in the ovary. After injection of eCG or reCG#5, all follicles with a diameter greater than 8 mm were confirmed. After administration of GnRH, ovulation was defined as the disappearance of follicles greater than 8 mm identified in the previous observation. After ovulation, the size and number of the corpus luteum (CL) produced by ovulation were measured (Adams et al., 1992).

[0157] On the other hand, as shown in Tables 4 and 5, blood samples were collected on the 14th and 17th days of treatment to determine the level of progesterone (P4) produced after ovulation.

[0158] Blood samples were obtained from all cows by jugular vein puncture to determine plasma progesterone levels. Blood was collected from each animal using a disposable needle (18G) in a 10 ml sterile glass tube with a rubber cap. Samples were always obtained in heparinized tubes and centrifuged immediately after collection. Plasma was frozen in duplicate (A or B) at -20°C and fully identified by cow tag number, processing date and time, and A or B.

[0159] The samples were processed by immunochemiluminescence (ECL1A, COBAS Module e601, Roche).

[0160] The mean number of follicles by size (8 to 10 mm, 10 to 12 mm, 12 to 14 mm, and >14 mm) and per treatment day (day 5, day 6, day 7, day 8, day 9, and day 10) were compared by ANOVA.

[0161] The mean number of ovulations per day for the treatments was compared using the same statistics.

[0162] In the same manner, P4 levels were compared on days 14 and 17 of the protocol. Treatment group effects, replicates, and their interactions were considered in all cases. Infostat software (Di Rienzo et al., 2018) was used.

[0163] result:

[0164] A cow from the eCG group in replicate 2 was withdrawn from the trial due to respiratory illness on treatment day 6. Cows were treated according to normal study center procedures under the supervision of the responsible veterinarian.

[0165] No differences were found in the number of follicles (by size) on any day of treatment (P > 0.1), with no repeat effect (P > 0.07) or group by repeat interaction (P > 0.4).

[0166] Table 6. Number of follicles according to follicle size and treatment day in cows treated with eCG (native; n=13) or reCG#5 (n=14) (mean ± SEM)

[0167]

[0168]

[0169] Group = P>0.10

[0170] Repeated = P > 0.07

[0171] Group*replication=P>0.4

[0172] These results are also shown in Figure 10 In the figure, you can see the information about the number of follicles (divided by size) during the treatment with the intravaginal device (days 5 to 7 of the protocol). As can be seen in the graph, there is no difference in the number of follicles present in the two groups.

[0173] In the same way, Figure 11 The number of follicles after the removal of the progesterone-containing device can be seen in Figure 4.

[0174] The total number of diagnosed ovulations (disappearance of follicles >8 mm) occurred on treatment days 9, 10, and 11 (only one cow in the eCG group ovulated on day 11). No difference in the number of ovulations was found between the two treatment groups (P = 0.83; Table 3). There was also no replicate effect (P = 0.06) or replicate*interaction (P = 0.85).

[0175] On days 14 and 17 of treatment, no difference in the mean P4 levels between the two treatment groups was found (P>0.7). No repeat effect was observed (P>0.9). The results are shown in the table below.

[0176] Table 7. P4 levels in cows treated with 2000 IU of eCG or its equivalent dose of eCG-Like on days 14 and 17 of treatment.

[0177] P4 Day 14 P4 Day 17 Mean ± SEM Mean ± SEM eCG (n=13) 45,92±8,67 76,45±16,29 eCG sample (n=14) 49,66±8,40 77,73±18,54

[0178] P>0.7

[0179] Figure 12 P4 levels and sampling days for the two groups are shown.

[0180] Based on the results obtained, we can conclude that reCG#5 injected into the test animals produces gonadotropin activity similar to that produced by natural eCG (Novormon). This is because the hyperstimulatory response observed in both groups of cows before and after the removal of the progesterone-containing device was similar, as was the number of ovulations observed after the device was removed.

[0181] On the other hand, since the follicle growth rates were similar in the two groups, we can infer that the half-lives of the two products are similar; however, specific trials to determine the half-life should be performed in the future.

