CN108676096B

CN108676096B - Recombinant porcine FSH-CTP fusion protein and preparation method and application thereof

Info

Publication number: CN108676096B
Application number: CN201810495854.XA
Authority: CN
Inventors: 罗昊澍; 师磊; 韩国
Original assignee: Beijing Weijiexin Biotechnology Co ltd
Current assignee: Beijing Weijiexin Biotechnology Co ltd
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2022-03-29
Anticipated expiration: 2038-05-22
Also published as: CN108676096A

Abstract

The invention provides a recombinant porcine FSH-CTP fusion protein, wherein the fusion protein is formed by directly or indirectly connecting a beta subunit of porcine FSH with a beta subunit carboxy terminal peptide CTP of human, primate or equine mammal chorionic gonadotropin, and combining an alpha subunit of the porcine FSH with the beta subunit of the porcine FSH through van der Waals force. The fusion protein can be prepared by utilizing a eukaryotic expression system based on a genetic engineering technology. Compared with the pig pituitary FSH, the pig FSH-CTP fusion protein provided by the invention has longer half-life and better drug effect, and can replace the pig pituitary FSH and pregnant mare serum gonadotropin on the current market to be used in the field of animal reproduction.

Description

Recombinant porcine FSH-CTP fusion protein and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedicine and animal reproduction, in particular to a recombinant porcine FSH-CTP fusion protein and a preparation method and application thereof.

Background

Porcine Follicle-stimulating hormone (pFSH) is a glycoprotein gonadotropin hormone secreted by the anterior pituitary, and pFSH can promote the growth of endometrium, ovary and Follicle of a sow; promoting the synthesis and secretion of estrogen; inducing the development of boar seminiferous tubule and maintaining spermatogenesis. pFSH is commonly used in the field of animal reproduction for the treatment of estrus synchronization, superovulation, embryo transfer, dam ovarian disease.

pFSH contains two subunits, α and β, the α subunit responsible for signal transcription and the β subunit involved in receptor binding for biological function. The α subunit of pFSH is identical to the α subunits of other pituitary glycoprotein hormones, such as porcine luteinizing hormone (pLH) and porcine thyrotropin (pTSH), and consists of 96 amino acid residues, contains 2N-linked glycosylation sites, and is located at N56 and N82. While the beta subunits of pFSH, pLH and pTSH are different, the pFSH beta subunit contains 109 amino acid residues and also 2N-linked glycosylation sites, located at N5 and N22.

Gonadotropins, which are common in the field of animal reproduction at present, mainly include porcine pituitary fsh (pfsh) and pregnant horse serum gonadotropin (PMSG). pFSH is mainly extracted and purified from pig pituitary, such as Folltropin-V (Belrnicke, Canada), and has low purity, low content, high cost, and great side effect due to the existence of LH. In addition, the half-life period is short (the half-life period in the body of the rat is about 5 hours), the injection is used for the synchronous estrus and superovulation of livestock for 3 to 5 days, and the drug input cost and labor force of a user are increased, so that the practical application is limited. PMSG is a glycoprotein hormone secreted by chorioallantoic cells of placenta of equine animals, has FSH (high) and LH (low) activities, long half-life (more than 120h in cattle), durable drug effect and can obtain ideal effect only by one-time injection. However, PMSG is mainly extracted from serum of pregnant mares, the source is limited, the product batch difference is large, and the mares are aborted and killed due to excessive blood collection.

Based on the inconvenience and limitation of the practical application of pig pituitary FSH and PMSG, a long-acting FSH preparation needs to be developed to prolong the half-life of pFSH and increase the residual time of pFSH in animals.

Disclosure of Invention

The invention aims to provide a recombinant porcine FSH-CTP fusion protein and a preparation method and application thereof.

The concept of the invention is as follows: by increasing the glycosylation degree of FSH, the molecular mass and the volume of FSH are increased, the renal clearance of glomeruli is reduced, and the action time of FSH in vivo is prolonged. CTP contains a plurality of O-linked glycosylation sites, can increase sialic acid glycosylation side chains and improve the molecular mass of glycoprotein. The closest natural protein molecules in terms of molecular structure, physicochemical properties and biological function can be obtained by expressing recombinant proteins using mammalian expression systems, in particular chinese hamster ovary Cells (CHO).

