Long-acting recombinant porcine FSH fusion protein and preparation method and application thereof
11299527 · 2022-04-12
Assignee
Inventors
Cpc classification
C07K19/00
CHEMISTRY; METALLURGY
C07K14/59
CHEMISTRY; METALLURGY
A61K38/24
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61P15/00
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
International classification
C07K14/59
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
Two long-acting recombinant porcine FSH (follicle-stimulating hormone) fusion proteins, comprising pFSH-Fc-1 and pFSH-Fc-2. α subunit/β subunit is directly or indirectly fused on a Fc fragment by means of a linking component; the β subunit/α subunit is combined with the α subunit/β subunit by means of a Van der Waals force or the linking component. The porcine FSH fusion proteins can be prepared by an eukaryotic expression system based on a gene engineering technology. The two porcine FSH fusion proteins have a good medicinal effect and have a longer half-life period as compared with that of a natural porcine FSH; the two porcine FSH fusion proteins do not generate an undesirable effect on animals and can replace pregnant mare serum gonadotropin (PMSG) to be used in animal breeding production. The two porcine FSH fusion proteins can be used for preparation of medicines in the field of animal breeding.
Claims
1. A long-acting recombinant porcine FSH fusion protein pFSH-Fc-1; wherein the pFSH-Fc-1 comprises two peptide chains and conforms to the equation (pFSHβ:pFSHα-L-Fc).sub.2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHα subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-1 is a divalent homodimer, and the amino acid sequence of the pFSHα-L-Fc is shown in SEQ ID NO: 6, and the amino acid sequence of the pFSHβ is shown in SEQ ID NO: 3.
2. An expression cassette or an expression vector, comprising a nucleic acid encoding the recombinant porcine FSH fusion protein according to claim 1.
3. A CHO cell for expressing of a fusion protein, the cell comprising the expression vector of claim 2.
4. A method for synchronizing estrus and superovulation in an animal comprising the step of administering at least one of recombinant porcine FSH fusion proteins according to claim 1, wherein the animal is a pig, a cow or a goat.
5. The method of claim 4, wherein the animal is a pig.
6. A method for treating anestrus in a pig by administering a fusion protein according to claim 1.
7. A cloning vector comprising a nucleic acid encoding a fusion protein according to claim 1.
8. A long-acting recombinant porcine FSH fusion protein pFSH-Fc-2 comprising two peptide chains and conforms to the equation: (pFSHα:pFSHβ-L-Fc).sub.2, wherein the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHβ subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that FSH-Fc-2 is a divalent homodimer, and the amino acid sequence of the pFSHβ-L-Fc is shown in SEQ ID NO: 8, and the amino acid sequence of the pFSHα is shown in SEQ ID NO:1.
9. An expression cassette or an expression vector, comprising a nucleic acid encoding the recombinant porcine FSH fusion protein according to claim 8.
10. A CHO cell for expressing a fusion protein, the cell comprising the expression vector of claim 9.
11. A cloning vector comprising a nucleic acid encoding a fusion protein according to claim 8.
12. A method for synchronizing estrus and superovulation in an animal comprising the step of administering at least one of recombinant porcine FSH fusion proteins according to claim 8, wherein the animal is a pig, a cow or a goat.
13. A method for treating anestrus in a pig by administering a fusion protein according to claim 8.
14. A method for preparing a recombinant porcine FSH fusion protein selected from the group consisting of: (a) a long-acting recombinant porcine FSH fusion protein pFSH-Fc-1; wherein the pFSH-Fc-1 comprises two peptide chains and conforms to the equation (pFSHβ:pFSHα-L-Fc).sub.2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHα subunit and the Fc fragment; Fc refers to a Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-1 is a divalent homodimer, and the amino acid sequence of the pFSHα-L-Fc is shown in SEQ ID NO: 6, and the amino acid sequence of the pFSHβ is shown in SEQ ID NO: 3; and (b) a long-acting recombinant porcine FSH fusion protein pFSH-Fc 2 wherein the pFSH-Fc-2 comprises two peptide chains and conforms to the equation (pFSHα:pFSHβ-L-Fc).sub.2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHβ subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-2 is a divalent homodimer, and the amino acid sequence of the pFSHβ-L-Fc is shown in SEQ ID NO: 8, and the amino acid sequence of the pFSHα is shown in SEQ ID NO: 1, the method comprising: artificially synthesizing genes encoding pFSHα, pFSHβ, pFSHα-L-Fc and pFSHβ-L-Fc, performing codon optimization, and cloning the codon optimized genes into eukaryotic expression vectors, respectively; simultaneously transforming the pFSHα recombinant vector and pFSHβ-L-Fc recombinant vector into eukaryotic cells, and expressing in eukaryotic cells, and isolating and purifying the target protein; or simultaneously transforming the pFSHβ recombinant vector and pFSHα-L-Fc recombinant vector into eukaryotic cells, and expressing in eukaryotic cells, and isolating and purifying the target protein.
