CN109970871A

CN109970871A - Long-acting reorganization FSH fusion protein, preparation method and its application in sow timing semen deposition

Info

Publication number: CN109970871A
Application number: CN201910349940.4A
Authority: CN
Inventors: 侯永敏; 吴俊辉; 李梅; 王安新; 雷瑶
Original assignee: Guangzhou Vesun Medical Technology Co Ltd
Current assignee: Guangzhou Vesun Medical Technology Co Ltd
Priority date: 2019-04-28
Filing date: 2019-04-28
Publication date: 2019-07-05

Abstract

The invention discloses long-acting reorganization FSH fusion protein, preparation method and its applications in sow timing semen deposition.The long-acting reorganization FSH fusion protein includes human follicle-stimulating element Fc fusion protein (hFSH-Fc), pig follicle-stimulating hormone Fc fusion protein (pFSH-Fc), human follicle-stimulating element CTP fusion protein (hFSH-CTP) and pig follicle-stimulating hormone CTP fusion protein (pFSH-CTP).The long-acting reorganization FSH fusion protein can significantly extend the Half-life in vivo of albumen, it is used in Suprapubic arch sling and replacement gilt, the ovarian follicle same period can be significantly induced to develop, the long-acting reorganization FSH fusion protein is used to promote sow estrus synchronization in the timing semen deposition program of replacement gilt and Suprapubic arch sling, moreover it is possible to significantly improve oestrus of sow rate, over-all conception rate and number born per litter.

Description

Long-acting reorganization FSH fusion protein, preparation method and its in sow timing semen deposition Using

Technical field

The present invention relates to molecular biology and field of veterinary.More particularly it relates to which long-acting reorganization FSH merges egg White, preparation method and its application in sow timing semen deposition.

Background technique

Animal reproduction technology plays a significant role to China's breeding level is improved, but compared with cultivating developed country, still The problems such as so low there is reproductive efficiency.Main cause is the breeding management mode on the existing pig farm in China, results in reproductive efficiency Problem low, control and prevention of disease is difficult, these also become the bottleneck of pig raising industry development.

In husbandry sector, batchization breeding is a very important breeding management means, it can be truly realized entirely Into out, effective prevention and control epidemic disease, the utilization rate for improving dam, the nonproductive number of days (NPD) for shortening dam solve industry bottleneck entirely, Greatly improve animal husbandry economy benefit.The breeding of sow batchization is around pig production by existing production model to different weeks batch Secondary production model transformation, and to the key process technology scheme of breeding production synchronization and batch under Cultivation pattern Carry out system research and application.It include the column home of timing insemination technique, same period childbirth technology and entirety in the technological system The multiple portions such as planning and designing.It wherein, is that its most important forms portion with the timing insemination technique that reproductive hormone is adjusted to means Point.Realize that batchization is bred it may first have to establish stable, efficient timing insemination technique.

Sow timing insemination technique is a breeding new technology based on the estrus synchronization of sow, same period ovulation, Under the conditions of not influencing the normal reproductive physiology of sow, follicular development and ovulation period are artificially controlled by exogenous hormone, realized Follicular development synchronizes and ovulation synchronizes, to more accurately judge the Best Times of semen deposition, is remarkably improved sow hair The reproductive performances such as feelings rate, over-all conception rate and total parturition rate.

For sow, estrus synchronization can be such that the Farrowing time concentrates, and concentrate and wean convenient for sow, all-in and all-out, Into next breeding cycle, the breeding potential of sow is effectively improved, reduces the propagation of cause of disease, is conducive to large-scale pig farm and carries out sternly The epidemic prevention system and guarantee Biosecurity system of lattice, can greatly optimize the utilization of resources on pig farm, ensure the biology peace on pig farm Entirely, maximize culture efficiency.

Exogenous gonadotropins (gonadotropins) play a key effect for induction oestrus of sow and estrus synchronization, It is the glycoprotein hormones for promoting sex hormone to generate and secrete, and mainly includes follicle-stimulating hormone (FSH), serum gonadotrophin (PMSG) etc..

Follicle-stimulating hormone is also known as follicular stimulating hormone (Follicle-stimulating hormone, abbreviation FSH), is hung down by brain Body secretion a kind of glycoprotein hormones, FSH by promote dam folliculus ovarii growth, promote follicular cell hyperplasia and Estrogen synthesis and secretion, continuous raised estrogen action cause while follicular development mature ovulation in cerebral cortex Dam heat.

Serum gonadotrophin (Pregnant Mare Serum Gonadotropin, abbreviation PMSG) is similar with FSH effect, it has There are the double activity of FSH (follicle-stimulating hormone) and LH (luteotropin), but based on the effect of FSH, there is obvious rush follicular development Effect.

Sow timing insemination technique process description: by exogenous gonadotropins (gonadotropins) (such as PMSG) Processing, can promote the synchronization of sow follicular development, to control sow timing estrus synchronization.Again (such as by ovulation induction hormone Gonadotropin-releasing hormone (GRH) GnRH) adjusting, sow ovulation can be made to synchronize, so that sow timing be induced to ovulate, it is fixed to realize When semen deposition.The reproductive hormone product of effect stability is the key that ensure that timing semen deposition synchronizes effect.

Currently, sow timing insemination technique is widely used in states such as America and Europes.The research and development of China's sow timing insemination technique with Using still in its infancy, main problem is to lack the stable estrus induction of quality and promotes the critical hormone of follicular development Product.

Replacement gilt timing semen deposition can be divided into according to the Estrus synchronization mode difference of sow and Suprapubic arch sling timing is defeated Smart (respectively referring to Fig. 1 a and Fig. 1 b).

It is at present PMSG for promoting the major hormone product of follicular development in conventional timing insemination technique process, with import Based on.PMSG is a kind of glycoprotein hormones, is secreted by the endometrium cup of pregnant horse, First Trimester blood middle concentration highest, in life In nature, PMSG has FSH (follicle-stimulating hormone) and LH (luteotropin) double activity concurrently to object, and can not separate.PMSG's partly declines Phase is longer, need to only inject 1 time, but because PMSG molecule contains higher hexose and sialic acid, half-life period is too long in animal body (120h) when being used for Superovulation on Animals, is easy to cause a variety of ill effects of dam, such as ovarian cyst, Early placental phase embryo Degeneration in advance.In addition, the PMSG of commercialization is extracted from the blood of pregnant mare and is obtained, unstable quality, purity and potency It is low, antibody can be generated when for other animals, there are certain immunogenicities, cannot use for a long time, have at high cost and tool There is potential cause pathogeny imcrobe infection.In addition, PMSG is prepared from pregnant mare acquisition serum, often because blood sampling excessively causes Abortion of mares, tire horse are dead, do not meet animal welfare.Moreover, the raising expense with horse increases year by year, influenced by raw material, Cost increases year by year, and the aquaculture cost of raiser and financial burden increasingly increase, and limits its extensive use.

PMSG acts on the receptor in target cell of ovary, including fsh receptor and LH receptor in jenny body.FSH for Jenny acts on ovary, promotes reach maturity ovulation and the corpus luteum generation of ovarian follicle, and LH promotes jenny ovulation.It is based on The mechanism of action of PMSG, FSH and LH, it can be seen that by directly using the effect of FSH and LH than using the effect of PMSG more Significantly.

The FSH product of existing listing is hypophysis FSH, and such as a-, odd (Canada) animal health Co., Ltd Bell is silent real Test the Folltropin- of room exploitationThe product is extracted from pig pituitary and is obtained, exist limited by raw material sources, purity and effect The problems such as valence is low, potential virus pollutes, and half-life period is too short, it is complicated that process is administered, metabolic half life is only 6 hours in vivo Left and right needs to inject 2 times daily, continuous to inject 3 days to maintain the lasting stimulation to ovarian follicle, in practical applications by very Big limitation.The follicle-stimulating hormone (FSH) extracted from animal hypophysis the disadvantages of needing multiple injection, cannot since it is there are half-life short Meets the needs of timing semen deposition and production of batch metaplasia.Hypophysis FSH product is seldom used currently on the market.

In addition, PMSG and hypophysis FSH exist, raw material sources are limited, purity and potency are low, unstable quality, production cost Height has the shortcomings that latent viral infection risk, becomes the bottleneck problem for restricting the popularization and application of sow timing insemination technique.

In human medicine, with advances in technology, pass through the recombinant protein medicine of mammalian cell expression and production It has been be used widely that, it is stable with raw material sources, reduce viral pollution risk, more compared with extracting product with biochemistry Add the significant advantages such as safe and effective and cost significant decrease.

Wherein, Chinese hamster ovary cell (Chinese Hamster Ovary Cell, CHO) is for outside eucaryote Source gene expression host cell the most successful, has more and more pharmaceutical proteins and obtains high efficient expression wherein, very much People has been listed with recombinant protein medicine.Compared with other expression systems, which is had many advantages, such as possesses complete translation Post-processing process, including glycosylation, hydroxylating, make the external source eukaryotic gene product of expression be able to maintain its natural structure and work Property, and it is extracellular to be secreted into expression product, is conducive to isolating and purifying for foreign protein.

The amino acid sequence homology of FSH albumen between different mammalian species is very high, for example, people's FSH α chain and β The amino acid sequence homology of chain and pig FSH α chain and β chain is respectively 83% and 96%, the amino acid sequence of people FSH and ox FSH Homology is up to 88%, prompts the FSH of separate sources in the potential application in mammalian reproduction field.Have at present and utilizes CHO Human medicine recombination hFSH (Human follicle-stimulating hormone, the abbreviation hFSH) listing of cell production, But still there are the following problems: firstly, the recombination hFSH expression quantity of existing method production is too low, preparation process is complicated, is produced into Ben Taigao；Secondly, its half-life short, needs frequent drug administration by injection.But field of veterinary is essentially sky to recombinant protein medicine at home White, veterinary drug only has a kind of non-vaccine recombinant protein medicine in the market at present is produced using Escherichia coli.Therefore, it utilizes Molecular biology and cell culture processes develop one that the FSH drug with bioactivity and longer half-life is this field Challenge.

More importantly yet there are no the correlation that long-acting reorganization FSH fusion protein is applied in sow timing semen deposition Report.