In order to achieve the purpose of the invention, the recombinant porcine FSH-CTP fusion protein provided by the invention is characterized in that a beta subunit of porcine FSH is directly or indirectly connected with a beta subunit carboxy terminal peptide CTP of chorionic gonadotropin, and an alpha subunit of porcine FSH is combined with the beta subunit of porcine FSH through van der Waals force.

Wherein the chorionic gonadotropin is derived from a human, primate, or equine mammal. Preferably from humans and horses.

Human CTP consists of 28 amino acid residues, containing 4O-linked glycosylation sites; equine CTP consists of 35 amino acid residues containing 12O-linked glycosylation sites.

The fusion protein is pFSH-hCTP or pFSH-eCTP.

pFSH-hCTP: contains two peptide chains, and meets the following equation: (pFSH α: pFSH β -hCTP)₂Wherein pFSH α refers to the α subunit of porcine FSH; colons represent porcine FSH α and β subunits connected by van der waals forces; pFSH β refers to the β subunit of porcine FSH; hCTP refers to human chorionic gonadotropin beta subunit carboxy terminal peptide; the bracketed outer subscript 2 indicates that the porcine FSH fusion protein is a bivalent homodimer.

Preferably, the fusion protein pFSH β -hCTP has the following pFSH β -hCTP:

i) a protein consisting of an amino acid sequence shown as SEQ ID NO. 3; or

ii) protein which is derived from i) and has the same function by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3; or

iii) protein composed of amino acid sequence with homology of more than 90% with the amino acid sequence shown in SEQ ID NO. 3 and with the same function.

pFSH-eCTP: contains two peptide chains, and meets the following equation: (pFSH α: pFSH β -eCTP)₂Wherein pFSH α refers to the α subunit of porcine FSH; colons represent porcine FSH α and β subunits connected by van der waals forces; pFSH β refers to the β subunit of porcine FSH; eCTP refers to equine chorionic gonadotropin beta subunit carboxy terminal peptide; the bracketed outer subscript 2 indicates that the porcine FSH fusion protein is a bivalent homodimer.

Preferably, the fusion protein pFSH beta-eCTP in pFSH-eCTP is:

iv) a protein consisting of the amino acid sequence shown in SEQ ID NO. 5; or

v) protein which is derived from iv) and has the same function and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 5; or

vi) a protein consisting of an amino acid sequence having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO. 5 and having the same function.

The amino acid sequence of the pFSH alpha is shown as SEQ ID NO. 1, or the protein which has homology of more than 90 percent with the SEQ ID NO. 1 and is composed of amino acid sequences with the same functions.

The modified protein, including two fusion proteins pFSH-hCTP, pFSH-eCTP or porcine FSH, is glycosylated, pegylated, acetylated or combined with BSA, etc., which belong to the protection scope of the invention.

The modified protein comprises two fusion proteins of pFSH-hCTP, pFSH-eCTP or fusion protein formed by fusing pig FSH protein with CTP or other proteins and does not change the activity of the pig FSH protein, and the modified protein belongs to the protection scope of the invention.

The invention also provides an expression cassette, an expression vector, a cloning vector, an engineering bacterium or a transgenic cell line, which comprises the nucleic acid for encoding the porcine FSH-CTP fusion protein.

The long-acting recombinant porcine FSH-CTP fusion protein can be prepared by the following method:

the fusion protein pFSH-hCTP is prepared as follows: optimizing and artificially synthesizing the encoding genes of pFSH alpha and pFSH beta-hCTP, respectively constructing the gene sequences of the optimized pFSH alpha and pFSH beta-hCTP in different expression cassettes, then connecting to the same expression vector, transforming host cells, expressing in the host cells, and separating and purifying target proteins; alternatively, the first and second electrodes may be,

and respectively constructing the optimized gene sequences of pFSH alpha and pFSH beta-hCTP on expression vectors, jointly transforming the obtained recombinant vectors into host cells, expressing the host cells, and separating and purifying the target protein.

