CN115991792A - Long-acting recombinant LH fusion protein and preparation method and application thereof - Google Patents

Abstract

Recombinant LH fusion proteins are disclosed, which refer to the joining of alpha and beta subunits of LH by Van der Waals forces, and optionally with a long-acting fragment. The LH fusion protein can be prepared by using a eukaryotic expression system based on genetic engineering technology. The drug effect of the LH fusion protein is higher than that of natural LH, the administration frequency is reduced, and single administration can be realized; can be used in animal reproduction production instead of chorionic gonadotropin (hCG).

Description

Long-acting recombinant LH fusion protein and preparation method and application thereof

Technical Field

The invention relates to the technical fields of biological medicine and animal breeding, in particular to a long-acting recombinant LH fusion protein and a preparation method and application thereof.

Background

Luteinizing Hormone (LH) is a gonadotropin, wherein porcine luteinizing hormone (Porcine luteinizing hormone, pLH for short), a gonadotropin secreted by porcine pituitary glands, is formed as a heterodimer by joining the α and β glycosylated subunits non-covalently. The alpha subunits of mammals are the same or similar, the beta subunits are composed of about 121 amino acids, and the beta subunits have great difference, not only have hormone-to-hormone specificity, but also have inter-species specificity, and are determinants of hormone biological activity and immune activity, so the beta subunits are called specific subunits and can be combined with target organ receptors.

According to the theory of the "two-cell-two gonadotrophin" proposed by Falck in 1959, luteinizing Hormone (LH) plays an important role in follicular development. Stimulating follicular membrane cells to synthesize androgens in the follicular phase, and providing substrates for estrogen synthesis; the oocyte is matured and ovulated before ovulation, and the luteal phase maintains luteal function. LH also plays an important role in the processes of oocyte resumption of meiosis, prostaglandin synthesis, progesterone synthesis, etc. In cattle and pigs, pLH has been shown to promote accelerated ovarian blood flow in females, induce follicular ovulation and promote corpus luteum formation on the basis of follicle stimulating effects; testosterone synthesis and secretion by testicular interstitial cells can be stimulated to males. Thus pLH has important value for livestock production and economic animal breeding.

In the field of animal reproduction, bovine, ovine and fish commonly use artificial synthetic luteinizing hormone releasing hormone (LH-RH, also known as GnRH) and analogues thereof, chorionic gonadotropin (HCG) or LH extracted from porcine pituitary tissue. GnRH and its analogues are capable of promoting release of high levels of LH and low levels of Follicle Stimulating Hormone (FSH) from the anterior pituitary of animals; HCG has high similarity with LH, can bind the same LH/HCG receptor (LHCGR) to exert biological effect, has short half-life of LH (about 30 min), can be rapidly metabolized in clinical use, and has long half-life of HCG (2-3 days), so that the HCG is used for simulating LH peak to induce ovulation.

At present, no recombinant veterinary LH product is marketed, and the livestock LH product mainly comprises LH extracted from pig pituitary gland, synthetic GnRH and analogues thereof and HCG extracted from pregnant woman urine. LH extracted from pig pituitary is difficult to separate pig FSH from LH, so that the purification difficulty is high, the productivity is low, the product batch difference is large, and the pig FSH is greatly limited in practical application; the synthetic GnRH and analogues thereof act on pituitary gonadotrophs to stimulate synthesis and secretion of luteinizing hormone LH and follicle stimulating hormone FSH, and the synthetic hormone molecules (formula weight, structure, elements and the like) are not identical to those of natural ones, so that the long-term large-dose use of the GnRH and analogues thereof can inhibit ovulation; HCG extracted from pregnant urine may cause ovarian hyperstimulation syndrome (ovarian hyperstimulation syndrome, OHSS).

Therefore, the problems that the half-life of pig pituitary LH is short, gnRH is easy to produce adverse effect, HCG possibly causes ovarian hyperstimulation syndrome and the like exist, the animal broad spectrum of the existing substances is poor, one protein cannot be used and is widely used in multiple species at the same time, and therefore, the long-acting animal source LH is required in the animal breeding field, particularly economic animals.

Disclosure of Invention

In order to solve the technical problems, the invention relates to a fusion protein which comprises an LH alpha subunit and an LH beta subunit, wherein the LH alpha subunit and the LH beta subunit are connected through van der Waals force or a connecting element.