15. The method of claim 14, wherein the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cells include 293 and CHO cells.
16. A fusion protein produced according to the method of claim 15.
17. A fusion protein produced according to the method of claim 14.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS
(4) The following Examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise indicated, the Examples are based on routine experimental conditions, such as Sambrook J & Russell D W, Molecular Cloning: a Laboratory Manual, 2001, or according to the conditions suggested by the manufacturer's instructions.
(5) The hFSH-hFc and pFSH-hFc described in the following Examples are both obtained by linking β subunits to hFc, and the construction method is the same as that of pFSH-Fc-2, and comprises: the gene sequences of human FSHα, human FSHβ, and human Fc were found after search, after codon optimization, the hFSHα, hFSHβ-L-hFc, pFSHα, and pFSHβ-L-hFc genes were artificially synthesized, and the hFSHa and hFSHβ-L-hFc, pFSHα and pFSHβ-L-hFc recombinant plasmids were transferred into 293 cells by transient transfection for expression, and purification was performed to obtain hFSH-hFc and pFSH-hFc.
Example 1: Preparation of pFSH-Fc-1 and pFSH-Fc-2 Proteins
(6) The GeneBank was searched for gene sequences of porcine FSHα (GenBank NM-214446.1), porcine FSHβ (GenBank NM-213875.1) and porcine Fc (GenBank BAE20056). Codon optimization was performed. The nucleotide sequence of pFSHα is represented by SEQ ID NO: 2; the nucleotide sequence of pFSHβ is represented by SEQ ID NO: 4; the sequence of pFSHα-L-pFc is represented by SEQ ID NO: 7; and the sequence of pFSHr3-L-pFc is represented by SEQ ID NO: 9.
(7) The artificially synthesized pFSHα, pFSHβ, pFSHα-L-pFc and pFSHβ-L-pFc genes were cloned into the vector pcDNA3.1, respectively. The recombinant vectors of pFSHβ and pFSHα-L-pFc, pFSHα and pFSHβ-L-pFc were respectively electrotransferred into 293 cells to express pFSH-Fc-1 and pFSH-Fc-2, and the proteins obtained by transient transfection and expression were purified and verified for the activity. After the activity was confirmed, the recombinant vectors of pFSHβ and pFSHα-L-pFc, pFSHα and pFSHβ-L-pFc were linearized and then electrotransferred into CHO cells to obtain stable cell lines of pFSH-Fc-1 and pFSH-Fc-2.