The fusion protein pFSH-eCTP is prepared as follows: optimizing and artificially synthesizing encoding genes of pFSH alpha and pFSH beta-eCTP, respectively constructing the optimized pFSH alpha and pFSH beta-eCTP gene sequences on different expression cassettes, then connecting the expression cassettes to the same expression vector, transforming host cells, expressing the host cells, and separating and purifying target proteins; alternatively, the first and second electrodes may be,

and respectively constructing the optimized pFSH alpha and pFSH beta-eCTP gene sequences on expression vectors, jointly transforming the obtained recombinant vectors into host cells, expressing the host cells, and separating and purifying the target protein.

Wherein, the optimized gene sequences of pFSH alpha, pFSH beta-hCTP and pFSH beta-eCTP are respectively shown as

SEQ ID NO

2, 4 and 6.

The expression vector is a eukaryotic expression vector and comprises but is not limited to pcDNA3.1, and the host cell is a eukaryotic cell and comprises but is not limited to 293 and CHO cells.

The invention also provides an ovulation-promoting medicine or a composition, and the active ingredient of the ovulation-promoting medicine or the composition is the recombinant porcine FSH-CTP fusion protein pFSH-hCTP and/or pFSH-eCTP.

The invention also provides any one of the following applications of the recombinant porcine FSH-CTP fusion protein:

1) promoting animal reproduction including estrus synchronization and superovulation;

2) application in preparing medicine for treating animal reproduction related diseases.

Wherein the animal is a mammal, including a pig, a cow, a sheep, a horse, or a dog; preferably pigs.

The invention also provides application of the long-acting recombinant porcine FSH-CTP fusion protein in the field of animal reproduction.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the recombinant porcine FSH-CTP fusion protein and the derivative protein or modified protein thereof have high purity of about 95 percent after purification, uniform quality, high safety factor and convenient large-scale production.

The half-life period of pFSH-hCTP in rats is about 25.7h, the half-life period of pFSH-eCTP is about 36.4h, the half-life period is longer than that of pig pituitary FSH (5h), only one injection is needed in one estrus cycle, continuous injection is not needed, and the administration cost and labor force are reduced.

And thirdly, the recombinant porcine FSH-CTP fusion protein, and the derived protein or modified protein thereof provided by the invention promote estrus synchronization and superovulation of sows, and further improve the farrowing performance of the sows, wherein the estrus synchronization effectiveness is more than 80%, the head average ovulation number is about 21-24, the head litter size is about 11-13, and the improved protein is superior to PMSG and pFSH.

The recombinant porcine FSH-CTP fusion protein, and the derived protein or modified protein thereof provided by the invention can also improve the estrus synchronization and superovulation of cattle and sheep, wherein the number of head-average embryos of Holstein cows is 5.6-6.9, and the estrus synchronization rate of pFSH-hCTP and pFSH-TP to the ewes reaches 93% and 80%, which is superior to that of PMSG and pFSH.

The recombinant porcine FSH-CTP fusion protein, and the derived protein or modified protein thereof have high protein purity, uniform quality and high safety, can effectively prolong the half-life of the porcine FSH, and reduce the administration frequency; the oestrus rate and ovulation rate of pigs, cattle, sheep and the like are improved, and the pFSH and PMSG can be replaced for application in the field of animal reproduction.

Drawings

FIG. 1 is an SDS-PAGE non-reduced electrophoretogram of pFSH-hCTP in example 1 of the present invention. Wherein, MK: a protein Marker; 1: clarifying the fermentation liquor; 2: capto MMC harvest; 3: butyl collecting liquid; 4: capto Q pool.

FIG. 2 is a SDS-PAGE non-reduced electrophoretogram of pFSH-eCTP in example 1 of the present invention. Wherein, MK: a protein Marker; 1: clarifying the fermentation liquor; 2: capto MMC harvest; 3: butyl collecting liquid; 4: capto Q pool.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The pFSH used in the examples below is a commercially available porcine pituitary follitropin product.

Example 1 preparation of pFSH-hCTP and pFSH-eCTP proteins

The gene library was searched for the gene sequences of porcine FSH alpha (GenBank NM-214446.1), porcine FSH beta (GenBank NM-213875.1), human CG beta (GenBank NM-000737.3), and equine CG beta (GenBank NM-001197093.1). Carrying out codon optimization: pFSH alpha nucleotide sequence shown in SEQ ID NO. 2; a pFSH beta-hCTP sequence shown as SEQ ID NO. 4; pFSH beta-eCTP sequence shown in SEQ ID NO 6.