The source of LH alpha and LH beta subunits of LH may be mammalian, including but not limited to porcine, bovine, ovine, equine, rabbit, monkey, dog, cat, murine, fish, further, murine may be rat, mouse. For example, a porcine LH alpha subunit amino acid sequence may be used

FPDGEFTMQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTML VPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKS(SEQ IDNO.1)

Porcine LH beta subunit amino acid sequence

SRGPLRPLCRPINATLAAENEACPVCITFTTSICAGYCPSMVRVLPAALPPVPQP

VCTYRELSFASIRLPGCPPGVDPTVSFPVALSCHCGPCRLSSSDCGGPRAQPLACDRPLLPGLLFL(SEQ ID NO.2)

The invention further provides a nucleotide sequence for expressing the protein, wherein the nucleotide sequence is obtained by researching species preference, mammal evolutionary tree and mammal gene sequence homology comparison and codon optimization of the inventor. Exemplary:

pig LH alpha subunit nucleotide sequence

tttcccgatggcgagttcaccatgcagggatgtcctgagtgcaaactgaaggagaacaagtactttagcaaactgggagcaccaatctaccagtgtatgggatgttgcttcagtagagcttatcccactccagctaggtccaagaagaccatgctggtgcctaagaacattaccagtgaggccacctgttgtgtggccaaggcattcaccaaagctactgtcatgggtaatgccagggtggagaaccataccgagtgccactgttctacatgttactaccacaaatct(SEQ ID NO.3)

Porcine LH beta subunit nucleotide sequence

agcagaggtccactcagacctctgtgcagacccataaacgctacactcgcagccgagaacgaggcatgtcctgtgtgcatcaccttcactacttcaatttgtgctggctactgccctagcatggttagggttctcccagccgcactgcctcctgtgcctcagcccgtctgtacctatcgcgaactgagcttcgcttctatcaggctccctggctgtccaccaggcgtcgatcccactgtgtccttcccagttgccctcagctgtcactgcggaccttgtagactgtcctcctccgactgtggaggaccacgcgcacagcctctggcttgcgaccggccactgctgcctgggctgctgtttctg(SEQ ID NO.4)

The present invention also provides a method for the long-acting of a protein such as LH protein or LH fusion protein by ligating the N-terminus or C-terminus of the above LH alpha subunit or LH beta subunit using a long-acting fragment, directly or indirectly via a Linker element which may be a Linker commonly used in fusion proteins, such as GSlinker, for example (GGGGS) ₃ ，(GGGGS) ₄ Etc.; the elements are connected to form a plurality of protein structures; the long-acting fragment is selected from protein fragments such as CTP, fc, serum albumin and the like. Preferred beta subunit carboxy terminal peptide CTP of human, primate or equine mammalian chorionic gonadotrophin may be selected, the protein structure including but not limited to (Van der Waals force linkage indicated by ": and" - "indicates chemical bond linkage) LHα: LHβ -CTP, LHβ: LHα -CTP.

For example, CTP is a human sequence, and the specific sequence may be SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO. 5)

Exemplary, the design sequence of the fusion protein pLH-CTP beta subunit is as follows

pLH-CTP beta subunit amino acid sequence

SRGPLRPLCRPINATLAAENEACPVCITFTTSICAGYCPSMVRVLPAALPPVPQPVCTYRELSFASIRLPGCPPGVDPTVSFPVALSCHCGPCRLSSSDCGGPRAQPLACDRPLLPGLLFL SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO.6)

Exemplary combinations:

pLH-CTP beta subunit nucleotide sequence

agcagaggtccactcagacctctgtgcagacccataaacgctacactcgcagccgagaacgaggcatgtcctgtgtgcat

caccttcactacttcaatttgtgctggctactgccctagcatggttagggttctcccagccgcactgcctcctgtgcctcagcc

cgtctgtacctatcgcgaactgagcttcgcttctatcaggctccctggctgtccaccaggcgtcgatcccactgtgtccttccc

agttgccctcagctgtcactgcggaccttgtagactgtcctcctccgactgtggaggaccacgcgcacagcctctggcttgc

gaccggccactgctgcctgggctgctgtttctgtccagctccagcaaagctcctcctccctcactgccttcaccttccagactgccagggccttccgatactcccatcctgccacaa(SEQ ID NO.7)

Exemplary "pLH" protein means a heterodimeric protein in which the pig LH alpha subunit shown in SEQ ID NO.1 and the pig LH beta subunit shown in SEQ ID NO.2 are linked by Van der Waals forces, and "pLH-CTP" protein means a heterodimeric protein in which the pig LH alpha subunit shown in SEQ ID NO.1 and the pLH-CTP beta subunit shown in SEQ ID NO.6 are linked by Van der Waals forces

Expression vector

The present invention also provides an expression vector that can be used to load cloned genes for expression of foreign proteins, including but not limited to pET20, pcdna3.1, pCMV, etc., preferably pcdna3.1.