(8) The stable cells of pFSH-Fc-1 and pFSH-Fc-2 were subjected to fermentation cultivation in a fermentor, and the fermentation broth was subjected to filtration to remove cells and cell debris using two-stage deep bed filtration membrane package, and then filtered through a 0.22 μm filter membrane to obtain clarified fermentation broth. The fermentation broth was firstly purified by Protein A affinity chromatography (MabSelect SuRe™, GE Healthcare): firstly, equilibrated to the baseline with an equilibration solution (50 mM glycine, 0.15 M NaCl, pH 7.2) and then eluted with an eluent (50 mM glycine, pH 3.0), and the eluate was collected. The solution collected from Protein A chromatography was further purified by Capto S cation exchange column (GE Healthcare) chromatography: the collected solution was adjusted to a pH of 6.5 with 1 M NaOH, the conductivity thereof was adjusted to 4.5 to 5.0 ms/cm with water, the collected solution was equilibrated with an equilibration solution (50 mM glycine, pH 6.5), loaded, and the flow-through effluent was collected. The solution collected from Capto S chromatography was finely purified by Capto Q anion exchange column (GE Healthcare) chromatography: the collected solution was adjusted to a pH of 8 with 1 M NaOH, and equilibrated to baseline with an equilibration solution (50 mM glycine pH 8.0), followed by eluting with eluent (50 mM glycine, 1 M KCl, pH 8.0), and the purified protein was collected. The purified protein of interest was subjected to SDS-PAGE gel electrophoresis (
Example 2: Activity Assay of pFSH-Fc-1 and pFSH-Fc-2 Proteins
(9) The activities of pFSH-Fc-1 and pFSH-Fc-2 were measured using a rat ovarian weight gain method (Steelman-Pohley method). The product of the present invention is intended to be used in place of PMSG in the field of animal breeding. Therefore, the activity of the sample was determined according to the Pharmacopoeia of China 2015 Edition “Serum Gonadotropin Bioassay”, with PMSG as a standard. The specific process for implementation was as follows: pFSH-Fc-1 (with an estimated value of specific activity of 10000 U/mg), pFSH-Fc-2 (with an estimated value of specific activity of 10000 U/mg) and PMSG were formulated into three doses of 40 IU (high), 20 IU (medium) and 10 IU (low), respectively. Female SD (Sprague Dawley) rats of 21-23 day-old, weighted 40-55 g were randomly divided into 9 groups, 6 in each group. Each rat was subcutaneously injected with 0.5 ml of the corresponding drug. After 6 days, the rats were sacrificed, weighed, and dissected, the ovaries were removed, and weighed and the ovary weight was converted into ovary weight per 100 g body weight (
Example 3: Pharmacokinetic Study of pFSH-Fc-1 and pFSH-Fc-2 Proteins
(10) Ten SD female rats of about 40 g were randomly divided into two groups: pFSH-Fc-1 group and pFSH-Fc-2 group. The corresponding drug was subcutaneously injected at 20 IU/kg body weight, 100 μl of blood was sampled at 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h after administration, respectively, centrifuged at 3000 rpm, and the serum was taken and stored at −80° C. for cryopreservation. The contents of pFSH-Fc-1 and pFSH-Fc-2 in the serum were measured using an FSH ELISA kit, and each blood sample was analyzed in triplicate. The half-life of pFSH-Fc-1 was calculated to be 57.2 h using Pksolver software, and the half-life of pFSH-Fc-2 was 63.4 h, both of which were higher than that of natural porcine FSH, and lower than that of PMSG.
Example 4: Use of pFSH-Fc-1 and pFSH-Fc-2 Proteins in Promoting Synchronization of Estrus and Superovulation in Primiparous and Multiparous Sows
(11) 100 primiparous Yorkshire sows and multiparous Yorkshire sows without estrus 2 weeks after weaning, weighed 85-100 kg with the same variety and similar signs were selected, respectively. They were randomly divided into 5 groups: pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups; each group was further divided into primiparous sow group and multiparous sow group. 1000 IU of the corresponding drugs were injected into the neck muscles at the back of ear of the donor pigs in each group, and 500 IU of HCG was injected 72 hours later. The estrus of each group of sows was observed after 5 days. Except for the oestrous No. 1 Yorkshire sow in the primiparous sow group was slaughtered and taken ovary for photographing, the oestrous sows of other groups were bred with the boars of the same variety for 3 times at intervals of 12 h each time. 36 hours after the first breeding, the donor pigs were subjected to surgery for ovums, and the number of ovums ovulated was counted (
(12) The results were shown in Table 1, The donor pigs in each group had good estrus, and the estrus rate of the primiparous and multiparous sows in the pFSH-Fc-1 group were 90% and 95%, respectively, which were higher than those of the PMSG group (60% and 65%), the hFSH-hFc group (65% and 70%) and the pFSH-hFc group (80% and 85%), and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.05). The estrus rates of the primiparous and multiparous sows in the pFSH-Fc-2 group were 100%, and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.01).