The artificially synthesized pFSH alpha, pFSH beta-hCTP and pFSH beta-eCTP genes were cloned into the vector pcDNA3.1, respectively. Recombinant vectors of pFSH alpha and pFSH beta-hCTP, pFSH alpha and pFSH beta-eCTP are respectively transferred into 293 cells to express pFSH-hCTP and pFSH-eCTP, and the transiently expressed protein is purified to verify the activity. After the activity exists, the recombinant vectors of pFSH alpha and pFSH beta-hCTP, pFSH alpha and pFSH beta-eCTP are linearized and then are electrotransferred into CHO cells to obtain the stable cell lines of pFSH-hCTP and pFSH-eCTP.

Inoculating the stable cell into a fermentation tank for fermentation culture, removing cells and cell debris from the fermentation liquor by a two-stage deep filtration membrane package, and filtering with a 0.22 μm filter membrane to obtain clear fermentation liquor. The clarified broth is first purified by weak cation exchange chromatography (e.g. Capto MMC, GE Healthcare): the sample was equilibrated with an equilibration solution (20mM ammonium acetate, pH5.0), and then eluted with an eluent (50mM glycine, 1M potassium chloride, pH8.0) to collect the eluate. The weak cation exchange chromatography pool is then purified by hydrophobic chromatography (e.g. Butyl, GE Healthcare): the sample was equilibrated with an equilibration solution (50mM glycine, 1M potassium chloride, pH8.0), and then eluted with an eluent (10mM PB, pH8.0) to collect the eluate. Finally, the hydrophobic chromatography pool is further purified by strong anion exchange (e.g. Capto Q, GE Healthcare) chromatography: the sample was equilibrated with an equilibration solution (50mM glycine, pH8.0), and then eluted with an eluent (50mM glycine, 1M KCl, pH8.0) to collect the purified target protein. The objective protein was subjected to SDS-PAGE gel electrophoresis (FIGS. 1 and 2).

Example 2 Activity assays of pFSH-hCTP and pFSH-eCTP proteins

The activities of pFSH-hCTP and pFSH-eCTP were measured by the rat ovarian weight increasing method (Steelman-Pohley method). The product of the invention can replace PMSG to be used in the field of animal reproduction, so the activity of the medicine is detected by referring to a 'blood gonadotropin bioassay' and PMSG is taken as a standard substance. The specific implementation is as follows: pFSH-hCTP (estimated specific activity 10000U/mg), pFSH-eCTP (estimated specific activity 10000U/mg) and PMSG are prepared into three doses of 40IU, 20IU and 10IU, namely high, medium and low doses. Female SD (Sprague Dawley) rats, 21-23 days old and 40-55g in weight, were randomly divided into 9 groups of 6 rats. Each rat was injected subcutaneously with 0.5ml of the corresponding drug, and after 6 days, the rat was sacrificed, weighed, dissected, and ovaries were removed, weighed, and converted into ovaries per 100g of the weight. The specific activity of pFSH-hCTP was calculated to be about 7900U/mg and pFSH-eCTP was calculated to be about 8700U/mg using the pharmacopoeia bioassay statistics BS2000 software of the institute of China.

Example 3 pharmacokinetic study of pFSH-hCTP and pFSH-eCTP proteins

About 10 female SD rats of about 40g were selected and randomly divided into two groups: the pFSH-hCTP group and the pFSH-eCTP group. Injecting the corresponding medicine with 20IU/kg body weight subcutaneously, taking 100 μ l of blood after administration of 0, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120 and 144h respectively, centrifuging at 3000rpm, and freezing and storing at-80 deg.C. Serum pFSH-hCTP and pFSH-eCTP were assayed in triplicate for each blood sample using the FSH ELISA kit. Pksolver software was used to calculate a half-life of pFSH-hCTP of 25.7h and a half-life of pFSH-eCTP of 36.4h, which is higher than that of pig pituitary FSH (pFSH has a half-life of about 5h in rats).