Stable transfer cell lines

The invention also provides a stable transfer cell line, including but not limited to HEK293, BHK, CHO cell line and the like, for introducing the expression vector.

Further, the method also comprises the processes of culturing the stable transfer cells expressing the exogenous protein, purifying and detecting the exogenous protein.

Dosage form

The present invention further provides compositions comprising the fusion proteins described above, including adjuvants including, but not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, emulsifiers, synergists, dosage forms including, but not limited to, injections, tablets, pills, capsules, suspensions, suppositories, emulsions, powder injection.

The compositions are administered to animals by means including, but not limited to, injection, oral administration, lavage, vaginal embolism, injection sites including, but not limited to, intramuscular, subcutaneous.

Use of the same

The present invention further provides uses of the above proteins, fusion proteins, in promoting animal reproduction, including use in promoting ovulation in the same period, superovulation, and embryo engineering to promote embryogenesis and development in animals including, but not limited to, pigs, cattle, sheep, horses, monkeys (rhesus, cynomolgus, etc.), dogs, cats, rabbits, mice (rats, mice), fish, etc., but preferably mammals. The LH fusion protein is used in an effective dose of 200-1000IU/kg for animals such as mice, rabbits, fish and the like, and 0.5-10IU/kg for animals such as pigs, cattle, sheep, horses, dogs, cats and the like. The frequency of use is single administration. And further, can prevent and treat the generation of ovarian cyst. The invention provides a plurality of functional verification experiments, and the administration is carried out in different modes, such as injection, vaginal embolism and the like, and the experiments prove that the functions of ovulation promotion and embryo development induction can be realized in the animals, and ovarian cyst can not be caused.

Further, the present invention also provides the use of a combination, but not necessarily, for example, of the fusion protein LH or LH-CTP in combination with other reproductive function promoting substances, drugs, etc., including but not limited to FSH, FSH-FC, FSH-CTP, porcine pituitary extract (e.g., commercially available as foltropin-v), prostaglandins, pregnant Mare Serum Gonadotropins (PMSG), etc.

The above substances may be used either before or after the use of the LH fusion protein, but are preferably used before; for example, PMSG and FSH can be used for follicular development, and can play a role in promoting reproductive function by being matched with the use of LH fusion protein. However, the higher the dose is, the better the dose is, and the higher the dose is, the ovarian related diseases, such as ovarian cyst, so that the selection of proper substances, medicines and combination use schemes can effectively ensure that animals maintain the healthy state of ovaries and promote reproductive function.

Furthermore, the invention also provides the application of the LH fusion protein in preventing and/or treating ovarian cyst in animals and the application in preparing a medicine for preventing and/or treating ovarian cyst.

A kit comprising the recombinant LH protein as described above, or the expression vector as described above, or the stably transfected cell line as described above.