(13) The number of ovums ovulated in each group of sows was higher than that in normal natural oestrous sows (8 to 14/pig), and the average number of ovums ovulated per pig of the primiparous and multiparous sows in the pFSH-Fc-1 group was 27.7 and 27.5, respectively, which were higher than those of the PMSG group (19.8 and 20.2), the hFSH-hFc group (22.1 and 21.6), and the pFSH-hFc group (25.7 and 26.2), and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.05). The average number of ovums ovulated per pig of the primiparous and multiparous sows in the pFSH-Fc-2 group was 29.9 and 29.1, respectively, which were higher than that of the PMSG group, the hFSH-hFc group and the pFSH-hFc group, and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.05).
(14) TABLE-US-00001 TABLE 1 Comparison of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc on synchronization of estrus and superovulation in primiparous and multiparous sows pFSH-Fc-1 group pFSH-Fc-2 group PMSG group primiparous multiparous primiparous multiparous primiparous multiparous Item sows sows sows sows sows sows Number of sows 20 20 20 20 20 20 tested Number of 18 19 20 20 12 13 oestrous sows Estrus rate 90%.sup.ACe 95%.sup.BDf 100%.sup.AACCe 100%.sup.BBDDf 60%.sup.a 65%.sup.b Number of 11 11 11 11 11 11 fertilized sows Number of 27.7 ± 2.10.sup.GIk 27.5 ± 2.11.sup.HJl 29.9 ± 2.66.sup.GJk 29.1 ± 2.30.sup.HJl 19.8 ± 2.04.sup.g 20.2 ± 2.13.sup.h ovums ovulated per pig pFSH-hFc group pFSH-hFc group primiparous multiparous primiparous multiparous Item sows sows sows sows Number of sows 20 20 20 20 tested Number of 13 14 16 17 oestrous sows Estrus rate 65%.sup.c 70%.sup.d 80%.sup.e 85%.sup.f Number of 11 11 11 11 fertilized sows Number of 22.1 ± 2.34.sup.i 21.6 ± 1.91.sup.j 25.7 ± 2.33.sup.k 26.2 ± 2.23.sup.l ovums ovulated per pig Note: The different capital and lower-case letters of the series superscript on the same line showed significant difference (P < 0.05), different double capital and lower-case letters showed highly significant difference (P < 0.01), and the same letter showed non-significant difference (P > 0.05).
Example 5: Application of pFSH-Fc-1 and pFSH-Fc-2 Proteins in Increasing Litter Size of SOWS
(15) 50 primiparous Yorkshire sows with the same variety and similar signs were selected. They were divided into 5 groups: pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups, each group of sows were injected with drugs according to the method of Example 4. During estrus, the oestrous sows were bred with the boars of the same variety for 3 times at intervals of 12 h each time. The litter size of each group of sows was recorded in detail.
(16) The results were shown in Table 2, the total number of litter sizes of sows in the pFSH-Fc-1 and pFSH-Fc-2 groups (123 and 125) were higher than that of the PMSG group (65), the hFSH-hFc group (68) and the pFSH-hFc group (99), and were significantly different from those of the PMSG and the hFSH-hFc groups (P<0.05). The average number of litter sizes per birth of the pFSH-Fc-1 and pFSH-Fc-2 groups (13.7 and 13.9) were also higher than that of the PMSG group (10.1), the hFSH-hFc group (10.5), and the pFSH-hFc group (12.4).
(17) TABLE-US-00002 TABLE 2 Comparison of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc on litter sizes of primiparous sows Number Total Average Number of number number of of sows farrowing of litter litter sizes Groups tested sows sizes per birth pFSH-Fc-1 group 10 9 123.sup.ABc 13.7 ± 1.25 pFSH-Fc-2 group 10 9 125.sup.ABc 13.9 ± 1.19 PMSG group 10 6 65.sup.a 10.1 ± 1.34 hFSH-hFc group 10 6 68.sup.b 10.5 ± 1.38 pFSH-hFc group 10 8 99.sup.c 12.4 ± 1.27 Note: The different capital and lower-case letters of the series superscript on the same column showed significant difference (P < 0.05), and the same letter showed non-significant difference (P > 0.05).