Example 4 use of pFSH-hCTP and pFSH-eCTP proteins to promote estrus synchronization and superovulation in primiparoused and multiparous sows

Respectively selecting 80 first-born large white sows and multiparous large white sows which do not have oestrus after weaning for 2 weeks, wherein the weight of the sows is 85-100kg, the breeds are the same, and the physical signs are close to each other. Randomized into 4 groups: pFSH-hCTP, pFSH-eCTP, PMSG and pFSH groups; the group is divided into a first-born sow group and a multiparous sow group. 1000IU of corresponding medicine is injected into the neck of donor pigs in the groups of pFSH-hCTP, pFSH-eCTP and PMSG through muscle injection, and 500IU of HCG is injected after 72h interval. Donor pigs in the pFSH group were intramuscularly injected with 400IU pFSH twice a day, 4 days, 100IU a day, and 500IU HCG 72h after the last injection. Oestrus was observed and recorded in groups of sows 5 days after pFSH-hCTP, pFSH-eCTP, PMSG or the first pFSH injection. The estrus sow and the congeneric boar are bred for 3 times, and the interval is 12h each time. After 36h of the first mating, the donor pigs were surgically egg-collected and the number of ovulations was calculated.

As a result, as shown in Table 1, the donor sows in each group showed good estrus, the estrus rates of the first-farrowing and multiparous sows in the pFSH-hCTP group were 80% and 85%, respectively, and the estrus rates of the first-farrowing and multiparous sows in the pFSH-eCTP group were 65% and 60%, respectively, which were higher than those in the PMSG group (50% and 55%) and the pFSH group (60% and 55%), and the pFSH-hCTP group differed significantly from those in the PMSG group and the pFSH group (P < 0.05).

The ovulation number of each group of sows is higher than that of normal naturally-estrous sows (8-14 sows/head), the head average ovulation number of the first-born sows and the multiparous sows of the pFSH-hCTP group is 23.7 and 22.9 respectively, the head average ovulation number of the first-born sows and the multiparous sows of the pFSH-eCTP group is 21.3 and 21.5 respectively, the head average ovulation number of the two groups of sows is higher than that of the PMSG group (19.1 and 19.4) and the pFSH group (20.6 and 20.3), the ratio difference between the first-born sows and the multiparous sows of the pFSH-hCTP group and the PMSG group and the ratio between the second-born sows of the PMSG group and the pFSH group is obvious (P <0.05), and the ratio difference between the first-born sows of the pFSH-eCTP group and the multiparous sows and the PMSG group is obvious (P < 0.05).

Example 5 use of pFSH-hCTP and pFSH-eCTP proteins to increase litter size in sows

Selecting 40 initial-born large white sows with the weight of 85-100kg, the same breed and similar physical signs. And (4) grouping: in the pFSH-hCTP, pFSH-eCTP, PMSG and pFSH groups, the sows were injected with the corresponding drugs according to the method described in example 4. After the sows are in estrus, the sows in estrus are selected to be bred with the congeneric boars for 3 times, and the interval of each time is 12 h. The oestrus rate and litter size of the sows in each group were recorded in detail.

Results the total litter size of the sows in the pFSH-hCTP group and pFSH-eCTP group as shown in Table 2 was 100 and 64, respectively, which were higher than those in the PMSG group (49) and the pFSH group (53), and the ratio of the pFSH-hCTP group to the PMSG group differed significantly (P < 0.05). The average litter sizes of the pFSH-hCTP group and the pFSH-eCTP group were 12.5 and 10.7, respectively, which were also higher than those of the PMSG group (9.8 head) and the pFSH group (8.8 head).

TABLE 2 comparison of pFSH-hCTP, pFSH-eCTP, PMSG and pFSH for litter size of gilts from farrowing

Note: the different case letters of the shoulder marks in the same column series indicate that the difference is significant (P <0.05), and the same letters indicate that the difference is not significant (P > 0.05).

Example 6 use of pFSH-hCTP and pFSH-eCTP proteins in the treatment of anestrus sows

60 anestrus white sows which are not estrualized for more than 21 days after weaning are selected, randomly divided into 4 groups of pFSH-hCTP, pFSH-eCTP, PMSG and pFSH, and the corresponding medicines are injected into the donor pigs according to the method of example 4. Observing the oestrus characteristics of the sows: such as red swelling and mucus in vulva; when pressed against the back, a static reaction occurs. And (5) mating the sow after estrus with the boar, and recording the conception condition.