Summary of the invention

Problems to be solved by the invention

The present invention is intended to provide a series of long-acting recombinant human follicle-stimulating hormone (Human follicle-stimulating Hormone, abbreviation hFSH) fusion protein and long-acting Recombinant Swine follicle-stimulating hormone (Porcine follicle-stimulating Hormone, abbreviation pFSH) fusion protein, preparation method and its application in sow timing semen deposition.

The solution to the problem

The present inventor is in depth studied in view of above-mentioned problems of the prior art, discovery by Fc merge, The long-actingization means such as CTP modify FSH, carry out Expression product, and the recombination that will be prepared by mammalian cell FSH fusion protein is applied in the timing semen deposition of sow, so as to complete the present invention.That is, the present invention is as described below:

Present invention firstly provides a kind of reorganization FSH fusion proteins, which is characterized in that the fusion protein is optional certainly:

(i) recombinate hFSH-Fc fusion protein amino acid sequence successively include from N-terminal to C-terminal signal peptide, hFSH β subunit, CTP, hFSH α subunit, peptide linker and Fc, wherein the Fc is IgG2-hFc variant or IgG1-hFc variant；Or,

(ii) amino acid sequence for recombinating pFSH-Fc fusion protein successively includes signal peptide, the Asia pFSH β from N-terminal to C-terminal Base, CTP, pFSH α subunit, peptide linker and Fc, wherein the Fc is times in pFc, IgG2-hFc variant or IgG1-hFc variant It is a kind of；Or,

(iii) recombination hFSH-CTP fusion protein is made of hFSH α subunit and hFSH β-CTP subunit, wherein hFSH β- The amino acid sequence of CTP subunit includes hFSH β subunit and CTP from N-terminal to C-terminal；Or,

(iv) recombination pFSH-CTP fusion protein is made of pFSH α subunit and pFSH β-CTP subunit, wherein pFSH β- The amino acid sequence of CTP subunit successively includes pFSH β subunit and CTP from N-terminal to C-terminal.

Wherein peptide linker described in (i) and (ii) contains 2-20 amino acid, and the peptide linker is present in hFSH α subunit Between IgG Fc variant, and peptide linker contains two or more amino for being selected from glycine, serine, alanine and threonine Acid；Preferably, the amino acid sequence of the peptide linker is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGly GlySer。

In some preferred embodiments, the amino acid sequence of CTP described in (i) described above and (ii) be from 33 amino acid residue sequences of HCG β chain carboxylic acid's base end, as shown in SEQ ID NO:20；Wherein described in (iii) and (iv) CTP amino acid sequence be 33 amino acid residues from hCG β chain carboxyl terminal, as shown in SEQ ID NO:20.Its In, amino acid sequence shown in SEQ ID NO:20 is nucleotide sequence coded as shown in SEQ ID NO:19.

In a further preferred embodiment, wherein the amino acid sequence of fusion protein described in (i) such as SEQ ID Shown in NO:2 or SEQ ID NO:4；The wherein amino acid sequence of fusion protein described in (ii) such as SEQ ID NO:6, SEQ ID Shown in NO:8 or SEQ ID NO:10；The amino acid for the hFSH β-CTP subunit for wherein including in fusion protein described in (iii) Sequence is as shown in SEQ ID NO:12, the amino acid sequence for the hFSH α subunit for including in the fusion protein such as SEQ ID NO: Shown in 14；The amino acid sequence such as SEQ ID NO:16 for the pFSH β-CTP subunit for wherein including in fusion protein described in (iv) Shown, the amino acid sequence for the pFSH α subunit for including in the fusion protein is as shown in SEQ ID NO:18.

The present invention also provides a kind of pharmaceutical compositions, which is characterized in that described pharmaceutical composition includes on a effective amount of Reorganization FSH fusion protein and pharmaceutically acceptable carrier described in text.

Wherein, " pharmaceutically acceptable carrier " refers to pharmaceutically acceptable substance, composition or carrier, such as: liquid Or solid-filling agent, diluent, excipient, solvent or packing substance, they carry or transport certain chemical substance.

The pharmaceutically acceptable carrier includes (but being not limited to): sucrose, mannitol, polysorbas20 (Tween 20), methionine, salt water, buffer, glucose, water, glycerol, and combinations thereof.Usual pharmaceutical preparation should be with administration mode phase Match, pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain glucose and other adjuvants Aqueous solution prepared by conventional method.The pharmaceutical composition preferably aseptically manufactures.Active constituent is given Dose is therapeutically effective amount.Pharmaceutical preparation of the invention may also be fabricated which sustained release preparation.

The effective quantity of fusion protein of the present invention can be with the mode of administration and the severity of disease to be treated etc. And change.Preferred a effective amount of selection can be determined depending on various factors by those of ordinary skill in the art (such as to be passed through Clinical test).The factor includes but is not limited to: the pharmacokinetic parameter of the fusion protein such as biological utilisation Rate, metabolism, half-life period etc.；Animal the severity of disease of being treated, the weight of animal, the immune state of animal, administration way Diameter etc..

The present invention also provides the preparation methods of reorganization FSH fusion protein described above, which is characterized in that

(1) when the reorganization FSH fusion protein is (i) or (ii), step includes:

A) expression vector of building coding recombination hFSH-Fc or pFSH-Fc fusion protein

The gene that coding hFSH-Fc or pFSH-Fc fusion protein is obtained using artificial synthesis, is inserted into mammal Fibrocyte expression vector obtains the expression plasmid containing hFSH-Fc or pFSH-Fc antigen-4 fusion protein gene；

B) recombination hFSH-Fc or pFSH-Fc fusion protein stablizes expression in mammalian host cell

Expression vector containing hFSH-Fc or pFSH-Fc fusion protein is transfected into mammalian host cell, screening is steady Surely the cell strain of hFSH-Fc or pFSH-Fc fusion is expressed；

C) concentration cultivation recombinates hFSH-Fc or pFSH-Fc fusion protein

D) purifying of hFSH-Fc or pFSH-Fc fusion protein is recombinated: including using Protein A affinity chromatography and hydrophobic Chromatographic column is purified, wherein the drainage column includes 4 Fast Flow of Butyl Sepharose, Octyl Sepharose 4 Fast Flow、Phenyl Sepharose 6 Fast Flow、Butyl-S Sepharose 6 Fast Flow, Butyl Sepharose 4B, Octyl Sepharose CL-4B, Phenyl Sepharose CL-4B, preferably Phenyl Sepharose 6 Fast Flow；

(2) when the reorganization FSH fusion protein is (iii) or (iv), step includes:

A) expression vector of building coding recombination hFSH-CTP or pFSH-CTP fusion protein

It is dynamic to be inserted into lactation for the gene that coding hFSH-CTP or pFSH-CTP fusion protein is obtained using artificial synthesis Object fibrocyte expression vector obtains the expression plasmid containing hFSH-CTP or pFSH-CTP antigen-4 fusion protein gene；

B) recombination hFSH-CTP or pFSH-CTP fusion protein stablizes expression in mammalian host cell

Expression vector containing hFSH-CTP or pFSH-CTP fusion protein is transfected into mammalian host cell, is screened Stablize the cell strain of expression hFSH-CTP or pFSH-CTP fusion；

C) concentration cultivation recombinates hFSH-CTP or pFSH-CTP fusion protein；

D) purifying of hFSH-CTP or pFSH-CTP fusion protein is recombinated: including using segmentation ammonium sulfate precipitation, precipitating multiple Molten and ultrafiltration is changed liquid, Anionic column chromatography and hydrophobic chromatography and is purified.

In preferred embodiments, the expression vector in preparation method described above is pCDNA3, pCMV/ ZEO, pIRES, pDR, pBK, pSPORT or pCMV-DHFR, preferably pCDNA3, more preferably after genetic modification pCDNA3；Wherein the cell transfecting method includes electroporation transfection method, calcium phosphate transfection, liposome transfection and plasm Fusion, preferably electroporation transfection method；Wherein the mammalian host cell include CHO, HEK293, BHK, NS0 and Sp2/0 cell, preferably Chinese hamster ovary celI, more preferably DHFR deficient CHO suspension cell (CHO DHFR^-)。

The present invention also provides reorganization FSH fusion proteins described above to prepare answering in animal reproduction field of medicament With.

Invention further provides the application of reorganization FSH fusion protein described above in sow timing semen deposition, Described in sow be Suprapubic arch sling or replacement gilt.

The effect of invention

Long-acting recombination follicle-stimulating hormone (FSH) fusion protein provided by the invention by mammalian cell animal expression and production, and Extend its half-life period by long-actingization means such as Fc fusion, CTP, has and promote the development of the sow ovarian follicle same period, regulate and control the sow same period The physiological action of heat, to realize sow timing semen deposition.With it is most common from pregnant mare serum in existing timing insemination technique The PMSG product of extraction is compared, and reorganization FSH fusion protein provided by the invention has potency and purity is high, and high with yield, The advantages that cost is controllable, virus-free infection, may replace PMSG for sow timing semen deposition, to improving sow reproductive efficiency, promote China's pig breeding industry modernization development is of great significance.

Specifically, long-acting reorganization FSH fusion protein provided by the invention has following excellent effect:

1. reorganization FSH fusion protein provided by the invention can significantly extend the Half-life in vivo of albumen.Wherein, reorganization FSH- Fc fusion protein is about 15 times of pig pituitary FSH half-life period；The half-life period of 23: PN: US20030211580 FIGURE: 3 claimed sequence fusion protein is the 5 of pig pituitary FSH Times or more.

2. long-acting reorganization FSH fusion protein is used for Suprapubic arch sling timing semen deposition, sow estrus synchronization can be remarkably promoted, it will The long-acting reorganization FSH fusion protein is for that can induce the ovarian follicle same period to develop in sow timing semen deposition program, moreover it is possible to significantly mention High oestrus of sow rate, over-all conception rate and number born per litter.

3. long-acting reorganization FSH fusion protein be used for replacement gilt timing semen deposition, can induce the sow ovarian follicle same period development and The process of heat, while oestrus of sow rate, over-all conception rate and number born per litter can also be significantly improved.

4. long-acting reorganization FSH fusion protein of the invention is applied to sow timing semen deposition, it can be achieved that saving without looking into feelings A large amount of labours.On the other hand, the fusion protein is used for sow timing semen deposition, it is contemplated that PSY (every sow of sow can be made The number pigs weaned provided every year) at least 1.0 or more are improved, to greatly improve the economic benefit of China's pig breeding industry.