[0182] The ovulation rates found in this study, using 2000 IU of eCG, are similar to those reported by other authors using the same dose in biologically similar cattle (Alfuraiji et al., 1993; Goulding et al., 1996).

[0183] No differences were found in P4 levels on days 14 and 17 of treatment, indicating that the function and number of corpora lutea present in each treatment group were equivalent to each other.

[0184] Finally, based on the similar responses obtained, we believe that reCG#5 used in this study could be used at a lower dose (400 IU) to improve pregnancy rates achieved in FTAI procedures in non-estrus beef and dairy cows. That is, it may have the same effect as natural eCG when used for this purpose. Sequence Listing <110> Syntex SA Abentin, Alejandro D. <120> Recombinant chorionic gonadotropin, preparation process, pharmaceutical composition and use thereof <130> MOM 234048 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 1063 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding NTP-βCTP-NTP-α <400> 1 atggagaccc tgcagggcct gctgctgtgg atgctgctga gcgtggggagg cgtgtgggct 60 ggcgatatcg gcctgaacat caccggcagc ggcctgaata tcacaggctc tggcctgaac 120 attaccggct ctggcctgaa tatcaccggc cctggctcta cagatatcag cagaggacca 180 ctgaggcctc tgtgccggcc tatcaacgcc accctggctg ctgagaagga ggcttgccca 240 atctgtatca cattcaccac aagcatctgc gccggctact gtccatccat ggtgcgcgtg 300 atgccagccg ctctgccagc tatccctcag ccagtgtgca cctatcggga gctgaggttc 360 gcttccatca ggctgccagg atgtccacct ggagtggacc ctatggtgtc ctttccagtg 420 gctctgtctt gccactgtgg cccctgccag atcaagacca cagactgtgg cgtgtttagg 480 gatcagccac tggcttgtgc tcctcaggct agctcctcta gcaaggatcc accctcccag 540 cccctgacca gcacatccac ccccacacct ggcgcttctc ggaggtcctc tcacccactg 600 cccatcaaga cctctggcag cggcctgaac atcacaggtt ctggcctgaa tattactggc 660 tctggcctga acatcaccgg ttccggcctg aatattaccg gcccaggctc cacatctttc 720 cccgacggcg agtttaccac acaggattgc cccgagtgta agctgaggga gaacaagtac 780 ttctttaagc tgggcgtgcc tatctatcag tgcaagggct gctgtttcag ccgggcctac 840 cctaccccag ctagaagccg caagacaatg ctggtgccta agaatatcac cagcgagtcc 900 acatgctgcg tggccaaggc ttttatccgg gtgaccgtga tgggcaacat caagctggag 960 aatcacaccc agtgctactg ttctacatgt tatcaccaca agatctgatt tcctgatgga 1020 gagtttacaa cgcaggattg cccagaaccc ccatcccaac ctc 1063 <210> 2 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Protein NTP-βCTP-NTP-α <400> 2 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Gly Asp Ile Gly Leu Asn Ile Thr Gly Ser Gly Leu 20 25 30 Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile 35 40 45 Thr Gly Pro Gly Ser Thr Asp Ile Ser Arg Gly Pro Leu Arg Pro Leu 50 55 60 Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro 65 70 75 80 Ile Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser 85 90 95 Met Val Arg Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val 100 105 110 Cys Thr Tyr Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys 115 120 125 Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys 130 135 140 His Cys Gly Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg 145 150 155 160 Asp Gln Pro Leu Ala Cys Ala Pro Gln Ala Ser Ser Ser Ser Lys Asp 165 170 175 Pro Pro Ser Gln Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala 180 185 190 Ser Arg Arg Ser Ser His Pro Leu Pro Ile Lys Thr Ser Gly Ser Gly 195 200 205 Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn 210 215 220 Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Pro Gly Ser Thr Ser Phe 225 230 235 240 Pro Asp Gly Glu Phe Thr Thr Gln Asp Cys Pro Glu Cys Lys Leu Arg 245 250 255 Glu Asn Lys Tyr Phe Phe Lys Leu Gly Val Pro With Tyr