As a result, as shown in Table 3, anestrus sows were sensitive to drug response, the oestrus rates of the sows in the pFSH-hCTP and pFSH-eCTP groups were 87% and 67%, respectively, which were higher than those in the PMSG group (53%) and the pFSH group (53%), and the pFSH-hCTP group differed significantly from those in the PMSG group and the pFSH group (P < 0.05). The conception rates of sows in the pFSH-hCTP and pFSH-eCTP groups were 85% and 80%, respectively, which were higher than those in the PMSG group (75%) and the pFSH group (63%).

TABLE 3 comparison of oestrus of pFSH-hCTP, pFSH-eCTP, PMSG and pFSH on induced anestrus sows

Example 7 use of pFSH-hCTP and pFSH-eCTP proteins to promote superovulation in cows

40 holstein cows with healthy constitution and no diseases at the age of 3-6 years are selected and randomly divided into pFSH-hCTP, pFSH-eCTP, PMSG and pFSH groups. Feeding 1kg of concentrate on the basis of the original feeding of each group of cows, and simultaneously performing intramuscular injection of VA, VD and VE. Each donor cow was embedded with progesterone pessary CIDR (containing 1.56 g/branch of progesterone). On Day of thrombolysis, Day 0, donor cattle of the pFSH-hCTP, pFSH-eCTP and PMSG groups were each intramuscularly injected with 1000IU of the corresponding drug (Day5) together with 0.5mg of chloroprostenol (PG), and thrombolysis was performed (Day 10). Donor cows in the pFSH group were administered 370IU pFSH intramuscularly twice daily beginning on days Day6 to Day9 at doses of 680/80 IU Day, 750/50 IU Day, 835/35 IU Day, 920/20 IU Day, 0.4mg PG injection and Day10 withdrawal concurrently with pFSH injection by Day9, respectively. Observing the oestrus condition of each group of donor cattle, and taking the situation that the donor cattle receives the bull to climb. The first insemination is carried out after the estrus is in a standing state for 12 hours, and the second insemination is carried out after 24 hours. Embryos were collected by non-surgical washing for 16 days and counted.

As a result, as shown in table 4, the effect of superovulation on donor cows in each administration group was significant (naturally, only one embryo was produced at a time by one cow), the number of embryos per cow in the pFSH-hCTP group and pFSH-eCTP group was 6.9 and 5.6, respectively, and higher than that in the pFSH group (4.4), and the ratio of the pFSH-hCTP group to PMSG group (5.6) and pFSH group was significantly different (P <0.05), and the ratio of the pFSH-eCTP group to pFSH group was significantly different (P < 0.05). The number of available embryos of cow heads in the pFSH-hCTP group and the pFSH-eCTP group is 5.4 and 4.1 respectively, which are higher than those of the PMSG group (3.9) and the pFSH group (3.1), the ratio difference of the pFSH-hCTP group to the PMSG group and the pFSH group is obvious (P <0.05), and the ratio difference of the pFSH-eCTP group to the pFSH group is obvious (P < 0.05). The number of unusable embryos on the cow heads in the pFSH-hCTP group and the pFSH-eCTP group is 1.5, which are lower than that in the PMSG group (1.7) and higher than that in the pFSH group (1.3).

TABLE 4 comparison of pFSH-hCTP, pFSH-eCTP, PMSG and pFSH for superovulation in Holstein cows

Example 8 application of pFSH-hCTP and pFSH-eCTP proteins to promotion of contemporaneous estrus in ewes

60 healthy disease-free female goats, 1.5-3 years old and 30-45kg in body weight, were selected and randomly divided into pFSH-hCTP, pFSH-eCTP, PMSG and pFSH groups. The donor sheep were placed progesterone pessaries on any Day of the estrus cycle and scored as 0 Day, and donor sheep of the pFSH-hCTP, pFSH-eCTP and PMSG groups were each intramuscularly injected with 300IU of the corresponding drug (Day10) and withdrawn (Day 12). The pFSH groups injected 25IU pFSH at Day10 and Day11, respectively. The oestrus expression of the ewes and the estrus test of the ewes is observed. The oestrus rate is calculated by taking the red vulva of the ewe, the mucus flowing out of the ewe and the crawling and crossing as oestrus.