Advantageous effects

1 fusion protein has strong mammal broad spectrum applicability

According to the invention, three fusion protein elements of LH alpha subunit, LH beta subunit and CTP from different species are creatively expressed through the comparison modes of species gene homology, evolutionary tree and the like on the sequence of LH protein and through codon preference design, and different structural fusion proteins are formed through different combinations, so that the invention can be widely used in various mammals through animal experiments, and superovulation and embryo engineering are promoted. Because of the broad spectrum of the veterinary preparation, the preparation can achieve multiple functions of synchronous ovulation, superovulation, promotion of reproduction and embryo engineering of multiple mammals by using one preparation, and can reduce the cost to the greatest extent in use. 2 the increased half-life reduces the frequency of use to provide a fusion protein that is useful in a broad spectrum in mammals to promote reproductive production in healthy animals, and although the methods of longevity exist in the prior art, not all of the methods are applicable to the fusion proteins of the present invention. Female follicular development and ovulation are a fine reproductive regulatory process. For LH, one of the main physiological effects is ovulation induction, in normal animals, a surge of LH occurs before ovulation to induce ovulation, and a surge of LH after ovulation is collapsed. In animal reproduction production application, because the arrival time of a natural LH peak cannot be predicted, the concentration of the drug is in the threshold range of the drug effect in a longer time by repeatedly administering LH or singly administering long-acting LH drugs, so as to achieve the purpose of ovulation induction. However, the long half-life of LH fusion proteins affects fertilized egg implantation. The inventor creatively and repeatedly verifies, discards long-term protein fragments such as Fc and albumin, finally selects a humanized CTP sequence to be combined with LH protein, properly improves the half-life of LH, reduces operation times, saves reagents, reduces cost, and avoids influencing the development and implantation of fertilized eggs due to overlong half-life. 3 promoting healthy animal reproduction, preventing and treating ovarian cyst, solving the problems of short pig pituitary LH half-life, bad effect easily produced by GnRH, possibility of ovarian hyperstimulation syndrome caused by HCG and the like in the prior art, providing a complete LH fusion protein for promoting animal reproduction and increasing production, and having better broad spectrum adaptability, high protein purity, better activity, proper half-life, no occurrence of animal follicular cyst and function of preventing and/or treating ovarian cyst. And further, the animal breeding promoting agent can be optionally combined with other animal breeding promoting substances, and the combination scheme can effectively ensure that the animal ovaries maintain a healthy state and promote the breeding function.

Drawings

FIG. 1 shows SDS-PAGE patterns of pLH and pLH-CTP.

SDS-PAGE electrophoresis of pLH and pLH-CTP in example 2, A and C are denaturing electrophoresis of pLH and pLH-CTP, respectively; b and D are non-denaturing electrophoresis charts of pLH and pLH-CTP, respectively. Electrophoresis shows that the purities of the pLH and the pLH-CTP are both about 95.0 percent.

FIG. 2 shows the ovulation pattern of pLH-CTP-induced mice.

Ovulation pattern of mice induced by pLH-CTP in example 4. The combination of the pLH-CTP fusion protein and the PMSG can effectively induce the superovulation of female mice, the ovulation quantity and the dosage are in dose-effect relation within a certain dosage range, and the average egg number of 5 IU/pLH-CTP fusion protein treatment group is the largest.

FIG. 3pLH-CTP ovarian B-ultrasound image of sow ovarian cyst

Ovarian B-ultrasound of treatment of sow ovarian cyst with pLH-CTP in example 8. The left image is an ovarian B ultrasonic image of a cyst sow before administration, and cyst follicles exist in the ovary (the diameter of the follicles is more than 9 mm). The right image is an ovarian B ultrasonic image of the cyst sow 6 days after administration, and the cyst follicles are seen to be resolved. Shows that the pLH-CTP can treat ovarian cyst.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 preparation of pLH and pLH-CTP fusion protein cell lines

And respectively carrying out codon optimization on protein sequences of pLH and pLH-CTP, wherein the nucleotide sequence of the pLH alpha subunit is shown as SEQ ID NO:3, the nucleotide sequence of the pLH beta subunit is shown as SEQ ID NO:4, and the nucleotide sequence of the pLH-CTP beta subunit is shown as SEQ ID NO:7 after the result of the codon optimization. Artificially synthesizing the nucleotide, and respectively constructing the nucleotide in pcDNA3.1 vectors to obtain recombinant expression vectors pcDNA3.1-pLH alpha, pcDNA3.1-pLH beta and pcDNA3.1-pLH beta-CTP; or pcDNA3.1-promoter 1-pLH alpha-promoter 2-pLH beta and pcDNA3.1-promoter 1-pLH alpha-promoter 2-pLH beta-CTP, linearizing the recombinant vector, and respectively electrotransferring into CHO cells to obtain stable transfer cell lines of pLH and pLH-CTP. The protein complex plhα can be naturally formed by van der waals forces after protein expression in a cell line: plhβ and plhα: plhβ -CTP.

EXAMPLE 2 purification of pLH and pLH-CTP fusion proteins

Fermenting and culturing stable-rotation cells expressing pLH and pLH-CTP in a fermentation tank, removing cells and cell fragments from fermentation liquor through a two-stage deep layer filtering membrane, and filtering with a 0.22 mu m filtering membrane to obtain clear fermentation liquor.