Example 6: Application of pFSH-Fc-1 and pFSH-Fc-2 Proteins in the Treatment of Anestrus Sows
(18) 100 anestrus Yorkshire sows without estrus for more than 21 days after weaning were randomly divided into 5 groups: pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups. 1000 IU of the corresponding drugs were respectively injected into the neck muscles at the back of ear of the donor pigs in each group, and 500 IU of HCG was injected 72 hours later. The estrus characteristics of sows were observed: such as redness and mucus in the vulva; and standing reflex occurs when the back is pressed. The oestrous sows were bred with boars according to Examples 4 and 5, and the impregnation conditions were recorded.
(19) The results were shown in Table 3. The anestrus sows were sensitive to drug reactions, and the estrus rates of the sows in the pFSH-Fc-1 and pFSH-Fc-2 groups (85% and 90%) were higher than those of the PMSG group (55%), the hFSH-hFc group (65%) and the pFSH-hFc group (75%), and were significantly different from that of the PMSG group (P<0.05). The pregnancy rates of the sows in the pFSH-Fc-1 and pFSH-Fc-2 groups (88.2% and 88.9%) were also higher than those of the PMSG group (63.6%), the hFSH-hFc group (61.5%) and the pFSH-hFc group (73.3%), and the difference was not significant (P>0.05).
(20) TABLE-US-00003 TABLE 3 Comparison of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc in inducing the estrus of the anestrus sows Number Number of of sows oestrous Groups tested sows Estrus rates Pregnancy rates pFSH-Fc- 20 17 85%.sup.Abc (17/20) 88.2%.sup.def (15/17) 1 group pFSH-Fc- 20 18 90%.sup.Abc (18/20) 88.9%.sup.def (16/18) 2 group PMSG group 20 11 55%.sup.a (11/20) 63.6%.sup.d (7/11) hFSH-hFc 20 13 65%.sup.b (13/20) 61.5%.sup.e (8/13) group pFSH-hFc 20 15 75%.sup.c (15/20) 73.3%.sup.f (11/15) group Note: The different capital and lower-case letters of the series superscript on the same column showed significant difference (P < 0.05), and the same letter showed non-significant difference (P > 0.05).
Example 7: Application of pFSH-Fc-1 and pFSH-Fc-2 Proteins in Promoting Superovulation of Cows
(21) 50 3-6 years old, healthy, disease-free Holstein cows were randomly divided into pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc, and pFSH-hFc groups. Each group of cows was fed 1 kg of concentrative feed based on the original feeding, at the same time, VA, VD and VE were intramuscularly injected. Each donor cow was implanted with progesterone vaginal plug CIDR (containing progesterone 1.56 g/vaginal plug). The day of plugging was recorded as Day 0, each group of donor cows was intramuscularly injected with 1000 IU of the corresponding drug (Day 5), and 0.5 mg of cloprostenol (PG) was injected, and then the plug was removed (Day 10), and the estrus was observed, the mounting of the bull accepted by donor cow shall prevail. The first insemination was performed 12 h after standing estrus, the second insemination was performed 24 h after standing estrus. Embryos were collected on Day 16 by non-surgically washing, and the number of embryos was counted.
(22) The results were shown in Table 4, the superovulation effects of donor cows in each administration group were remarkable (naturally, one cow generates only one embryo at a time), and the average numbers of embryos per cow in the pFSH-Fc-1 group and the pFSH-Fc-2 group (7.9 and 8.7) were higher than that of the PMSG group (5.7), the hFSH-hFc group (6.1), and the pFSH-hFc group (7.3), and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.05). The average numbers of available embryos per cow in the pFSH-Fc-1 and the pFSH-Fc-2 groups (6.1 and 7.4) were higher than those in the PMSG group (3.5), the hFSH-hFc group (3.9) and the pFSH-hFc group (5.7), and were significantly different from those of the PMSG group and the hFSH-hFc group (P<0.05). The average numbers of unavailable embryos per cow in the pFSH-Fc-1 and pFSH-Fc-2 groups (1.8 and 1.3) were lower than those in the PMSG group (2.2) and the hFSH-hFc group (2.2), and that in the pFSH-Fc-2 group was less than that in the pFSH-hFc group (1.6), and the difference between the pFSH-Fc-2 group and the PMFG group as well as the hFSH-hFc group was significant (P<0.05).