Gln Cys Lys 260 265 270 Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Arg Lys 275 280 285 Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Serving Thr Cys Cys Val 290,295,300 Ala Lys Ala Phe Ile Arg Val Thr Val Met Gly Asn Ile Lys Leu Glu 305 310 315 320 Asn His Thr Gln Cys Tyr Cys Ser Thr Cys Tyr His Lys Ile 325 330 335 <210> 3 <211> 1023 <212> DNA <213> The NTP-βCTP-CTP-α sequence <400> 3 atggagaccc tgcaggcct gctgctgtgg atgctgctgt ctgtgggagg cgtgtgggct 60 ggcgatatcg gcctgaacat caccggctcc ggctgaata tcacaggctc tggctgaac 120 atcaccggaa gcggcctgaa tatcaccgga ccaggcagca cagatatctc ccggggacca 180 ctgaggcctc tgtgcagacc tatcaacgcc acactggccg ctgagaagga ggcttgccca 240 atctgtatca ccttcaccac atccatctgc gccggctact gtccttctat ggtgagagtg 300 atgccagccg ctctgccagc tatcccacag cccgtgtgca cctatagaga gctgcgcttc 360 gctagcatca gactgccagg atgtccacct ggagtggacc ctatggtgtc ttttccagtg 420 gctctgagct gccactgtgg cccctgccag atcaagacca cagactgtgg cgtgtttcgc 480 gatcagccac tggcttgtgc tcctcaggct agctcctcta gcaaggaccc accaagccag 540 ccactgacca gcacatccac cccaacaccc ggcgcttctc ggaggtcctc tcaccctctg 600 ccaatcaaga caagcgcccc ccaggcctct tcctctagca aggatcctcc atcccagcct 660 ctgacctcta caagcacccc aacacctgga gcttccagac gctcctctca cccactgccc 720 atcaagacct ctttcccaga cggcgagttt accacacagg attgccccga gtgtaagctg 780 cgcgagaaca agtacttctt taagctgggc gtgcctatct atcagtgcaa gggctgctgt 840 ttcagccggg cctaccctac accagctcgg tccaggaaga ccatgctggt gccaaagaat 900 atcacctccg agtctacatg ctgcgtggcc aaggctttta tccgggtgac cgtgatgggc 960 aacatcaagc tggagaatca cacccagtgc tactgttcca catgttatca ccacaagatc 1020 tga 1023 <210> 4 <211> 340 <212> PRT <213> Artificial sequence <220> <223> Recombinant protein NTP-βCTP-CTP-α <400> 4 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Gly Asp Ile Gly Leu Asn Ile Thr Gly Ser Gly Leu 20 25 30 Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile 35 40 45 Thr Gly Pro Gly Ser Thr Asp Ile Ser Arg Gly Pro Leu Arg Pro Leu 50 55 60 Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro 65 70 75 80 Ile Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser 85 90 95 Met Val Arg Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val 100 105 110 Cys Thr Tyr Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys 115 120 125 Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys 130 135 140 His Cys Gly Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg 145 150 155 160 Asp Gln Pro Leu Ala Cys Ala Pro Gln Ala Ser Ser Ser Ser Lys Asp 165 170 175 Pro Pro Ser Gln Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala 180 185 190 Ser Arg Arg Ser Ser His Pro Leu Pro Ile Lys Thr Ser Ala Pro Gln 195 200 205 Ala Ser Ser Ser Ser Lys Asp Pro Pro Ser Gln Pro Leu Thr Ser Thr 210 215 220 Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser Ser His Pro Leu Pro 225 230 235 240 Ile Lys Thr Ser Phe Pro Asp Gly Glu Phe Thr Thr Gln Asp Cys Pro 245 250 255 Glu Cys Lys Leu Arg Glu Asn Lys Tyr Phe Phe Lys Leu Gly Val Pro 260 265 270 Ile Tyr Gln Cys Lys Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 275 280 285 Ala Arg Ser Arg Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu 290 295 300 Ser Thr Cys Cys Val Ala Lys Ala Phe Ile Arg Val Thr Val Met Gly 305 310 315 320 Asn Ile Lys Leu Glu Asn His Thr Gln Cys Tyr Cys Ser Thr Cys Tyr 325 330 335 His His Lys Ile 340 <210> 5 <211> 906 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding NTP-βCTP-α <400> 5 atggagaccc tgcagggcct gctgctgtgg atgctgctgt ccgtgggagg cgtgtgggct 60 ggcgatatcg gcctgaacat caccggctct ggcctgaata tcacaggatc tggcctgaac 120 attaccggat ccggcctgaa tatcaccggc cctggctcca cagatatcag ccggggacca 