Detailed description of the invention

Fig. 1 a shows the program of replacement gilt timing semen deposition, and Fig. 1 b shows the program of Suprapubic arch sling timing semen deposition.

Fig. 2 a shows the single-stranded structural schematic diagram of hFSH-Fc and pFSH-Fc, and Fig. 2 b shows hFSH-Fc and pFSH-Fc The structural schematic diagram of dimerization.

Fig. 3 a and Fig. 3 b respectively illustrate constructed coding hFSH-vIgG2-hFc and hFSH-vIgG1-hFc fusion egg White eukaryon expression plasmid map.

Fig. 4 a, Fig. 4 b and Fig. 4 c show constructed coding pFSH-pFc, pFSH-vIgG2-hFc and pFSH-vIgG1- The eukaryon expression plasmid map of hFc fusion protein.

Fig. 5 shows the SDS- of recombination fusion protein hFSH-vIgG2-hFc, hFSH-vIgG1-hFc and pig pituitary FSH PAGE electrophoretogram.Wherein, recombination fusion protein hFSH-vIgG2-hFc, hFSH-vIgG1-hFc and pig pituitary FSH are located at The swimming lane of the 1st, the 2nd and the 3rd of electrophoretogram, the 4th swimming lane are Marker.

Fig. 6 shows the SEC-HPLC map of recombination fusion protein hFSH-vIgG2-hFc and hFSH-vIgG1-hFc.Its In, 1 is the SEC-HPLC map of hFSH-vIgG1-hFc, and 2 be the SEC-HPLC map of hFSH-vIgG2-hFc.

Fig. 7 shows pig pituitary FSH and recombination fusion protein pFSH-pFc, pFSH-vIgG2-hFc and pFSH-vIgG1- The SDS-PAGE electrophoresis of hFc.Wherein, pig pituitary FSH and recombination fusion protein pFSH-pFc, pFSH-vIgG2-hFc, pFSH- VIgG1-hFc is located at the 1st swimming lane and 3-5 swimming lane of electrophoretogram, and the 2nd swimming lane is Marker.

Fig. 8 shows the SEC- of recombination fusion protein pFSH-pFc, pFSH-vIgG2-hFc and pFSH-vIgG1-hFc HPLC map.Wherein, 1 be pFSH-vIgG2-hFc SEC-HPLC map, 2 be pFSH-pFc SEC-HPLC map, 3 are The SEC-HPLC map of pFSH-vIgG1-hFc.

Fig. 9 shows the single-stranded structural schematic diagram of hFSH-CTP and pFSH-CTP.

Figure 10 shows the eukaryon expression plasmid map of constructed coding hFSH-CTP fusion protein.

Figure 11 shows the eukaryon expression plasmid map of constructed coding pFSH-CTP fusion protein.

Figure 12 shows the SDS-PAGE electrophoresis of pig pituitary FSH, recombination fusion protein hFSH-CTP and pFSH-CTP.Its In, pig pituitary FSH, recombination fusion protein hFSH-CTP and pFSH-CTP are located at the 1st swimming lane and the 3-4 swimming of electrophoretogram Road, the 2nd swimming lane are Marker.

Figure 13 shows the SEC-HPLC map of hFSH-hFc.Wherein, 1 be recombination fusion protein hFSH-CTP SEC- HPLC map, 2 be the SEC-HPLC map of recombination fusion protein pFSH-CTP.

Figure 14 shows the Drug-time curve of each reorganization FSH-Fc fusion protein and pig pituitary FSH.

Figure 15 shows the Drug-time curve of each 23: PN: US20030211580 FIGURE: 3 claimed sequence fusion protein and pig pituitary FSH.

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, according to normal conditions such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

The preparation of 1 reorganization FSH-Fc fusion protein of embodiment

The expression vector of 1.1 building coding recombination hFSH-Fc fusion proteins

Gene order design is optimized based on Chinese hamster ovary celI preference codon, synthesizes warp using artificial synthesized method Cross the fusion base containing encoded signal peptide and hFSH albumen β chain maturation peptide fragment, CTP and hFSH protein alpha chain maturation peptide fragment of optimization Cause obtains hFSH matter between the EcoRV restriction enzyme site in synthesized DNA fragmentation insertion transfer vector such as pUC57 Grain (i.e. phFSH), uses the correctness of DNA sequencing method validation insetion sequence.

Expression vector is transformed according to pcDNA3.0.First by the NeoR/KanR (neomycin/block that of pcDNA3.0 script Mycin) remove, change anti-apoptotic genes expression BIRC2 into, BIRC2 gene is by relying on E3 ubiquitin in conjunction with the Caspase-3/7 of activation The activity of ligase mediates Caspase-3/7 degradation, to inhibit Apoptosis.Again by ampicillin therein and its table Remove up to structure, be inserted into new kalamycin resistance gene, by T7 promoter inducing expression.Again in the EcoRI of multiple cloning sites IRES and DHFR gene is inserted into after restriction enzyme site.DHFR gene can make engineering cell in dihydrofolate reductase inhibitor MTX In the presence of survive and growth, and can by be continuously improved MTX concentration so that the target gene near external source DHFR gene obtains Amplification, to improve the copy number of target gene.Artificial synthesized IRES element, that is, internal ribosome entry site, for starting Expression of the DHFR in Chinese hamster ovary celI.This improved plasmid is named as pUH3.0.

The artificial synthesized coding flexible peptide linker containing BamHI (5 ' end) and EcoRI (3 ' end) restriction enzyme site respectively The fusion L-vIgG2-hFc of (Linker, referred to as " L ") and Fc variant (including vIgG2-hFc and vIgG1-hFc) segment with L-vIgG1-hFc.The fusion segment of acquisition is inserted respectively into BamHI in transfer vector PUC19 and EcoRI is restricted Between restriction enzyme site, the plasmid pL-vIgG2-hFc and pL-vIgG1-hFc of the variant containing Fc is respectively obtained.L- is verified by DNA sequencing The gene order correctness of vIgG2-hFc and L-vIgG1-hFc.

To prepare hFSH-L-Fc fusion, with restriction enzyme SpeI and BamHI double digestion phFSH, gel electrophoresis Glue recycling encoded signal peptide and the fusion segment of hFSH albumen β chain maturation peptide fragment, CTP and hFSH α chain maturation peptide fragment afterwards, warp The said gene segment of purifying is inserted respectively into the end 5'- of peptide linker in pL-vIgG2-hFc and pL-vIgG1-hFc plasmid, T4 Ligase connection respectively constitutes phFSH-L-vIgG2-hFc and phFSH-L-vIgG1-hFc plasmid.Constructed fusion by HFSH β, CTP, hFSH α, peptide linker and Fc variant gene composition, single-stranded structure is as shown in Figure 2 a, dimerization domain such as Fig. 2 b It is shown.

Restriction enzyme SpeI/EcoRI double digestion phFSH-L-vIgG2-hFc and phFSH-L-vIgG1-hFc plasmid, DNA gel purifies to obtain hFSH-L-vIgG2-hFc and hFSH-L-vIgG1-hFc segment.By purified hFSH-L-Fc segment It is inserted between the corresponding restriction enzyme site of improved mammalian cell expression plasmid pUH3.0 described above, final acquisition, which contains, melts Expression plasmid pUH3.0-hFSH-vIgG2-hFc, the pUH3.0-hFSH-vIgG1-hFc for closing gene, they are referred to as The structure difference of pUH3.0-hFSH-Fc plasmid, the plasmid is as shown in Figure 3a and Figure 3b shows.Wherein, the pUH3.0-hFSH- The nucleotide sequence and amino acid sequence for the hFSH-L-vIgG2-hFc for including in vIgG2-hFc plasmid are respectively such as SEQ ID NO: Shown in 1-2；The nucleotide sequence and ammonia for the hFSH-L-vIgG1-hFc for including in the pUH3.0-hFSH-vIgG1-hFc plasmid Base acid sequence is respectively as shown in SEQ ID NO:3-4.Needed for the plasmid albumen of expression alien gene containing mammal cell with high efficient Promoter CMV；Plasmid resistant gene containing kanamycin can carry out resistance screening in bacterium.In addition, when host is thin When born of the same parents are DHFR gene expression deficiencies, the dihyrofolate reductase of the mouse contained by improved pUH3.0 expression vector (DHFR) gene makes it in the presence of amethopterin (MTX), can coamplification pFSH-Fc fusion and DHFR gene.

With the α chain and β chain of CTP peptide fragment connection hFSH, can correctly be folded convenient for two chains.(preferably by peptide linker Flexible joint) bioactivity of albumen can be improved in the coupling that carries out hFSH and Fc segment, for the purpose of the present invention, preferably length It is the peptide linker of about 20 or less (but cannot be less than 2) amino acid, certainly by the peptide linker of 1 Amino acid profile also at this In the protection scope of invention, preferably using the peptide linker for containing or being selected from by 2 or more following Amino acid profile: glycine, silk Propylhomoserin, alanine and threonine.The peptide linker of the embodiment of the present invention contains Gly-Ser peptide component, amino acid sequence GlyS erGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。

The expression vector of 1.2 building coding recombination pFSH-Fc fusion proteins

" hFSH involved in the expression vector of the coding recombination hFSH-Fc fusion protein will be constructed in 1.1 sections Albumen β maturation peptide fragment " and the encoding gene of " hFSH protein alpha maturation peptide fragment " replace with respectively " pFSH albumen β maturation peptide fragment " and The encoding gene of " pFSH protein alpha maturation peptide fragment ", according to same construction method in 1.1 sections, building obtain contain fusion Expression plasmid pUH3.0-pFSH-pFc, pUH3.0-pFSH-vIgG2-hFc and pUH3.0-pFSH-vIgG1-hFc, by them It is referred to as pUH3.0-pFSH-Fc plasmid, the structure of plasmid is respectively as shown in Fig. 4 a, Fig. 4 b and Fig. 4 c.Wherein, the pUH3.0- The nucleotide sequence and amino acid sequence for the pFSH-L-pFc for including in pFSH-pFc plasmid are respectively such as SEQ ID NO:5-6 institute Show；The nucleotide sequence and amino acid for the pFSH-L-vIgG2-hFc for including in the pUH3.0-pFSH-vIgG2-hFc plasmid Sequence is respectively as shown in SEQ ID NO:7-8；The pFSH-L- for including in the pUH3.0-pFSH-vIgG1-hFc plasmid The nucleotide sequence and amino acid sequence of vIgG1-hFc is respectively as shown in SEQ ID NO:9-10.