(23) TABLE-US-00004 TABLE 4 Comparison of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc on superovulation of Holstein cows Average Average Average numbers of numbers of numbers of available unavailable embryos embryos embryos Groups per cow per cow per cow pFSH-Fc-1 group 7.9 ± 0.70.sup.ABc 6.1 ± 0.94.sup.DEf 1.8 ± 0.40.sup.ghi pFSH-Fc-2 group 8.7 ± 0.78.sup.ABc 7.4 ± 0.91.sup.DEf 1.3 ± 0.46.sup.GHi PMSG group 5.7 ± 0.64.sup.a 3.5 ± 0.81.sup.d 2.2 ± 0.60.sup.g hFSH-hFc group 6.1 ± 0.70.sup.b 3.9 ± 0.70.sup.e 2.2 ± 0.87.sup.h pFSH-hFc group 7.3 ± 0.95.sup.c 5.7 ± 0.95.sup.f 1.6 ± 0.70.sup.i Note: The different capital and lower-case letters of the series superscript on the same column showed significant difference (P < 0.05), and the same letter showed non-significant difference (P > 0.05).
Example 8 Application of pFSH-Fc-1 and pFSH-Fc-2 Proteins in Promoting Synchronization of Estrus in Female Goats
(24) 75 1.5-3 years old, weighed 30-45 kg, healthy, disease-free goats were randomly divided into pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc groups. The progesterone vaginal plug was implanted on the donor goat at any day in the estrus cycle and the day was recorded as Day 0. Each donor goat was intramuscularly injected with 300 IU of the corresponding drug (Day 10), and then the plug was removed (Day 12). The estrus performance of the female goat was observed, and a teaser goat was used to judge whether the female goat was oestrous. The situations of redness and mucus in the vulva of the female goat and the acceptance of mounting were considered as estrus, and the estrus rate was calculated.
(25) The results were shown in Table 5, the estrus of female goats in each group was obvious, and the estrus rates of female goats in the pFSH-Fc-1 group and the pFSH-Fc-2 group were both 93.3%, which were higher than those of the PMSG group (60%), the hFSH-hFc group (73.3%) and the pFSH-hFc group (80.0%), and were significantly different from that of the PMSG group (P<0.05).
(26) TABLE-US-00005 TABLE 5 Comparison of pFSH-Fc-1, pFSH-Fc-2, PMSG, hFSH-hFc and pFSH-hFc on estrus of female goats Number Number of female of oestrous goats female Groups tested goats Estrus rates pFSH-Fc-1 group 15 14 93.3%.sup.Abc (14/15) pFSH-Fc-2 group 15 14 93.3%.sup.Abc (14/15) PMSG group 15 9 60.0%.sup.a (9/15) hFSH-hFc group 15 11 73.3%.sup.b (11/15) pFSH-hFc group 15 12 80.0%.sup.c (12/15) Note: The different capital and lower-case letters of the series superscript on the same column showed significant difference (P < 0.05), and the same letter showed non-significant difference (P > 0.05).
Example 9: Detection of Antigenic Immunity of pFSH-Fc-1 and pFSH-Fc-2 Proteins
(27) The antigenic immunities of the pFSH-Fc-1 and pFSH-Fc-2 proteins were detected by detecting the anti-drug antibody (ADA) of the drug in the serum of the sample by using the Bridging-ELISA method. 40 primiparous Yorkshire sows were selected and divided into 4 groups: pFSH-Fc-1 group, pFSH-Fc-2 group, hFSH-hFc group and pFSH-hFc group. 1000 IU of the drugs were injected into the neck muscle at the back of ear of each donor pig, and administered once every three days for a total of 5 weeks (13 times of administration), and the day for the first administration was recorded as Day 0. Blood was collected at the following times: before the first administration (Day −2), before the third administration (Day 6), before the fifth administration (Day 12), and before the sixth to last administration (Day 15, 18, 21, 24, 27, 30, 33 and 36), and the third day after the last administration (Day 39). The blood was centrifuged, the serum was collected, and the OD.sub.490nm value of ADA in the serum was detected in the ELISA plate coated with pFSH-Fc-1, pFSH-Fc-2, hFSH-hFc or pFSH-hFc, respectively. Positive control samples were prepared with anti-pFSH-Fc-1, anti-pFSH-Fc-2, anti-hFSH-hFc or anti-pFSH-hFc rabbit monoclonal antibody diluted with 100% mixed pig serum, respectively; and negative control (N) samples were prepared with serum of a primiparous Yorkshire sow injected with the same amount of PBS buffer. The threshold value (SCP) was used as the judgment value to distinguish whether the test sample was positive or negative. The SCP was calculated to be 1.15 by using JMP® statistical analysis, that is, SCP 1.15 was judged to be positive, and the sample contained ADA; otherwise it was negative and the sample did not contain ADA.