180 ctgaggcctc tgtgccggcc tatcaacgcc acactggccg ctgagaagga ggcttgccca 240 atctgtatca ccttcaccac atctatctgc gccggctact gtccaagcat ggtgcgcgtg 300 atgccagccg ctctgccagc tatccctcag ccagtgtgca cctatcggga gctgaggttc 360 gcttctatca ggctgccagg atgtccacct ggagtggacc ctatggtgag ctttccagtg 420 gctctgtcct gccactgtgg cccttgccag atcaagacca cagactgtgg cgtgtttagg 480 gatcagccac tggcttgtgc tcctcaggct agctcctcta gcaaggatcc acccagccag 540 cccctgacca gcacatccac cccaacacct ggagctagcc ggaggtcctc tcacccactg 600 cccatcaaga catccttccc agacggcgag tttaccacac aggattgccc cgagtgtaag 660 ctgagggaga acaagtactt ctttaagctg ggcgtgccaa tctatcagtg caagggctgc 720 tgtttctcca gagcctaccc tacaccagct agatctcgca agaccatgct ggtgcccaag 780 aatatcacct ctgagagcac atgctgcgtg gccaaggctt ttatccgggt gaccgtgatg 840 ggcaacatca agctggagaa tcacacccag tgctactgtt ctacatgtta tcaccacaag 900 atctga 906 <210> 6 <211> 301 <212> PRT <213> artificial sequence <220> <223> NTP-βCTP-α protein complex <400> 6 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Gly Asp Ile Gly Leu Asn Ile Thr Gly Ser Gly Leu 20 25 30 Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile 35 40 45 Thr Gly Pro Gly Ser Thr Asp Ile Ser Arg Gly Pro Leu Arg Pro Leu 50 55 60 Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro 65 70 75 80 Ile Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser 85 90 95 Met Val Arg Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val 100 105 110 Cys Thr Tyr Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys 115 120 125 Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys 130 135 140 His Cys Gly Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg 145 150 155 160 Asp Gln Pro Leu Ala Cys Ala Pro Gln Ala Ser Ser Ser Ser Lys Asp 165 170 175 Pro Pro Ser Gln Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala 180 185 190 Ser Arg Arg Ser Ser His Pro Leu Pro Ile Lys Thr Ser Phe Pro Asp 195 200 205 Gly Glu Phe Thr Thr Gln Asp Cys Pro Glu Cys Lys Leu Arg Glu Asn 210 215 220 Lys Tyr Phe Phe Lys Leu Gly Val Pro Ile Tyr Gln Cys Lys Gly Cys 225 230 235 240 Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Arg Lys Thr Met 245 250 255 Leu Val Pro Lys Asn Ile Thr Ser Glu Ser Thr Cys Cys Val Ala Lys 260 265 270 Ala Phe Ile Arg Val Thr Val Met Gly Asn Ile Lys Leu Glu Asn His 275 280 285 Thr Gln Cys Tyr Cys Ser Thr Cys Tyr His His Lys Ile 290 295 300 <210> 7 <211> 891 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding NTP-β-NTP-α <400> 7 atggagaccc tgcagggcct gctgctgtgg atgctgctgt ccgtgggagg cgtgtgggct 60 ggcgatatcg gcctgaacat caccggctct ggcctgaata tcacaggatc tggcctgaac 120 attaccggat ccggcctgaa tatcaccggc cctggctcca cagatatctc tcggggacca 180 ctgaggcctc tgtgcagacc aatcaacgcc accctggccg ctgagaagga ggcttgcccc 240 atctgtatca cattcaccac aagcatctgc gccggctact gtccatccat ggtgagagtg 300 atgccagccg ctctgccagc tatcccacag cccgtgtgca cctatagaga gctgcgcttc 360 gcttctatca gactgccagg atgtccacct ggagtggacc ctatggtgag ctttccagtg 420 gccctgtcct gccactgtgg cccttgccag atcaagacca cagactgtgg cgtgtttcgc 480 gatcagccac tggcttgtgg ctctggcctg aacatcacag gcagtggcct gaatattacc 540 ggctccggcc tgaacattac cggttccggc ctgaatatta ctggaccagg cagcacatcc 600 ttccctgacg gcgagtttac cacacaggat tgccccgagt gtaagctgcg cgagaacaag 660 tacttcttta agctgggcgt gcctatctat cagtgcaagg gctgctgttt ctctcgggct 720 taccctaccc cagctcggag caggaagaca atgctggtgc ccaagaatat cacctctgag 780 agcacatgct gcgtggccaa ggcttttatc cgggtgaccg tgatgggcaa catcaagctg 840 gagaatcaca cccagtgcta ctgttccaca tgttatcacc acaagatctg a 891 <210> 8 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Protein NTP-β-NTP-α <400> 8 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Gly