The stable expression of 1.3 reorganization FSH-Fc fusion proteins in mammalian cells

The expression plasmid that will be constructed in the expression plasmid pUH3.0-hFSH-Fc and 1.2 sections that are constructed in above-mentioned 1.1 section PUH3.0-pFSH-Fc is each separately transfected into DHFR deficient CHO host cell (CHO-DHFR^-), Fig. 2 b shows recombination dimerization Change the schematic diagram of hFSH-Fc fusion protein and pFSH-Fc fusion protein.It is transfected using electroporation method, uses 960 μ Fd The Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) of capacitor, its electric field is set It is set to 250V, 2 × 10 in cuvette⁷~5 × 10⁷The Plasmid DNA that 10 μ g are linearized with PvuI is added in a cell.Turning Culture medium two days later, is changed to the grown cultures containing 1 μM of MTX kanamycins or 100 μM of MSX (as used GS selection markers) by dye Base can be obtained at 2-4 weeks through resistance since there are anti-apoptotic element, cloning efficiency is increased substantially relative to general carrier The transfectant of primary dcreening operation.Using the method for ELISA, with the expression of anti-hFSH antibody test hFSH-Fc and pFSH-Fc.Utilize DHFR The expression that selected marker improves recombination dimerizing protein is expanded, is trained thus in the growth containing progressive concentration MTX It supports in base, the recombination dimerizing protein gene transfected with DHFR gene coamplification.It can be in up to 10 μ with limiting dilution method subclone The transfectant grown in M MTX or 1mM MSX (such as with GS selection markers) culture medium.Since there are anti-apoptotic element, Ke Longxiao Rate is increased substantially relative to general carrier, can be obtained subclone at 2-4 weeks.The secretion rate of subclonal cell line is made into one Step analysis.It screens secretion level and is more than about 10 (being preferably about 20) μ g/10⁶The cell strain of (i.e. million) a cell/24 hour, is obtained High expression recombination hFSH-Fc fusion protein must be stablized and recombinate the cell strain of pFSH-Fc fusion protein.

1.4 recombination hFSH-Fc fusion proteins and the production and purifying for recombinating pFSH-Fc fusion protein

Using high yield cell strain obtained in above-mentioned 1.3 section, serum-free domestication culture is carried out first in culture dish, so After be transferred in shaking flask the domestication culture that suspend, carry out the screening of culture medium simultaneously during domestication, different ingredients be added Observe the biochemical indicators such as the growth conditions of cell, the activity of growth tendency and expression product and sialic acid, preferred cell training The condition of supporting are as follows: 100 μM of Cu are added in basal medium²⁺, feeding culture medium addition 2mM ManNAc (N- acetyl group-D- amino Mannose), this method can make to recombinate hFSH-Fc fusion protein and recombinate the degree of glycosylation increase of pFSH-Fc fusion protein, saliva Liquid acid content improves about 20%.After taming successfully, cell amplification is carried out, sufficient amount is arrived in amplification, carries out the monitoring of 7L bioreactor Culture, is more than 1 × 10 in cell density⁷Start to be cooled to 33 DEG C of cultures when a/mL, batch growth cycle is 20 days.Take 1mL Chinese hamster ovary celI culture supernatant, the loading after 0.45 μm of syringe filters filters, using 20 μm of ProteinA chromatographic columns of POROS A It is flat with mobile phase A (PBS solution, pH7.0) punching after loading, it is eluted with Mobile phase B (100mM glycine solution, pH3.5), Flow velocity is 2ml/min, and column temperature is room temperature, Detection wavelength 280nm.It is measured and is recombinated by above-mentioned ProteinA-HPLC method The expression quantity of hFSH-Fc fusion protein and recombination pFSH-Fc fusion protein, as the result is shown: recombination hFSH-vIgG2-hFc and again The cumulative production of group hFSH-vIgG1-hFc cell strain expression is respectively 2.08g/L, 0.86g/L；Recombination pFSH-vIgG2-hFc, Recombination pFSH-vIgG1-hFc, recombination pFSH-pFc cell strain expression cumulative production be respectively 1.84g/L, 0.89g/L, 0.75g/L。

Recombinate hFSH-Fc fusion protein and recombinate pFSH-Fc fusion protein purifying the following steps are included:

1) Protein A affinity chromatography: centrifugation collects supernatant, according to the present invention the characteristic of albumen coupling Fc segment, utilizes Supernatant is loaded onto the Protein A column of phosphate buffer saline (PBS) balance by affinity chromatography method；Fusion to be reorganized Protein binding washs the column after ProteinA, with PBS, until OD₂₈₀Value is lower than 0.01, then the acetic acid for being 4.0 with 20mM pH The recombination hFSH-Fc fusion protein and recombination pFSH-Fc fusion protein of sodium buffer elution of bound, finally use the 1M of pH10.0 Tris-HCl buffer neutralization activity collection liquid.The hFSH-Fc albumen and pFSH-Fc purity of purifying can reach 98% or more.

2) above-mentioned Protein A activity collection liquid hydrophobic chromatography: is changed to 20mM Tris-HCl- with hyperfiltration process 1.5M NaCl (pH8.0) buffer, by the sample pipetting volume to 20mM Tris-HCl-1.5M NaCl (pH8.0) it is equilibrated Phenyl-6 Fast Flow column is first eluted with identical equilibrium liquid, then with 20mM Tris-HCl-1.35M NaCl (pH8.0) Elution is finally eluted with 20mM Tris-HCl-0.5M NaCl (pH8.0) buffer.

ELISA as the result is shown recombination hFSH-Fc fusion protein and recombination pFSH-Fc fusion protein in Chinese hamster ovary celI Successful expression.As shown in figure 5 and figure 7, SDS-PAGE gel electrophoresis map under the reducing conditions, pig pituitary FSH (commercial product) and Recombination hFSH-Fc fusion protein (including hFSH-vIgG2-hFc and hFSH-vIgG1-hFc) of the invention, recombination pFSH-Fc melt Hop protein (including pFSH-pFc, pFSH-vIgG2-hFc and pFSH-vIgG1-hFc) shows phase in 42kDa and 128kDa respectively Corresponding target protein hybridising band with anti-hFSH antibody test and combines ProteinA-HPLC analysis method, demonstrates this hair Contain FSH albumen and Fc segment in bright obtained recombination hFSH fusion protein and recombination pFSH fusion protein.Utilize above-mentioned purifying The purifying hFSH-Fc fusion protein and pFSH-Fc fusion protein that technique obtains by SEC-HPLC detect its purity, as Fig. 6 with It is in Fig. 8 the results show that the hFSH-Fc fusion protein and pFSH-Fc fusion protein purity of purifying can reach 98% or more.

The preparation of 2 23: PN: US20030211580 FIGURE: 3 claimed sequence fusion protein of embodiment

The expression vector of 2.1 building coding recombination hFSH-CTP fusion proteins

Gene order design is optimized based on Chinese hamster ovary celI preference codon, synthesizes warp using artificial synthesized method That crosses optimization contains coding hFSH albumen β chain maturation peptide fragment, CTP, bGH termination signal, CMV promoter and hFSH α chain mature peptide The DNA fragmentation of section, both ends have SpeI and EcoRI restriction enzyme site.Such as by synthesized DNA fragmentation insertion transfer vector Between EcoRV restriction enzyme site in pUC57, hFSH-CTP plasmid (i.e. phFSH-CTP) is obtained, DNA sequencing side is used The correctness of method verifying insetion sequence.

With restriction enzyme SpeI and EcoRI double digestion phFSH plasmid, glue recycles the DNA piece cut after gel electrophoresis Section.The improved mammalian cell expression plasmid pUH3.0 of restriction enzyme SpeI/EcoRI double digestion is used again, it will be above-mentioned The DNA fragmentation of digestion after purification is connected in the carrier after linearizing with T4 ligase, final to obtain the expression containing fusion Plasmid pUH3.0-hFSH-CTP uses the correctness of DNA sequencing method validation insetion sequence.Constructed fusion by The DNA fragmentation composition of hFSH β, CTP, bGH termination signal, CMV promoter and hFSH α, single-stranded structure is as shown in figure 9, described The structure of plasmid is as shown in Figure 10.

Promoter CMV needed for the plasmid albumen of expression alien gene containing mammal cell with high efficient；The plasmid contains card That mycin resistant gene can carry out resistance screening in bacterium.In addition, when host cell is DHFR gene expression deficiency When, dihyrofolate reductase (DHFR) gene of the mouse contained by improved pUH3.0 expression vector makes it in ammonia first butterfly It, can coamplification pFSH-CTP fusion and DHFR gene in the presence of purine (MTX).

Two subunits of FSH α and β are started expression by two CMV promoters respectively, and β subunit is terminated by bGH termination signal Transcription, and IRES element is connected behind α subunit to transcribe DHFR gene, and transcription is terminated by SV40 termination signal.

FSH β subunit carboxyl terminal is connected to a CTP peptide fragment.CTP is human chorionic gonadotrophin (hCG) albumen β sub- One of base c-terminus includes the peptide segment structure of 33 amino acid, positioned at 113~145 of hCG β protein protomer.Have 4 on CTP A glycosylation modified site potential O- is respectively occurring at S121, S127, S132 and S138, and O- is glycosylation modified to be increased Add the ratio for improving sialic acid content in protein molecular, it is significant to extend the half-life period of albumen in vivo.Wherein, the hFSH egg White β chain maturation peptide fragment and the nucleotide sequence and amino acid sequence of CTP segment are respectively as shown in SEQ ID NO:11-12；It is described The nucleotide and amino acid sequence of hFSH α subunit are respectively as shown in SEQ ID NO:13-14.