(28) The results were shown in Table 6, the SCPs of the donor pig samples in pFSH-Fc-1 group and pFSH-Fc-2 group were all less than 1.15, and no ADA was detected, indicating that no anti-drug antibody was generated in the Yorkshire sows injected with 1000 IU of pFSH-Fc-1 or pFSH-Fc-2. In the hFSH-hFc group, the SCPs in the serum samples of Nos. 1, 2, 4, 7 and 9 sows were greater than 1.15 on Day 27 and after Day 27 (first detected on Day 27, and last detected on Day 39), and the SCPs in the serum samples of other sows were greater than 1.15 on Day 30 and after Day 30 (first detected on Day 30, and last detected on Day 39), indicating that ADA was detected in all sows in the hFSH-hFc group, i.e., a positive rate of 100%. In the pFSH-hFc group, the SCPs in the serum samples of Nos. 2, 3, 6, 7 and 9 sows were greater than 1.15 on Day 30 and after Day 30 (first detected on Day 30, and last detected on Day 39), the SCPs in the serum samples of Nos. 1, 5, and 8 sows were greater than 1.15 on Day 36 and after Day 36 (first detected on Day 36, and last detected on Day 39), and the SCPs in all serum samples of Nos. 4 and 10 sows were less than 1.15, indicating that ADA was detected in 8 sows in the pFSH-hFc group, i.e., a positive rate of 80%. The above-mentioned results indicate that the Yorkshire sows injected with 1000 IU of hFSH-hFc or pFSH-hFc generate anti-drug antibody.
(29) TABLE-US-00006 TABLE 6 Detection of antigenic immunity of pFSH-Fc-1, pFSH-Fc-2, hFSH-hFc and pFSH-hFc to Yorkshire sows Groups pFSH-Fc-1 pFSH-Fc-2 hFSH-hFc pFSH-hFC Administration 1000IU/pig 1000IU/pig 1000IU/pig 1000IU/pig dosage Time for the — — Day 27 Day 30 first detection Time for the — — Day 39 Day 39 last detection Number of 10 10 10 10 individuals in each group Number of 0 0 10 8 positive individuals Individual 0% 0% 100% 80% positive rate
(30) Although the present invention is described in detail using general description and specific embodiments hereinbefore, it is obvious to a person skilled in the art that some modifications or improvements can be made based on the present invention. Hence all these modifications or improvements made on the basis of not deviating from the spirit of the present invention fall into the protection scope claimed in the present invention.
INDUSTRIAL APPLICABILITY
(31) The present invention provides two long-acting recombinant porcine FSH (follicle-stimulating hormone) fusion proteins, comprising pFSH-Fc-1 and pFSH-Fc-2. α subunit/β subunit is directly or indirectly fused with a Fc fragment by means of a linking component; the β subunit/α subunit binds with the α subunit/β subunit by means of a Van der Waals force or the linking component. The porcine FSH fusion proteins can be prepared by an eukaryotic expression system based on a gene engineering technology. The two porcine FSH fusion proteins provided by the present invention have a good medicinal effect and have a longer half-life period as compared with that of a natural porcine FSH; do not generate an undesirable effect in animals, do not have immunogenicity in the sow, do not cause anti-drug antibody, and can replace pregnant mare serum gonadotropin (PMSG) used in animal breeding production, can effectively promote the reproduction rate of animals, especially economic animals, and have good economic value and application prospects.
COMPUTER READABLE SEQUENCE LISTING
(32) The material contained in the ASCII text file entitled, “CNKH27US_AmendedSequence_Listing4” created on Jul. 16, 2021, having a size of 14 kB is hereby incorporated by reference herein in its entirety.