The purification process of the pLH fermentation broth is as follows: the first step was purified by cation exchange chromatography (nano 50 SP), equilibrated with equilibration solution (70 mM PB, pH 7.0) to baseline, loaded, equilibrated with equilibration solution to baseline, eluted with eluent (70mM PB,0.1M NaCl,pH7.0), and the eluent was collected. The second step was purification by hydrophobic chromatography (GE Capto Phenyl ImpRes) using 3M NaCl to adjust the conductance of the collection solution to match that of the equilibration solution and 1M HCl to adjust the pH to 7.0. Equilibrated with equilibration solution (0.5M NaCl,10mM Tris-HCl, pH 7.0), loaded, equilibrated again to baseline with equilibration solution, then eluted with eluent (10 mM Tris-HCl, pH 7.0), and the eluent was collected. And thirdly, adopting a molecular sieve (Chromdex 200 PG) column for fine purification, balancing by using a balancing solution (PBS), loading, eluting by using the balancing solution, and collecting to obtain the purified protein. SDS-PAGE gel electrophoresis (FIGS. 1A and 1B) of the purified target protein showed a protein purity of 95.0% or higher.

The purification process of the pLH-CTP fermentation broth is as follows: the first step was purification by hydrophobic chromatography (GE Capto Phenyl ImpRes) using 3M (NH) ₄ ) ₂ SO ₄ After adjusting the conductance of the broth to match that of the equilibration solution, the pH was adjusted to 7.0 with 1M HCl, and the pH was adjusted to a pH of about 7.5M (NH) ₄ ) ₂ SO ₄ Equilibrated to baseline with 10mM Tris-HCl, pH 7.0), then eluted with eluent (10 mM Tris-HCl, pH 7.0) and the eluent was collected. And in the second step, a molecular sieve (Chromdex 200 PG) column is adopted for fine purification, a balancing solution (PBS buffer solution) is used for balancing the hydrophobic chromatography eluent, the sample is loaded, and then the balancing solution is used for eluting, so that the purified protein is obtained after collection. SDS-PAGE gel electrophoresis (FIGS. 1C and 1D) of the purified target protein showed a protein purity of 95.0% or higher.

EXAMPLE 3 Activity detection of pLH and pLH-CTP fusion proteins

The specific activities of pLH and pLH-CTP are determined by adopting a rat seminal vesicle weight gain method by referring to the four "1217 luteinizing hormone bioassay method" of Chinese pharmacopoeia.

The national standard of the urotropin is taken as a standard of a test sample, and 16, 8 and 4IU/ml of standard/test sample diluent with high, medium and low 3 concentrations are respectively prepared according to the estimated titers of the urotropin mark titer (203 IU/branch), pLH (the specific activity is estimated to be 8000 IU/mg) and pLH-CTP (the specific activity is estimated to be 8000 IU/mg). 54 male SD (Sprague Dawley) rats of 19-23 days of age (or 40-55g in weight) of the same origin were randomly divided into 9 groups of 6 rats each. Each mouse was subcutaneously injected with 0.5ml of a standard solution or a test solution at a concentration for about the same time each day, 1 time each day, 4 times continuously, after 24 hours from the last 1 time of injection, the animals were sacrificed, weighed, dissected, the whole prostate was removed, the seminal vesicles were peeled off from the junction of the anterior leaflet and the seminal vesicles, the attached tissues were removed, surrounding liquid was sucked with filter paper, and the weight was directly weighed and converted into the seminal vesicle weight of 10g each body weight. The result of software calculation of pharmacopoeia bioassay statistics BS2000 by using the center inspection shows that the specific activity of pLH is about 9121IU/mg, which is 23 times of the specific activity requirement (400 IU/mg) of LH in human pharmacopoeia urotropin, and the specific activity of pLH-CTP is about 7453IU/mg, which is 19 times of the specific activity requirement (400 IU/mg) of LH.

Example 4 use of pLH-CTP fusion protein in the superovulation process in mice

40 immature female mice of 3 weeks of age were selected, and each female mouse was divided into 5 groups after intraperitoneal injection of 5IU of pregnant horse serum gonadotropin (PMSG), including a positive control group, a negative control group and 3 groups of pLH-CTP fusion protein treated groups, each group of 8 female mice. Ovulation induction treatment was performed 46-48 hours after PMSG injection, 5IU (200. Mu.l) of hCG was intraperitoneally injected into each female mouse of the positive control group, 200. Mu.l of physiological saline was intraperitoneally injected into each female mouse of the negative control group, and 2.5 IU/mouse (167 IU/kg), 5 IU/mouse (333 IU/kg), 10 IU/mouse (667 IU/kg) of pLH-CTP fusion protein was respectively injected into each female mouse of the 3-group pLH-CTP fusion protein-treated group. Egg taking is carried out 14-16 h after ovulation promotion treatment, and the number of eggs is counted and counted.