The expression vector of 2.2 building coding recombination pFSH-CTP fusion proteins

It will be constructed in 2.1 sections involved in the expression vector of the coding recombination hFSH-CTP fusion protein " hFSH albumen β maturation peptide fragment " and the encoding gene of " hFSH protein alpha maturation peptide fragment " replace with " pFSH albumen β mature peptide respectively The encoding gene of section " and " pFSH protein alpha maturation peptide fragment " is obtained according to construction method same in 2.1 sections building containing fusion The expression plasmid pUH3.0-pFSH-CTP of gene, the structure of plasmid are as shown in figure 11.Wherein, the pFSH albumen β chain mature peptide The nucleotide sequence and amino acid sequence of section and CTP segment are respectively as shown in SEQ ID NO:15-16；The pFSH α subunit Nucleotide and amino acid sequence are respectively as shown in SEQ ID NO:17-18.

The stabilization table of 2.3 recombination hFSH-CTP fusion proteins and recombination pFSH-CTP fusion protein in mammalian cells It reaches

The expression plasmid pUH3.0-hFSH-CTP and pUH3.0-pFSH-CTP that construct in above-mentioned 2.1 and 2.2 sections are transfected Enter DHFR deficient CHO host cell (CHO-DHFR^-), Fig. 9 shows recombination hFSH-CTP fusion protein and recombination pFSH- The schematic diagram of CTP fusion protein single-stranded structure.It is transfected using electroporation method, uses the Gene of 960 μ Fd capacitors Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA), sets 250V for its electric field, 2 × 10 in cuvette⁷~5 × 10⁷The Plasmid DNA that 10 μ g are linearized with PvuI is added in a cell.It is transfecting two days later, it will Culture medium is changed to the growth medium containing 100 μ g/mL kalamycin resistance marker gene, obtains the transfectant through resistance primary dcreening operation. Using the method for ELISA, with the expression of anti-FSH-antibody detection hFSH-CTP and pFSH-CTP.Selectivity mark is expanded using DHFR The expression that note gene improves recombination dimerizing protein uses DHFR thus in the growth medium containing progressive concentration MTX The recombinant protein gene of gene coamplification transfection.It can be grown in up to 6 μM/mL MTX culture medium with limiting dilution method subclone Transfectant.The secretion rate of subclonal cell line is further analysed.It screens secretion level and is more than about 3 (being preferably about 5) μ g/ 10⁶The cell strain of (i.e. million) a cell/24 hour obtains and stablizes high expression recombination hFSH-CTP fusion protein and recombination The cell strain of pFSH-CTP fusion protein.

2.4 recombination hFSH-CTP fusion proteins and the production and purifying for recombinating pFSH-CTP fusion protein

Using high yield cell strain obtained in above-mentioned 2.3 section, serum-free domestication culture is carried out first in culture dish, so After be transferred in shaking flask the domestication culture that suspend, carry out the screening of culture medium simultaneously during domestication, different ingredients be added Observe the biochemical indicators such as the growth conditions of cell, the activity of growth tendency and expression product and sialic acid, preferred cell training The condition of supporting are as follows: 100 μM of Cu are added in basal medium²⁺, feeding culture medium addition 2mM ManNAc (N- acetyl group-D- amino Mannose), this method can be such that the degree of glycosylation for recombinating hFSH-CTP fusion protein increases, and sialic acid content improves about 15%. After taming successfully, cell amplification is carried out, sufficient amount is arrived in amplification, carries out the monitoring culture of 7L bioreactor, is more than 1 in cell density ×10⁷Start to be cooled to 33 DEG C of cultures when a/mL, batch growth cycle is 20 days.It is measured and is recombinated with ProteinA-HPLC method The expression quantity of hFSH-CTP fusion protein and recombination pFSH-CTP fusion protein, the results show that recombination hFSH-CTP cell strain table The cumulative production reached reaches 300mg/L, and the cumulative production of recombination pFSH-CTP cell strain expression reaches 200mg/L.

It is described recombination hFSH-CTP fusion protein and recombinate pFSH-CTP fusion protein purifying the following steps are included:

1) it is segmented ammonium sulfate precipitation: cell liquid 9000G is centrifuged, collect supernatant, be slowly added to ammonium sulfate solids particle simultaneously It is stirred continuously dissolution, supernatant is collected in centrifugation when ammonium sulfate concentrations reach 15%, continuously add ammonium sulfate solids particle to 35%, Precipitating is collected by centrifugation.

2) precipitating is redissolved and liquid is changed in ultrafiltration: above-mentioned deposit sample is redissolved in 20mmol, the phosphate buffer of pH7.5 (PBS), redissolution process is slowly stirred, and is controlled temperature < 10 DEG C, is prevented denaturing samples.It is completely dissolved to solid, sample solution becomes After transparent, liquid is changed in ultrafiltration, replaces and arrives 20mmolPBS, in the buffer of 10mmol NaCl, pH7.5.

3) above-mentioned Ultrafiltration buffer Anionic column chromatography: is loaded onto 20mmolPBS, 10mmol NaCl, pH7.5 balance GE Q-Sepharose Fast Flow (article No.: the 17-0510-01) chromatographic column crossed uses balance after destination protein combination Liquid continues to wash, until OD₂₈₀Lower than 0.01AU, then with 20mmolPBS, the elution mesh of 100mmol NaCl, pH7.5 Albumen, collect.

4) above-mentioned anion Q column activity collection liquid ultrafiltration hydrophobic chromatography: is changed to 20mM Tris-HCl-1.8M NaCl (pH8.0) buffer, by the sample pipetting volume to the GE equilibrated with 20mM Tris-HCl-1.8M NaCl (pH8.0) PHENYL SEPHAROSE 6FF (article No.: 17-0973-05) chromatographic column is first eluted with identical equilibrium liquid, then uses 20mM Tris-HCl-1.5M NaCl (pH8.0) elution, is finally eluted with 20mM Tris-HCl-0.8M NaCl (pH8.0) buffer.

Using the method for ELISA, with anti-FSH-antibody and anti-CTP antibody detect respectively recombination fusion protein hFSH-CTP and Expression of the pFSH-CTP in Chinese hamster ovary celI carries out SDS-PAGE gel electrophoresis map as shown in figure 12 under the reducing conditions, and pig is hung down Body FSH (commercial product) and recombination hFSH-CTP fusion protein of the invention, recombination pFSH-CTP fusion protein are respectively in 43kDa Show corresponding target protein hybridising band with 50kDa, demonstrate the obtained recombination hFSH-CTP fusion protein of the present invention and It recombinates and contains FSH albumen and CTP peptide fragment in pFSH-CTP fusion protein.Figure 13 is the purifying obtained using above-mentioned purifying process HFSH-CTP fusion protein and pFSH-CTP fusion protein detect its purity by SEC-HPLC, the results show that the hFSH- of purifying CTP albumen and pFSH-CTP purity of protein can respectively reach 98% or more.

The pharmacokinetics of 3 reorganization FSH-Fc fusion protein of embodiment measures

Test is divided into recombination hFSH-vIgG2-hFc, hFSH-vIgG1-hFc, pFSH-vIgG2- provided by the invention HFc, pFSH-vIgG1-hFc, pFSH-pFc fusion protein administration group and pig pituitary FSH (commercial product) administration group, every group of selection The male SD rat of 220 ± 10g of weight 5, is given by the dosage of 30IU/kg be subcutaneously injected respectively.

Pig pituitary FSH group upon administration 1h, 2h, 4h, 6h, 8h, 12h, for 24 hours, 36h, 60h take blood, recombinate hFSH-vIgG2- HFc, hFSH-vIgG1-hFc, pFSH-vIgG2-hFc, pFSH-vIgG1-hFc, pFSH-pFc fusion protein difference are upon administration 0h, 2h, 6h, 12h, for 24 hours, 32h, 48h, 56h, 72h, 80h, 96h, 104h, 120h take blood, and under conditions of 4 DEG C of 3000rpm After being centrifuged 5min, serum, -20 DEG C of preservations are drawn.Each time point blood plasma is measured using ELISA kit (BIOCHECK, the U.S.) Middle FSH immunocompetence.Using kinetica4.4 software, each group main pharmacokinetic parameter is calculated by statistics moments method.Each group Pharmacokinetic curve is as shown in figure 14, and half-life period, the results are shown in Table 1.

The results show that pig pituitary FSH is about 3.45h in rat intracorporal elimination half-life period；And dosage is waited to recombinate hFSH- The elimination of vIgG2-hFc, hFSH-vIgG1-hFc, pFSH-vIgG2-hFc, pFSH-vIgG1-hFc, pFSH-pFc fusion protein Half-life period respectively may be about 56.22h, 55.63h, 53.98h, 57.16h, 56.73h, be about 15 times of pig pituitary FSH half-life period.

The half-life period of table 1 reorganization FSH-Fc fusion protein and pig pituitary FSH

The pharmacokinetics of 4 23: PN: US20030211580 FIGURE: 3 claimed sequence fusion protein of embodiment measures

Test is divided into recombination hFSH-CTP fusion protein administration group provided by the invention, recombination pFSH-CTP fusion protein Administration group and pig pituitary FSH (commercial product) administration group, the male SD rat of every group of 220 ± 10g of selection weight 5 are pressed respectively The dosage of 30IU/kg, which is given, to be subcutaneously injected.

Pig pituitary FSH group upon administration 1h, 2h, 4h, 6h, 8h, 12h, for 24 hours, 36h, 60h take blood, recombination hFSH-CTP melts Hop protein administration group and recombination pFSH-CTP fusion protein administration group upon administration 0h, 2h, 4h, 8h, 12h, 18h, for 24 hours, 32h, 48h, 56h, 72h, 80h, 96h, 104h, 120h take blood, and draw serum after centrifugation 5min under conditions of 4 DEG C of 3000rpm ,- 20 DEG C of preservations.FSH immunocompetence in each time point blood plasma is measured using ELISA kit (BIOCHECK, the U.S.).It uses Kinetica4.4 software calculates each group main pharmacokinetic parameter by statistics moments method.Each group pharmacokinetic curve is as schemed Shown in 15, half-life period, the results are shown in Table 2.

The results show that pig pituitary FSH is about 3.45h in rat intracorporal elimination half-life period；And isodose recombination hFSH- The elimination half-life period of CTP fusion protein and recombination pFSH-CTP fusion protein respectively may be about 18.58h and 19.18h, and the two disappears Except half-life period is close, their half-life period is 5 times of pig pituitary half-life period or more.