As shown in Table 5, the estrus of the ewes in each group was significant, and the estrus rates of the ewes in the pFSH-hCTP group and the pFSH-eCTP group were 93% and 80%, respectively, which were higher than those in the PMSG group (67%) and the pFSH group (67%).

TABLE 5 comparison of oestrus of the goat with pFSH-hCTP, pFSH-eCTP, PMSG and pFSH

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing Weijixin Biotechnology Ltd

<120> recombinant porcine FSH-CTP fusion protein and preparation method and application thereof

<130> KHP181112396.2

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> PRT

<213> pig (Sus scrofa)

<400> 1

Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Val Ile Leu Ala Ile Leu Ser

1 5 10 15

Val Phe Leu Gln Ile Leu His Ser Phe Pro Asp Gly Glu Phe Thr Met

20 25 30

Gln Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys

35 40 45

Leu Gly Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala

50 55 60

Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn

65 70 75 80

Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys Ala

85 90 95

Thr Val Met Gly Asn Ala Arg Val Glu Asn His Thr Glu Cys His Cys

100 105 110

Ser Thr Cys Tyr Tyr His Lys Ser

115 120

<210> 2

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggactact acaggaagta cgccgccgtg atcctggcca tcctgtccgt gttcctgcag 60

atcctgcact ccttccctga cggcgagttc accatgcagg gctgccccga gtgcaagctg 120

aaggagaaca agtacttctc caagctgggc gcccccatct accagtgcat gggctgctgc 180

ttctcccggg cttaccctac ccctgcccgg tccaagaaga ccatgctggt gcccaagaac 240

atcacctccg aggccacctg ttgcgtggcc aaggccttca ccaaggccac cgtgatgggc 300

aacgccaggg tggagaacca caccgagtgc cactgcagca cctgctacta ccacaagtcc 360

<210> 3

<211> 157

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile

1 5 10 15

Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys

20 25 30

Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly

35 40 45

Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn

50 55 60

Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys

65 70 75 80

Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val

85 90 95

Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys

100 105 110

Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys

115 120 125

Glu Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser

130 135 140

Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

145 150 155

<210> 4

<211> 471

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaagtccc tgcagttctg cttcctgttc tgctgctgga aggccatctg ctgcaattcc 60

tgcgagctga ccaacatcac catcaccgtg gagaaggagg agtgcaactt ctgcatcagc 120

atcaacacca cctggtgcgc tggctactgc tacaccaggg acctggtgta caaggacccc 180

gccaggccca acatccagaa gacctgcacc ttcaaggagc tggtctacga gaccgtcaag 240

gtgcctggct gtgcccacca cgctgactcc ctgtacacct accccgtcgc taccgagtgc 300

cactgcggaa agtgcgactc cgactccacc gactgcacag tgaggggcct cggccctagc 360

tactgctcct tctccgagat gaaggagtcc agctcctcca aggctccccc tcctagcctg 420

ccttcccctt ccaggctgcc tggcccttcc gataccccca tcctgcccca a 471

<210> 5

<211> 164

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile

1 5 10 15

Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys

20 25 30

Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly

35 40 45

Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn

50 55 60

Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys

65 70 75 80

Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val

85 90 95

Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys

100 105 110

Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys

115 120 125

Glu Ser Ser Ser Ser Lys Asp Pro Pro Ser Gln Pro Leu Thr Ser Thr

130 135 140

Ser Thr Pro Thr Pro Gly Ala Ser Arg Arg Ser Ser His Pro Leu Pro

145 150 155 160

Ile Lys Thr Ser

<210> 6

<211> 492

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaagtccc tgcagttctg cttcctgttc tgctgctgga aggccatctg ctgcaactcc 60