As a result of the experiment, it was found that the negative control group had no ovum to be discharged, the female mice of the hCG positive control group had an average ovulation of 34.4.+ -. 10.7, whereas the female mice of the 2.5 IU/pLH-CTP fusion protein-only treated group had an average ovulation of 38.8.+ -. 11.8, the female mice of the 5 IU/pLH-CTP fusion protein-only treated group had an average ovulation of 51.5.+ -. 14.5, and the female mice of the 10 IU/pLH-CTP fusion protein-only treated group had an average ovulation of 43.0.+ -. 16.9 (Table 1 and FIG. 1). Experimental results show that the combination of the pLH-CTP fusion protein and the PMSG can effectively induce the superovulation of female mice, and the average egg-obtaining number of 5 IU/only the pLH-CTP fusion protein treated group is the maximum in a dose range in a dose-effect relationship, which indicates that the optimal ovulation-promoting dose of 5 IU/only the mice is shown.

TABLE 1 number of superovulated ova in different treated mice

Group of	Number of mice (Only)	hCG/pLH-CTP dosage	Number of ovulation homoenergetic (piece/piece)
				Negative control group	8	200 μl/physiological saline	0 c
Positive control group	8	hCG 5 IU/only	34.4±10.7 b
				pLH-CTP-2.5 group	8	2.5 IU/min	38.8±11.8 ab
pLH-CTP-5 group	8	5 IU/only	51.5±14.5 a
				pLH-CTP-10 group	8	10 IU/only	43.0±16.9 ab

Note that: shoulder marks differ by letters indicating significant differences (P < 0.05), and the same letters indicate insignificant differences (P > 0.05).

EXAMPLE 5pLH-CTP induces superovulation in sow

Pre-estrus ternary hybridization (Changbai×Da Baidu lock) sows of 16 days old and 140-150 days old were selected and randomly divided into a negative control group, an hCG group, a pLH-CTP low dose group and a pLH-CTP high dose group, each group of 4 sows. 250IU (4 IU/mg) of pLH-CTP was intramuscular injected into the sow in the group of the low dosage of pLH-CTP, 500IU (8 IU/mg) of pLH-CTP was intramuscular injected into the sow in the group of the high dosage of pLH-CTP, an equal volume of normal saline was intramuscular injected into the sow in the group of the negative control, and 500IU (1 ml) of hCG was intramuscular injected into the sow in the group of the hCG. Sows were sacrificed 5 days after injection, ovaries and oviducts were removed in pre-warmed saline and rapidly transported to the laboratory. Oocytes were obtained by flushing the oviduct with sterilized PBS and the number of oocytes was recorded.

The results showed (Table 2) that when normal saline was administered to the sow, only 2 sows were ovulated due to insufficient gonadotrophin in the pre-primordial period, and the number of ovulations was very small. The administration of 500IU hCG to sows, the total ovulation number of sows is 51, the ovulation number is 12.8+/-4.3/head, and the number of ovulations is higher than that of negative control groups, which shows that the administration of hCG can induce the sows to superovulate, the ovaries of the sows are detected, the surface of the ovaries is visible to the red body just after ovulation, however, a small number of non-ovulated cyst enlargement follicles exist on the surface of the ovaries of 1 head sows, the diameter of the follicles is larger than 10mm, which shows that hCG can induce the sows to ovulate, but the ovarian cyst of the sows can be caused. The sow is given 250IU pLH-CTP and 500IU pLH-CTP, the ovulation number is obviously increased, the ovulation number (13.5+/-1.3 pieces/head) of the sow in the 250IU pLH-CTP group is slightly higher than that of the sow in the 500IU hCG group, no obvious difference exists, the ovulation number (19.8+/-4.3 pieces/head) of the sow in the 500IU pLH-CTP group is obviously higher than that of the sow in the 500IU hCG group and the 250IU pLH-CTP group, the ovaries of the two groups of sows are inspected, the surface of the ovaries of the two groups of sows can see the red body after ovulation, and no large follicle exists, so that the ovulation is completely indicated.

The experiment shows that the administration of pLH-CTP to sows can induce the sows to ovulate, the ovulation number of the sows is more than that of hCG, the sows are safe, and follicular cyst of the sows can not be caused.