The half-life period of table 2 23: PN: US20030211580 FIGURE: 3 claimed sequence fusion protein and pig pituitary FSH

Embodiment 5 recombinates application of the hFSH-Fc fusion protein in replacement gilt timing semen deposition

Healthy replacement gilt 200,220-225 age in days, weight 90-120kg, all sows complete vaccine immunity, turn It is raised to big column, administration and semen deposition breeding forward to positioning rail are raised.It is randomly divided into four groups: recombination hFSH-vIgG2-hFc administration Group, recombination hFSH-vIgG1-hFc administration group, PMSG administration group and blank control group, every group of head number are 50.

Testing program: on-test the 1st day, all sows started to feed Altrenogest, continuously feeding 18 days, daily 20mg stops being spaced 42h after feeding, is administered processing respectively to the sow of three administration groups, recombination hFSH-vIgG2-hFc and again Group hFSH-vIgG1-hFc administration group by 250 units/head while cooperating 300 units of HCG/head administered intramuscular respectively； PMSG administration group is according to 1000 units/head administered intramuscular.100 μ g/ head of 80h intramuscular injection GnRH is spaced after administration, respectively It is timed artificial insemination breeding with 40h for 24 hours after injecting GnRH.Blank control group only feeds Altrenogest, continuously feeds 18 It, daily 20mg does not do other drug treatment.

Stop feeding to start to observe and record heat situation in sow one week after Altrenogest (test the 19th day) and (occur quiet vertical anti- Should be heat), count Oestrus rate.The pregnant inspection of progress B ultrasound in 25 days to 28 days after breeding counts over-all conception rate；Remember within 114 days after gestation Record childbirth and farrowing situation, count female Litter Size in Pigs.

Estrus synchronization result: control group replacement gilt heat is randomly distributed in test the 22nd day to 28 days, will look into daily Feelings and breeding；Recombination hFSH-vIgG2-hFc and the replacement gilt heat integrated distribution for recombinating hFSH-vIgG1-hFc administration group exist It tests the 22-23 days；The replacement gilt heat of PMSG administration group is distributed in test the 21-23 days.Illustrate to be melted with recombination hFSH-Fc Oestrus of sow concentration degree significantly improves after hop protein drug treatment.Prompt can concentrate oestrus to breed, without routinely Feelings and breeding are looked into daily, labour and semen deposition cost can be saved.

Each group replacement gilt timing semen deposition the results are shown in Table 3.The result shows that: compared with blank control group, recombinate hFSH- VIgG2-hFc, Oestrus rate, the over-all conception rate for recombinating tri- administration groups of hFSH-vIgG1-hFc and PMSG significantly improve (P < 0.01), number born per litter is without significant difference.Wherein recombinate the Oestrus rate and over-all conception rate ratio of hFSH-vIgG2-hFc administration group PMSG administration group is high, but without significant difference.Illustrate that present invention recombination hFSH-Fc can significantly improve replacement gilt Oestrus rate, total Conception rate, number born per litter are also improved.

Table 3 recombinates hFSH-Fc fusion protein application effect in replacement gilt timing semen deposition

Note: over-all conception rate uses χ²It examines, different shoulder marks indicate that difference is extremely significant (P < 0.01), and identical shoulder mark indicates poor Different not significant (P > 0.05).

Embodiment 6 recombinates application of the hFSH-Fc fusion protein in Suprapubic arch sling timing semen deposition

A tire Suprapubic arch sling is selected, total 320, lactation number of days 21-28 days is first raised on big column, administration and semen deposition breeding Forward to positioning rail is raised.

Be divided into 4 groups: blank control group, recombination hFSH-vIgG2-hFc administration group, recombination hFSH-vIgG1-hFc administration group, PMSG administration group, every group each 80.

Test first day, 17:00's sow weans in the afternoon, and the drug treatment for 24 hours after wean recombinates hFSH-vIgG2-hFc Administration group and recombination hFSH-vIgG1-hFc administration group according to 250 units/head while cooperating 300 units of HCG/head intramuscular injection Administration；PMSG administration group is according to 1000 units/head administered intramuscular；Blank control group is not administered.72h intramuscular injection after administration 100 μ g/ head of GnRH is distributed in interval later and is timed semen deposition breeding with 40h for 24 hours.Blank control group is not done at any administration Reason carries out looking into feelings daily and semen deposition is bred according to conventional manual's semen deposition scheme.

(test the 2nd day) starts to observe and record heat situation in sow one week and (standing reaction occurs i.e. the 2nd day after wean For heat), count Oestrus rate；The pregnant inspection of progress B ultrasound in 25 days to 28 days after breeding counts conception rate；Record childbirth and production after 114 days Young situation counts number born per litter.

As a result: observation each group heat situation, one tire sow of control group are tested the 3rd day to 7 days, have oestrus of sow at random, often It will look into feelings and breeding；Recombinate the one of hFSH-vIgG2-hFc and recombination hFSH-vIgG1-hFc administration group, PMSG administration group Tire sow is concentrated on day 4 with heat in 5 days, is illustrated with the latter tire Suprapubic arch sling hair of recombination hFSH-Fc fusion protein drug treatment Feelings concentration degree significantly improves, and wean to estrus time shortened in 6 days.Prompt can concentrate oestrus to breed, without pressing It is conventional to look into feelings and breeding daily, labour and semen deposition cost can be saved.

The timing semen deposition of each group Suprapubic arch sling the results are shown in Table 4, the results showed that, compared with blank control group, recombinate hFSH- VIgG2-hFc and recombination hFSH-vIgG1-hFc, PMSG administration group can significantly improve over-all conception rate (P < 0.01), and every flat Equal litter size also improves；It wherein recombinates the over-all conception rate of hFSH-L-vIgG2-hFc and every average litter size is given higher than PMSG Medicine group, but there was no significant difference.In summary, illustrate that present invention recombination hFSH-Fc can significantly improve Suprapubic arch sling Oestrus rate And conception rate, shorten wean to estrus time interval, number born per litter is also improved.

Table 4 recombinates hFSH-Fc fusion protein application effect in Suprapubic arch sling timing semen deposition

Note: Oestrus rate, over-all conception rate, total parturition rate use χ²It examines, different shoulder marks indicate extremely significant (the P < of difference 0.01), identical shoulder mark indicates that difference is not significant (P > 0.05).

Embodiment 7 recombinates application of the pFSH-Fc fusion protein in replacement gilt timing semen deposition

Healthy replacement gilt 300,220-225 age in days, weight 120-130kg, all sows complete vaccine immunity, turn It is raised to big column, administration and semen deposition breeding forward to positioning rail are raised.It is randomly divided into five groups: recombination pFSH-vIgG2-hFc administration Group, recombination pFSH-vIgG1-hFc administration group, recombination pFSH-pFc, PMSG administration group, blank control group, every group each 60.

Testing program: on-test the 1st day, all sow feeding Altrenogests, continuous feeding 18 days, daily 20mg stopped It is spaced 42h after feeding, processing, recombination pFSH-vIgG2-hFc administration group and recombination pFSH- are administered respectively to four administration groups VIgG1-hFc, recombination pFSH-pFc administration group according to 250 units/head while cooperating 300 units of HCG/head intramuscular injection respectively Administration；PMSG administration group is according to 1000 units/head administered intramuscular.All sows are spaced 80h after above-mentioned administration time 100 μ g/ head of intramuscular injection GnRH is timed artificial insemination breeding with 40h for 24 hours after injecting GnRH.Blank control group Altrenogest is only fed, continuous feeding 18 days, daily 20mg does not do other drug treatment.

Stop feeding to start to observe and record heat situation in sow one week after Altrenogest (test the 19th day) and (occur quiet vertical anti- Should be heat), count Oestrus rate；The pregnant inspection of progress B ultrasound in 25 days to 28 days after breeding counts conception rate；It records within 114 days after gestation Childbirth and farrowing situation, count number born per litter.

Estrus synchronization result: control group replacement gilt heat is randomly distributed in test the 22nd day to 28 days, will look into daily Feelings and breeding；Recombination pFSH-vIgG2-hFc administration group, recombination pFSH-vIgG1-hFc administration group concentrate on heat in 22-23 days； Recombination pFSH-pFc administration group, PMSG administration group concentrate on heat in 21-23 days.Illustrate with recombination pFSH-Fc fusion protein administration Oestrus of sow concentration degree significantly improves after processing.Prompt can concentrate oestrus breed, without routinely look into daily feelings and Breeding, can save labour and semen deposition cost.

The timing semen deposition of each group replacement gilt the results are shown in Table 5, the results showed that, recombination pFSH-vIgG2-hFc administration group and The Oestrus rate and over-all conception rate for recombinating pFSH-vIgG1-hFc administration group, recombination pFSH-pFc administration group and PMSG administration group are aobvious It writes and is higher than blank control group (P < 0.01), number born per litter is without significant difference；Wherein recombinate pFSH-vIgG2-hFc administration group Oestrus rate, over-all conception rate, number born per litter are higher than PMSG administration group, but there was no significant difference.Illustrate present invention recombination pFSH-Fc The Estrus synchronization of replacement gilt can be significantly improved, number born per litter is also improved.

Table 5 recombinates pFSH-Fc fusion protein application effect in replacement gilt timing semen deposition

Note: without miscarriage, superseded and death during sow gestation.Oestrus rate, over-all conception rate, total parturition rate use χ²Inspection It tests, different shoulder marks indicate that difference is extremely significant (P < 0.01), and identical shoulder mark indicates that difference is not significant (P > 0.05).

Embodiment 8 recombinates application of the pFSH-Fc fusion protein in Suprapubic arch sling timing semen deposition

A tire Suprapubic arch sling 300 is selected, lactation number of days 21-28 days is first raised on big column, administration and semen deposition breeding forward It is raised to positioning rail.Be divided into 5 groups: blank control group, recombination pFSH-vIgG2-hFc administration group, recombination pFSH-vIgG1-hFc to Medicine group, recombination pFSH-pFc administration group, PMSG administration group, every group each 60.