tgcgagctga ccaacatcac catcaccgtg gagaaggagg agtgcaactt ctgcatcagc 120

atcaacacca cctggtgcgc cggctactgc tacaccaggg acctggtgta caaggaccct 180

gccaggccca acatccagaa gacctgcacc ttcaaggagc tggtgtacga gaccgtgaag 240

gtgcccggct gtgcccacca tgccgattcc ctgtacacct accccgtggc taccgagtgc 300

cactgcggca agtgcgactc cgacagcacc gattgcaccg tgaggggcct gggaccctcc 360

tactgctcct tcagcgagat gaaggagtcc agctcctcca aggaccctcc ttcccagccc 420

ctgacctcca ccagcacccc tacacctggc gcttccagga ggtcctccca ccctctgccc 480

atcaagacct cc 492

Claims

1. The recombinant porcine FSH-CTP fusion protein is characterized in that a beta subunit of porcine FSH is directly or indirectly connected with a beta subunit carboxy terminal peptide CTP of chorionic gonadotrophin, and an alpha subunit of porcine FSH is combined with the beta subunit of porcine FSH through van der Waals force;

wherein the chorionic gonadotropin is from a human or horse;

the fusion protein is pFSH-hCTP or pFSH-eCTP;

pFSH-hCTP: contains two peptide chains, and meets the following equation: (pFSH α: pF)SHβ-hCTP)₂Wherein pFSH α refers to the α subunit of porcine FSH; colons represent porcine FSH α and β subunits connected by van der waals forces; pFSH β refers to the β subunit of porcine FSH; hCTP refers to human chorionic gonadotropin beta subunit carboxy terminal peptide; the bracketed outer subscript 2 represents the porcine FSH fusion protein as a bivalent homodimer;

pFSH-eCTP: contains two peptide chains, and meets the following equation: (pFSH α: pFSH β -eCTP)₂Wherein pFSH α refers to the α subunit of porcine FSH; colons represent porcine FSH α and β subunits connected by van der waals forces; pFSH β refers to the β subunit of porcine FSH; eCTP refers to equine chorionic gonadotropin beta subunit carboxy terminal peptide; the bracketed outer subscript 2 represents the porcine FSH fusion protein as a bivalent homodimer;

the amino acid sequence of pFSH beta-hCTP in the fusion protein pFSH-hCTP is shown in SEQ ID NO. 3;

the amino acid sequence of pFSH beta-eCTP in the fusion protein pFSH-eCTP is shown in SEQ ID NO. 5.

2. An expression cassette, an expression vector, a cloning vector, an engineered bacterium, or a transgenic cell line comprising a nucleic acid encoding the fusion protein of claim 1.

3. The method for preparing the fusion protein according to claim 1, wherein the fusion protein pFSH-hCTP is prepared as follows: optimizing and artificially synthesizing the encoding genes of pFSH alpha and pFSH beta-hCTP, respectively constructing the gene sequences of the optimized pFSH alpha and pFSH beta-hCTP in different expression cassettes, then connecting to the same expression vector, transforming host cells, expressing in the host cells, and separating and purifying target proteins; alternatively, the first and second electrodes may be,

respectively constructing the optimized gene sequences of pFSH alpha and pFSH beta-hCTP on expression vectors, jointly transforming the obtained recombinant vectors into host cells, expressing the host cells, and separating and purifying target proteins;

respectively constructing the optimized pFSH alpha and pFSH beta-eCTP gene sequences on expression vectors, jointly transforming the obtained recombinant vectors into host cells, expressing the host cells, and separating and purifying target proteins;

wherein, the optimized gene sequences of pFSH alpha, pFSH beta-hCTP and pFSH beta-eCTP are respectively shown as SEQ ID NO 2, 4 and 6.

4. The method of claim 3, wherein the expression vector is pcDNA3.1; the host cell is CHO or 293 cell.

5. An ovulation-promoting drug or composition, characterized in that the active ingredient thereof is a fusion protein as claimed in any one of claims 1 to 3.

6. The fusion protein of claim 1, the medicament or composition of claim 5, for any one of the following uses:

1) for the preparation of a medicament for promoting animal reproduction;

2) the application in the preparation of the medicine for treating animal reproduction related diseases;

the promotion of animal reproduction comprises estrus synchronization and superovulation.

7. The use of claim 6, wherein the animal is a mammal, including a pig, a cow, or a sheep.

8. The use of claim 7, wherein the animal is a pig.