Asp Ile Gly Leu Asn Ile Thr Gly Ser Gly Leu 20 25 30 Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile 35 40 45 Thr Gly Pro Gly Ser Thr Asp Ile Ser Arg Gly Pro Leu Arg Pro Leu 50 55 60 Cys Arg Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro 65 70 75 80 Ile Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser 85 90 95 Met Val Arg Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val 100 105 110 Cys Thr Tyr Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys 115 120 125 Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys 130 135 140 His Cys Gly Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg 145 150 155 160 Asp Gln Pro Leu Ala Cys Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly 165 170 175 Leu Asn Ile Thr Gly Ser Gly Leu Asn Ile Thr Gly Ser Gly Leu Asn 180 185 190 Ile Thr Gly Pro Gly Ser Thr Ser Phe Pro Asp Gly Glu Phe Thr Thr 195 200 205 Gln Asp Cys Pro Glu Cys Lys Leu Arg Glu Asn Lys Tyr Phe Phe Lys 210 215 220 Leu Gly Val Pro Ile Tyr Gln Cys Lys Gly Cys Cys Phe Ser Arg Ala 225 230 235 240 Tyr Pro Thr Pro Ala Arg Ser Arg Lys Thr Met Leu Val Pro Lys Asn 245 250 255 Ile Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ala Phe Ile Arg Val 260 265 270 Thr Val Met Gly Asn Ile Lys Leu Glu Asn His Thr Gln Cys Tyr Cys 275 280 285 Ser Thr Cys Tyr His His Lys Ile 290 295 <210> 9 <211> 1062 <212> DNA <213> Synthetic sequence <220> <223> Nucleotide sequence encoding CTP-β-CTP-CTP-α <400> 9 atggagaccc tgcagggcct gctgctgtgg atgctgctgt ccgtgggagg cgtgtgggct 60 ggcgtgttcc gggaccagcc tctggcttgc gctccacagg cttccagctc ttccaaggat 120 cccccttctc agccactgac ctctacatcc accccaacac caggagcttc caggcggagc 180 tctcaccctc tgccaatcaa gacctccagc agaggaccac tgaggcctct gtgcaggccc 240 atcaacgcca ccctggctgc tgagaaggag gcttgcccta tctgtatcac attcaccaca 300 tccatctgcg ctggctactg tcctagcatg gtgcgcgtga tgccagccgc tctgccagct 360 atcccacagc ccgtgtgcac ctatagggag ctgcggttcg ctagcatcag gctgcctgga 420 tgtccaccag gagtggaccc aatggtgagc tttcctgtgg ccctgtcttg ccattgtggc 480 ccatgccaga tcaagaccac agactgtggc gtgtttagag atcagccact ggcctgtgct 540 ccacaggctt cttccagctc taggaccct ccaagccagc ccctgaccag cacatctacc 600 cctaccccag gagctagcag acgctccagc catccactgc caatcaagac ctctgcccct 660 caggcctctt ccagctctaa agacccccct tctcagcccc tgacctccac aagcacccca 720 acacctggag cttccaggcg gtccagccat ccactgccca tcaagacaag cttccctgac 780 ggcgagttta ccacacagga ttgcccagag tgtaagctgc gggagaacaa gtacttcttt 840 aagctgggcg tgcccatcta tcagtgcaag ggctgctgtt tctccagggc ctaccctacc 900 ccagctagat cccgcaagac aatgctggtg cctaagaata tcacctctga gtccacatgc 960 tgcgtggcca aggcttttat ccgcgtgacc gtgatgggca acatcaagct ggagaatcac 1020 acccagtgct actgttctac atgttatcac cataagatct ga 1062 <210> 10 <211> 353 <212> PRT <213> Recombinant protein CTP-β-CTP-CTP-α <400> 10 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Gly Val Phe Arg Asp Gln Pro Leu Ala Cys Ala Pro 20 25 30 Gln Ala Ser Ser Ser Ser Lys Asp Pro Pro Ser Gln Pro Leu Thr Ser 35 40 45 Thr Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser Ser His Pro Leu 50 55 60 Pro Ile Lys Thr Ser Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro 65 70 75 80 Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Ile Cys Ile 85 90 95 Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg 100 105 110 Val Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val Cys Thr Tyr 115 120 125 Arg Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys Pro Pro Gly 130 135 140 Val Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys His Cys Gly 145 150 155 160 Pro Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg Asp Gln Pro 165 170 175 Leu Ala Cys Ala Pro Gln Ala Ser Ser Ser Ser Lys Asp Pro Pro Ser 180 185 190 Gln Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg 195 200 205 Ser Ser His Pro Leu Pro Ile Lys Thr Ser Ala Pro Gln Ala Ser Ser 210 215 220 Ser Ser Lys Asp Pro Pro Ser Gln Pro Leu Thr Ser Thr Ser Thr Pro 225 230 235 240 Thr Pro Gly Ala Ser Arg Arg Ser Ser His Pro Leu Pro Ile Lys Thr 245 250 255 Ser Phe Pro Asp Gly Glu Phe Thr Thr Gln Asp Cys Pro Glu Cys Lys 260 265 270 Leu Arg Glu Asn Lys Tyr Phe Phe Lys Leu Gly Val Pro Ile Tyr Gln 275 280 285 Cys Lys Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser 290 295 300 Arg Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ser Thr Cys 305 310 315 320 Cys Val Ala Lys Ala Phe Ile Arg Val Thr Val Met Gly Asn Ile Lys 325 330 335 Leu Glu Asn His Thr Gln Cys Tyr Cys Ser Thr Cys Tyr His His Lys 340 345 350 Ile <210> 11 <211> 915 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding β-CTP-CTP-α <400> 11 atggagaccc tgcagggcct gctgctgtgg atgctgctgt ctgtgggagg cgtgtgggct 60 tccaggggac cactgcggcc tctgtgcaga cctatcaacg ccaccctggc cgctgagaag 120 gaggcttgcc caatctgtat cacattcacc acaagcatct gcgctggcta ctgtccctct 180 atggtgagag tgatgccagc cgctctgcca gctatcccac agcccgtgtg cacctataga 240 gagctgcgct tcgcttctat cagactgcca ggatgtccac ctggagtgga ccctatggtg 300 tcctttccag tggccctgag ctgccactgt ggaccatgcc agatcaagac cacagactgt 360 ggcgtgtttc gcgatcagcc actggcttgt gctcctcagg cttccagctc ttccaaggac 420 ccaccctccc agcctctgac ctccacaagc accccaacac ccggcgcttc taggcggagc 480 tctcaccctc tgccaatcaa gacctccgcc cccaggcct ctagctcttc caaggatcct 540 ccaagccagc ctctgacctc tacatccacc cccacacctg gcgctagcag acgcagctct 600 catccactgc ccatcaagac atctttccca gacggcgagt ttaccacaca ggattgcccc 660 gagtgtaagc tgcgcgagaa caagtacttc tttaagctgg gcgtgcctat ctatcagtgc 720 aagggctgct gtttctccag ggcctaccct accccagcta ggagccggaa gacaatgctg 780 gtgccaaaga atatcaccag cgagtctaca tgctgcgtgg ccaaggcttt tatccgggtg 840 accgtgatgg gcaacatcaa gctggagaat catacccagt gctactgttc cacatgttat 900 shit tctga 915 <210> 12 <211> 304 <212> PRT <213> artificial sequence <220> <223> β-CTP-CTP-α complex protein <400> 12 Met Glu Thr Leu Gln Gly Leu Leu Leu Trp Met Leu Leu Ser Val Gly 1 5 10 15 Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Arg Pro Ile 20 25 30 Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Ile Cys Ile Thr 35 40 45 Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser Met Val Arg Val 50 55 60 Met Pro Ala Ala Leu Pro Ala Ile Pro Gln Pro Val Cys Thr Tyr Arg 65 70 75 80 Glu Leu Arg Phe Ala Ser Ile Arg Leu Pro Gly Cys Pro Pro Gly Val 85 90 95 Asp Pro Met Val Ser Phe Pro Val Ala Leu Ser Cys His Cys Gly Pro 100 105 110 Cys Gln Ile Lys Thr Thr Asp Cys Gly Val Phe Arg Asp Gln Pro Leu 115 120 125 Ala Cys Ala Pro Gln Ala Ser Ser Ser Ser Lys Asp Pro Pro Ser Gln 130 135 140 Pro Leu Thr Ser Thr Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser 145 150 155 160 Ser His Pro Leu Pro Ile Lys Thr Ser Ala Pro Gln Ala Ser Ser Ser 165 170 175 Ser Lys Asp Pro Pro Ser Gln Pro Leu Thr Ser Thr Ser Thr Pro Thr 180 185 190 Pro Gly Ala Ser Arg Arg Ser Ser His Pro Leu Pro Ile Lys Thr Ser 195 200 205 Phe Pro Asp Gly Glu Phe Thr Thr Gln Asp Cys Pro Glu Cys Lys Leu 210 215 220 Arg Glu Asn Lys Tyr Phe Phe Lys Leu Gly Val Pro Ile Tyr Gln Cys 225 230 235 240 Lys Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Arg 245 250 255 Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ser Thr Cys Cys 260 265 270 Val Ala Lys Ala Phe Ile Arg Val Thr Val Met Gly Asn Ile Lys Leu 275 280 285 Glu Asn His Thr Gln Cys Tyr Cys Ser Thr Cys Tyr His His Lys Ile 290 295 300