It tests the 1st day, 17:00's sow weans in the afternoon, and the drug treatment for 24 hours after wean recombinates pFSH-vIgG2-hFc Administration group and recombination pFSH-vIgG1-hFc administration group, recombination pFSH-pFc administration group are matched according to 250 units/head, simultaneously respectively Close 300 units of HCG/head administered intramuscular；PMSG administration group is according to 1000 units/head administered intramuscular.72h after administration 100 μ g/ head of intramuscular injection GnRH is timed artificial insemination's breeding respectively at interval later with 40h for 24 hours.Blank control group is not Any drug treatment is done, carries out looking into feelings daily according to conventional manual's semen deposition scheme and semen deposition is bred.

As a result: observation each group heat situation, one tire sow of control group have oestrus of sow from test the 3rd day to 7 days at random, Feelings and breeding will be looked into daily；Recombinate pFSH-vIgG2-hFc administration group, recombination pFSH-vIgG1-hFc administration group, recombination PFSH-pFc administration group, the tire sow concentration of PMSG administration group with heat in 5 days, illustrate with recombination pFSH-Fc fusion on day 4 The latter tire Suprapubic arch sling heat concentration degree of protein medicine-feeding processing significantly improves, and wean to estrus time shortened in 6 days.Prompt can It is concentrating oestrus to breed, without routinely looking into feelings and breeding daily, labour and semen deposition cost can be saved.

The timing semen deposition of each group Suprapubic arch sling the results are shown in Table 6, the results showed that, compared with blank control group, recombinate pFSH- VIgG2-hFc administration group and recombination pFSH-vIgG1-hFc administration group, recombination pFSH-pFc administration group, PMSG administration group can Over-all conception rate (P < 0.01) is significantly improved, every average litter size is improved；Wherein recombinate pFSH-vIgG2-hFc administration group Over-all conception rate and every average litter size be higher than PMSG administration group, but there was no significant difference.In summary, illustrate present invention weight Group pFSH-Fc can significantly improve replacement gilt Estrus synchronization, shorten wean to estrus time interval, number born per litter Also it is improved.

Table 6 recombinates pFSH-Fc fusion protein application effect in Suprapubic arch sling timing semen deposition

Note: without miscarriage, superseded and death during sow gestation.Oestrus rate, over-all conception rate, total parturition rate are examined using χ 2 It tests, different shoulder marks indicate that difference is extremely significant (P < 0.01), and identical shoulder mark indicates that difference is not significant (P > 0.05).

Embodiment 9 recombinates application of the hFSH-CTP fusion protein in replacement gilt timing semen deposition

Healthy replacement gilt 150,220-225 age in days, weight 120-130kg, all sows complete vaccine immunity, turn It is raised to big column, administration and semen deposition breeding forward to positioning rail are raised.It is randomly divided into three groups, recombination hFSH-CTP administration group, PMSG administration group and blank control group, every group each 50.

Testing program: on-test the 1st day, replacement gilt fed Altrenogest, and continuous feeding 18 days, daily 20mg stops Interval 42h is administered processing after feeding, recombination hFSH-CTP administration group according to 1200IU/ and meanwhile cooperate 300 unit of HCG/ Head administered intramuscular；PMSG administration group is according to 1000 units/head administered intramuscular；Blank control group is not administered.Then exist It is spaced 80h intramuscular injection GnRH100 μ g after administration, is timed artificial insemination breeding with 40h for 24 hours after injecting GnRH. Blank control group only feeds Altrenogest, and continuous feeding 18 days, daily 20mg does not do other drug treatment.

Stop feeding to start to observe and record heat situation in sow one week after Altrenogest (test the 19th day) and (occur quiet vertical anti- Should be heat), count Oestrus rate；The pregnant inspection of progress B ultrasound in 25 days to 28 days after breeding counts conception rate；Childbirth is recorded after 114 days With farrowing situation, number born per litter is counted.

Estrus synchronization result: control group replacement gilt heat is randomly distributed in test the 22nd day to 28 days, will look into daily Feelings and breeding；The replacement gilt heat integrated distribution of hFSH-CTP administration group is recombinated in test the 22-23 days；PMSG administration group Replacement gilt heat is distributed in test the 21-23 days.Illustrate with oestrus of sow after recombination hFSH-CTP fusion protein drug treatment Concentration degree significantly improves.Prompt can concentrate oestrus to breed, and without routinely looking into feelings and breeding daily, can save labour With semen deposition cost.

The timing semen deposition of each group replacement gilt the results are shown in Table 7, the results showed that, it recombinates hFSH-CTP administration group and PMSG gives The Oestrus rate and over-all conception rate of medicine group are all remarkably higher than blank control group (P < 0.01), and there was no significant difference for number born per litter.Its Estrus synchronization, the number born per litter of middle recombination hFSH-CTP administration group are higher than PMSG group, but there was no significant difference.Explanation The present invention, which recombinates hFSH-CTP, can significantly improve replacement gilt Oestrus rate and over-all conception rate, and number born per litter is also improved.

Table 7 recombinates hFSH-CTP fusion protein application effect in replacement gilt timing semen deposition

Embodiment 10 recombinates application of the hFSH-CTP in Suprapubic arch sling timing semen deposition

One tire Suprapubic arch sling, sow kind are that new U.S.A is binary boar, and lactation number of days 21-25 days is first raised on big column, given Medicine and semen deposition breeding forward to positioning rail are raised.Three groups are randomly divided into, blank control group, recombination hFSH-CTP administration group, PMSG Administration group, every group each 90.

It tests the 1st day, all sows in the afternoon 17:00 wean, the drug treatment for 24 hours after wean recombinates hFSH-CTP Administration group is according to 1200IU/ while cooperating 300 units of HCG/head administered intramuscular；PMSG administration group is single according to 1000 Position/head administered intramuscular.The then 100 μ g/ head of 72h intramuscular injection GnRH after above-mentioned administration time, later respectively at interval Semen deposition breeding is timed with 40h for 24 hours.Blank control group does not do any drug treatment, daily according to conventional manual's semen deposition scheme It carries out looking into feelings and semen deposition breeding.

As a result: observation each group heat situation, one tire sow of control group have oestrus of sow from test the 3rd day to 7 days at random, Feelings and breeding will be looked into daily；Recombinate hFSH-CTP administration group, a tire sow of PMSG administration group concentrates and sent out on day 4 with 5 days Feelings illustrate to be significantly improved with the recombination latter tire Suprapubic arch sling heat concentration degree of hFSH-CTP fusion protein drug treatment, and wean is arrived Estrus time shortened in 6 days.Prompt can concentrate oestrus to breed, and without routinely looking into feelings and breeding daily, can save Save labour and semen deposition cost.

The timing semen deposition of each group Suprapubic arch sling the results are shown in Table 8, the results showed that, compared with blank control group, weight of the present invention Group hFSH-CTP administration group can significantly improve Estrus synchronization (P < 0.01), and number born per litter is also improved, but without aobvious Write difference.Wherein the Estrus synchronization, number born per litter of recombination hFSH-CTP administration group are higher than PMSG administration group, but without aobvious It writes difference (P > 0.05).In summary, illustrate that present invention recombination hFSH-CTP can significantly improve replacement gilt Oestrus rate, total Conception rate and total parturition rate, number born per litter are also improved.

Table 8 recombinates hFSH-CTP fusion protein application effect in Suprapubic arch sling timing semen deposition

Note: sow during pregnancy without miscarriage, death and eliminate.Over-all conception rate uses χ²It examines, different shoulder marks indicate Difference is extremely significant (P < 0.01), and identical shoulder mark indicates that difference is not significant (P > 0.05).

Embodiment 11 recombinates application of the pFSH-CTP in replacement gilt timing semen deposition

Healthy replacement gilt 180,220-225 age in days, weight 120-130kg, after all sows complete vaccine immunity, It is first raised on big column, administration and semen deposition breeding forward to positioning rail are raised.Stochastic averagina is divided into three groups: recombination pFSH-CTP administration Group, PMSG administration group, blank control group, every group 60.

Administration group testing program: test the 1st day, sow starts simultaneously at feeding Altrenogest, continuous feeding 18 days, daily 20mg stops interval 42h after feeding and is administered processing, and recombination pFSH-CTP administration group is according to 1200IU/ while cooperating HCG300 Unit/head administered intramuscular；PMSG administration group is according to 1000 units/head administered intramuscular.80h muscle note is spaced after administration 100 μ g of GnRH is penetrated, is timed artificial insemination breeding with 40h for 24 hours after injecting GnRH.Blank control group only feeds alkene Third pregnant element, continuous feeding 18 days, daily 20mg does not do other drug treatment.

Estrus synchronization result: control group replacement gilt heat is randomly distributed in test the 22nd day to 28 days, will look into daily Feelings and breeding；The replacement gilt heat integrated distribution of pFSH-CTP administration group, PMSG administration group is recombinated in test the 21-23 days. Illustrate to be significantly improved with oestrus of sow concentration degree after recombination pFSH-CTP fusion protein drug treatment.Prompt can concentrate oestrus It breeds, without routinely looking into feelings and breeding daily, labour and semen deposition cost can be saved.

The timing semen deposition of each group replacement gilt the results are shown in Table 9, the results showed that, it recombinates pFSH-CTP administration group and PMSG gives The Estrus synchronization of medicine group is all remarkably higher than blank control group (P < 0.01), and number born per litter is without significant difference；Two are given There was no significant difference for result between medicine group.Illustrate the present invention recombination pFSH-CTP can significantly improve replacement gilt Oestrus rate and Conception rate, number born per litter are also improved.

Table 9 recombinates pFSH-CTP fusion protein application effect in replacement gilt timing semen deposition

Embodiment 12 recombinates application of the pFSH-CTP in Suprapubic arch sling timing semen deposition

One tire Suprapubic arch sling, sow strain are that new U.S.A is binary boar, lactation number of days 21-25 days.All sows are completed It after vaccine immunity, is first raised on big column, administration and semen deposition breeding forward to positioning rail are raised.Three groups are randomly divided into, blank control Group, recombination pFSH-CTP administration group, PMSG administration group, long run test 3 batches, every group each 80.

It tests the 1st day, all sows in the afternoon 17:00 wean, the drug treatment for 24 hours after wean recombinates hFSH-CTP Administration group is according to 1200IU/ while cooperating 300 units of HCG/head administered intramuscular；PMSG administration group is single according to 1000 Position/head administered intramuscular.72h intramuscular injection GnRH100 μ g/ head after administration, be spaced later for 24 hours with 40h programmed breeding Breeding.Blank control group does not do any drug treatment, carries out looking into feelings daily according to conventional manual's semen deposition scheme and semen deposition is bred.