TABLE 2 different treatments induce the number of superovulations in pre-estrus sows

Note that: the shoulder marks with different cases and letters indicate significant differences (P < 0.01), the same letters indicate significant differences (P < 0.05), and no same letters indicate insignificant differences (P.gtoreq.0.05).

Example 6 application of pLH-CTP fusion protein in bovine embryo engineering

The 15-head Siemens young cows are selected and divided into 3 groups, including a positive control group (Folltropin-v, pituitary pig FSH and LH mixture) and a 2-group experimental group (pFSH-FC fusion protein is combined with different doses of pLH-CTP fusion protein for treatment, and the pFSH-FC fusion protein is synthesized by Beijing Weijie biological technology Co., ltd.) and 5 cows are selected from each group. 15 cows are simultaneously embedded with vaginal suppository (CIDR) containing progestin, which is marked as day0, exogenous hormone treatment is started at day5, and a positive control group is injected with commercial product Folltropin-v extracted from pig pituitary at day5, the total dosage is 200mg, and the injection is continuously carried out for 4 days according to the proportion of 4:3:2:1, 2 injection is carried out every day, and 8 injection is carried out in total; the experimental group was injected only once at day5, with 150. Mu.g of pFSH-FC fusion protein, in combination with 400IU and 800IU of pLH-CTP fusion protein, respectively, for a total of 2 groups. Each group was thrombolytic at day7 and injected once every morning and afternoon with 0.6mg prostaglandin PG, oestrus and artificial insemination were observed at day9, secondary artificial insemination was performed at day10, embryo washout at day16, and embryo counts were collected.

The experimental result shows that the negative control group does not obtain embryos, the positive control group has 9.6+/-2.1 embryos at the head, and the number of available embryos at the head is 6.6+/-2.0. The experimental results show that the number of embryos obtained by injecting 150 mug pFSH-FC and 400IU pLH-CTP fusion protein once at the head of the experimental group is 8.6+/-1.4, and the number of available embryos at the head is 5.8+/-1.5; the experimental groups of 150. Mu.g pFSH-FC and 800IU pLH-CTP fusion protein each had 11.8.+ -. 2.6 embryos and 7.8.+ -. 2.1 embryos for each head (Table 3). The ovaries of the cattle were examined rectally and no anovulatory follicles were found. The pFSH-FC fusion protein and pLH-CTP fusion protein can effectively promote superovulation of cows, and the experimental groups of 150 mug pFSH-FC and 800IU pLH-CTP fusion proteins can obtain embryo numbers which are higher than those of other groups, so that the pFSH-FC fusion protein and pLH-CTP fusion protein can be applied to embryo engineering of cows.

TABLE 3 effects of different treatments on superovulation in cattle

Example 7 application of pLH-CTP fusion protein to improving conception effect of ewes

60 adult female Hu sheep were selected and randomly divided into a blank control group, a pFSH-Fc group, a pFSH-Fc+pLH-CTP low dose group and a pFSH-Fc+pLH-CTP high dose group. The donor sheep were placed with progesterone vaginal suppositories and designated Day0 during the estrus cycle, the corresponding drug was simultaneously intramuscularly injected with the withdrawal of the suppository (Day 13), 10 μg pFSH-Fc for the pFSH-Fc group of ewes, 10 μg pFSH-Fc and 80IU LH-CTP for the pFSH-fc+lh-CTP low dose group of ewes, 10 μg pFSH-Fc and 160IU LH-CTP for the pFSH-fc+lh-CTP high dose group of ewes, and an equal volume of normal saline for the empty control group of ewes. The ram tries the estrus after the administration, and observes the estrus expression of the ewe within 3 days, the ewe is accepted as estrus, artificial insemination is performed at regular time 36h and 48h after the administration, and the day is examined 30 days after the hybridization.

The results show (Table 4) that administration of 10. Mu.g pFDH-Fc and 80/160IU pLH-CTP increased the conception rate of the ewes from the group of the Hu, and in quantitative relationship, the conception rate of the ewes from the group of 10. Mu.g pFDH-Fc and 80IU pLH-CTP was 53.3%, slightly higher than that of the group of pFDH-Fc (33.3%) and 10. Mu.g pFDH-Fc single drug group (46.7%), the conception rate of the ewes from the group of 10. Mu.g pFDH-Fc and 160IU pLH-CTP was 80.0%, and significantly higher than that of the blank group (P < 0.01), and higher than that of the group of 10. Mu.g pFDH-Fc single drug.