Claims

1. A recombinant single-chain chorionic gonadotropin polypeptide, characterized in that: The polypeptide has an amino acid sequence encoding the β chain and α chain of equine chorionic gonadotropin, and the amino acid sequence is connected to one or more sequences including a glycosylation site, wherein the glycosylation site includes an N-glycosylation sequence (NTP) in the N-terminal region of the β chain, wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:

6.

2. A DNA molecule, characterized in that It encodes the recombinant single-chain chorionic gonadotropin polypeptide according to claim 1.

3. The DNA molecule according to claim 2, characterized in that It has a sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:

5.

4. An expression vector, characterized in that: It comprises the DNA molecule according to claim 2 or 3.

5. A pharmaceutical composition, characterized in that It comprises the recombinant single-chain chorionic gonadotropin polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

6. Use of the recombinant single-chain chorionic gonadotropin polypeptide according to claim 1 or the pharmaceutical composition according to claim 5 for preparing a drug for treating a disease related to mammalian reproduction or ovulation.

7. The use according to claim 6, wherein the disorder related to mammalian reproduction or ovulation is excessive ovulation, blocked ovulation, ovarian hypofunction, postpartum estrus induction or testicular hypofunction.

8. The use according to claim 6 or 7, wherein the mammal is selected from the group consisting of cattle, pigs, sheep, dogs, rabbits, deer, goats and rats.

9. A method for preparing a recombinant single-chain chorionic gonadotropin polypeptide, characterized in that: It comprises transfecting the vector according to claim 4 into mammalian cells.

10. The method according to claim 9, characterized in that The mammalian cells are CHO cells.

11. The method of claim 10, further comprising purifying the recombinant single-chain chorionic gonadotropin by immunoaffinity.

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