The results show that the use of 10 mug pFSH-Fc and 160IU pLH-CTP can induce the oestrus of the ewe, improve the conception rate of the ewe, and further be used in the synchronous oestrus-timed insemination of the ewe, and improve the reproduction production efficiency of the ewe.

TABLE 4 Effect of different treatments on improving conception of ewes

Group of	pLH-CTP dose	Number of heads	Conception rate
				Blank control group	0	15	33.3％ a (5/15)
pFSH-Fc group	0	15	46.7％ b (7/15)
				pFSH-Fc+pLH-CTP low dose group	80IU	15	53.3％(8/15)
pFSH-Fc+pLH-CTP high dose group	160IU	15	80.0％ A (12/15)

Note that: the different case letters of the shoulder marks indicate significant differences (P < 0.01), and no identical letters indicate insignificant differences (P.gtoreq.0.05).

Example 8 application of pLH-CTP fusion protein in treating ovarian cyst of sow

Selection of 5-head weaning oestrus without normal ovulation, abdominal B-ultrasound detection of the presence of follicular swelling (follicular diameter greater than 10 mm) in a multiparous sow, intramuscular injection of 1500IU pLH-CTP, daily abdominal B-ultrasound monitoring of the ovarian status of the sow after administration, results showing that 3 days after administration, cyst follicular begins to subside and 60% (3/5) of the follicular swelling in the sow completely subsides 6-7 days after administration (FIG. 3). Shows that the pLH-CTP can treat ovarian cyst.

Claims (12)

1. A recombinant LH protein comprising an LH α subunit and an LH β subunit, wherein the LH α subunit and LH β subunit are linked by van der waals forces or a linking element.

2. The recombinant LH protein of claim 1, further comprising a long-acting fragment selected from at least one of CTP, fc, serum albumin.

3. The recombinant LH protein of claim 2, which is structurally preferred that the LH α subunit or LH β subunit is linked to a long-acting fragment CTP; preferably, the CTP may be an amino acid sequence shown in SEQ ID No. 5; the CTPs are directly or indirectly connected through a connecting element.

4. The recombinant LH protein of claim 1 or 2 or 3, wherein the amino acid sequence of the α subunit of LH is shown in SEQ ID No.1 and the amino acid sequence of the β subunit of LH is shown in SEQ ID No. 2; the amino acid sequence of the fusion protein pLH beta-CTP subunit formed by the beta subunit of LH and the long-acting fragment CTP is shown in SEQ ID NO. 6.

5. The recombinant LH protein of claim 4, wherein the nucleotide sequence of the α subunit of LH is shown in SEQ ID No.3 and the nucleotide sequence of the β subunit of LH is shown in SEQ ID No. 4; the nucleotide sequence of the fusion protein pLH beta-CTP subunit formed by the beta subunit of LH and the long-acting fragment CTP is shown as SEQ ID NO. 7.

6. An expression vector capable of expressing the nucleotide sequence of claim 5.

7. A stably transfected cell line capable of transfecting the expression vector of claim 6.

8. Use of the recombinant LH protein of claims 1-5, the expression vector of claim 6, the stably transfected cell line of claim 7 for promoting reproductive function in an animal; further, uses in reproductive function of the animals include embryo engineering to promote synchronous estrus, induce superovulation, and promote embryo development.

9. The use as claimed in claim 8, wherein the LH fusion protein is administered at a dose of 0.5-1000IU/kg, and an effective dose of 200-1000IU/kg for murine, rabbit, fish, and 0.5-10IU/kg for porcine, bovine, ovine, equine, canine, feline; the frequency of use is single administration or more.

10. A method for promoting reproductive function in animals comprising combining the recombinant LH protein of claims 1-5, the expression vector of claim 6, the stably transfected cell line of claim 7 with other substances that promote reproductive correlation; such other proliferation-promoting substances include FSH, FSH-FC, FSH-CTP, PMSG (haemopoietin), porcine pituitary extract, prostaglandins, etc.

11. Use of the recombinant LH protein of claims 1-5, the expression vector of claim 6, the stably transfected cell line of claim 7 in the manufacture of a medicament for the prevention and treatment of ovarian cysts in animals.

12. A kit comprising the recombinant LH protein of claims 1-5, or the expression vector of claim 6, or the stably transfected cell line of claim 7.

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