As a result: observation each group heat situation, one tire sow of control group have oestrus of sow from test the 3rd day to 7 days at random, Feelings and breeding will be looked into daily；Recombinate pFSH-CTP administration group, a tire sow of PMSG administration group concentrates and sent out on day 4 with 5 days Feelings illustrate to be significantly improved with the recombination latter tire Suprapubic arch sling heat concentration degree of pFSH-CTP fusion protein drug treatment, and wean is arrived Estrus time shortened in 6 days.Prompt can concentrate oestrus to breed, and without routinely looking into feelings and breeding daily, can save Save labour and semen deposition cost.

The timing semen deposition of each group Suprapubic arch sling the results are shown in Table 10, the results showed that, compared with blank control group, weight of the present invention Group pFSH-CTP and PMSG administration group can significantly improve Oestrus rate and over-all conception rate (P < 0.01), and number born per litter is without aobvious It writes difference (P > 0.05)；There was no significant difference for result between two administration groups.In summary, illustrate present invention recombination pFSH- CTP can significantly improve Suprapubic arch sling Estrus synchronization, shorten wean to estrus time interval, number born per litter also mentions It is high.

Table 10 recombinates pFSH-CTP fusion protein application effect in Suprapubic arch sling timing semen deposition

Note: sow during pregnancy without miscarriage, death and eliminate.Over-all conception rate χ²It examines, different shoulder marks indicate poor Heteropolar significant (P < 0.01), identical shoulder mark indicate that difference is not significant (P > 0.05).

Sequence table

<110>Guangzhou Wei Sheng Pharmaceutical Technology Co., Ltd

<120>long-acting reorganization FSH fusion protein, preparation method and its application in sow timing semen deposition

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1485

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60

tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120

agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180

ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240

agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300

tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360

tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420

gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480

ctgccacagg cccccgatgt gcaggactgc cctgaatgta ctctgcagga gaaccccttc 540

ttttctcagc ccggcgctcc tatcctgcag tgtatgggat gctgttttag tagagcatat 600

cctaccccac tgcgctcaaa gaaaacaatg ctggtccaga agaatgtgac aagcgaatct 660

acttgctgtg tggctaaatc ctacaaccgc gtgaccgtga tgggcggctt caaggtggag 720

aatcacacag catgccattg ttctacttgc tactaccata agagtggatc cggtggcggt 780

tccggtggag gcggaagcgg cggtggagga tcagtggagt gccctccatg tccagcaccc 840

cctgtcgcag gtccatctgt gttcctgttt ccacccaagc ctaaagacac tctgatgatc 900

tcccgcaccc cagaagtcac ctgtgtggtc gtggatgtga gccatgaaga ccccgaggtc 960

cagttcaatt ggtacgtgga tggcgtcgag gtgcacaacg ctaagacaaa acctagagaa 1020

gagcagttca actctacctt tcgcgtcgtg agtgtgctga cagtcgtgca ccaggactgg 1080

ctgaatggca aggagtataa gtgcaaagtg agcaacaaag gactgcctgc ctcaatcgaa 1140

aagactattt ccaagaccaa aggacagcca agagagcccc aggtgtacac cctgcctcca 1200

agccgcgaag agatgactaa aaatcaggtc tctctgacct gtctggtgaa ggggttttat 1260

cctagtgata tcgccgtgga atgggagtca aacggtcagc cagagaacaa ttacaagacc 1320

acacccccta tgctggacag cgatgggtct ttctttctgt atagcaaact gacagtggac 1380

aagtctcggt ggcagcaggg taacgtcttc tcttgcagtg tgatgcacga agcactgcac 1440

aatcattaca cccagaagtc actgtcactg agcccaggaa aatga 1485

<210> 2

<211> 494

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala

1 5 10 15

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

20 25 30

Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp

100 105 110

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

115 120 125

Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro

130 135 140

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

145 150 155 160

Leu Pro Gln Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln

165 170 175

Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met

180 185 190

Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys

195 200 205

Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val

210 215 220

Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu

225 230 235 240

Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly

245 250 255

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val

260 265 270

Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

305 310 315 320

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val

340 345 350

Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser

370 375 380

Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

485 490

<210> 3

<211> 1497

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60

tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120

agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180

ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240

agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300

tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360

tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420

gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480

ctgccacagg cccccgatgt gcaggactgc cctgaatgta ctctgcagga gaaccccttc 540

ttttctcagc ccggcgctcc tatcctgcag tgtatgggat gctgttttag tagagcatat 600

cctaccccac tgcgctcaaa gaaaacaatg ctggtccaga agaatgtgac aagcgaatct 660

acttgctgtg tggctaaatc ctacaaccgc gtgaccgtga tgggcggctt caaggtggag 720

aatcacacag catgccattg ttctacttgc tactaccata agagtggatc cggtggcggt 780

tccggtggag gcggaagcgg cggtggagga tcagacaaaa ctcacacatg cccaccgtgc 840

ccagcacctg aagtcgcggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 900

accctcatga tctcccggac acctgaggtc acatgcgtgg tggtggacgt gagccacgaa 960

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020

aagccgcggg aggagcagta caacagcacg taccgggtgg tcagcgtcct caccgtcctg 1080

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140

gcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1200

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1260

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1320

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1497

<210> 4

<211> 498

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala

1 5 10 15

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

20 25 30

Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp

100 105 110

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

115 120 125

Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro

130 135 140

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

145 150 155 160

Leu Pro Gln Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln

165 170 175

Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met

180 185 190

Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys

195 200 205

Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val

210 215 220

Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu

225 230 235 240

Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly

245 250 255

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

260 265 270

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

305 310 315 320

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

405 410 415

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

485 490 495

Gly Lys

<210> 5

<211> 1509

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60

tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120

attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180

gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240

gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300

cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360

tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420

ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480

ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540

aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600

agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660

agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720

cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780

ggtggcggtt ccggtggagg cggaagcggc ggtggaggat caaagaccaa accaccatgt 840

cccatatgcc caggctgtga agtggccggg ccctcggtct tcatcttccc tccaaaaccc 900

aaggacaccc tcatgatctc ccagaccccc gaggtcacgt gcgtggtggt ggacgtcagc 960

aaggagcacg ccgaggtcca gttctcctgg tacgtggacg gcgtagaggt gcacacggcc 1020

gagacgagac caaaggagga gcagttcaac agcacctacc gtgtggtcag cgtcctgccc 1080

atccagcacc aggactggct gaaggggaag gagttcaagt gcaaggtcaa caacgtagac 1140

ctcccagccc ccatcacgag gaccatctcc aaggctatag ggcagagccg ggagccgcag 1200

gtgtacaccc tgcccccacc cgccgaggag ctgtccagga gcaaagtcac cgtaacctgc 1260

ctggtcattg gcttctaccc acctgacatc catgttgagt ggaagagcaa cggacagccg 1320

gagccagagg gcaattaccg caccaccccg ccccagcagg acgtggacgg gaccttcttc 1380

ctgtacagca agctcgcggt ggacaaggca agatgggacc atggagaaac atttgagtgt 1440

gcggtgatgc acgaggctct gcacaaccac tacacccaga agtccatctc caagactcag 1500

ggtaaatga 1509

<210> 6

<211> 502

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

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 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser

130 135 140

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

145 150 155 160

Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys

165 170 175

Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr

180 185 190

Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg

195 200 205

Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr

210 215 220

Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala

225 230 235 240

Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His

245 250 255

Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu Val

275 280 285

Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys

355 360 365

Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala Pro

370 375 380

Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser Lys Val

405 410 415

Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His Val

420 425 430

Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr

435 440 445

Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys

465 470 475 480

Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile

485 490 495

Ser Lys Thr Gln Gly Lys

500

<210> 7

<211> 1494

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60

tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120

attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180

gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240

gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300

cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360

tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420

ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480

ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540

aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600

agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660

agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720

cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780

ggtggcggtt ccggtggagg cggaagcggc ggtggaggat cagtggagtg ccctccatgt 840

ccagcacccc ctgtcgcagg tccatctgtg ttcctgtttc cacccaagcc taaagacact 900

ctgatgatct cccgcacccc agaagtcacc tgtgtggtcg tggatgtgag ccatgaagac 960

cccgaggtcc agttcaattg gtacgtggat ggcgtcgagg tgcacaacgc taagacaaaa 1020

cctagagaag agcagttcaa ctctaccttt cgcgtcgtga gtgtgctgac agtcgtgcac 1080

caggactggc tgaatggcaa ggagtataag tgcaaagtga gcaacaaagg actgcctgcc 1140

tcaatcgaaa agactatttc caagaccaaa ggacagccaa gagagcccca ggtgtacacc 1200

ctgcctccaa gccgcgaaga gatgactaaa aatcaggtct ctctgacctg tctggtgaag 1260

gggttttatc ctagtgatat cgccgtggaa tgggagtcaa acggtcagcc agagaacaat 1320

tacaagacca caccccctat gctggacagc gatgggtctt tctttctgta tagcaaactg 1380

acagtggaca agtctcggtg gcagcagggt aacgtcttct cttgcagtgt gatgcacgaa 1440

gcactgcaca atcattacac ccagaagtca ctgtcactga gcccaggaaa atga 1494

<210> 8

<211> 497

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser

130 135 140

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

145 150 155 160

Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys

165 170 175

Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr

180 185 190

Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg

195 200 205

Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr

210 215 220

Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala

225 230 235 240

Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His

245 250 255

Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

275 280 285

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

290 295 300

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

305 310 315 320

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

325 330 335

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

340 345 350

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

355 360 365

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys

370 375 380

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

385 390 395 400

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

405 410 415

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

420 425 430

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

435 440 445

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

450 455 460

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

465 470 475 480

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

485 490 495

Lys

<210> 9

<211> 1506

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60

tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120

attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180

gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240

gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300

cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360

tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420

ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480

ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540

aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600

agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660

agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720

cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780

ggtggcggtt ccggtggagg cggaagcggc ggtggaggat cagacaaaac tcacacatgc 840

ccaccgtgcc cagcacctga agtcgcgggg ggaccgtcag tcttcctctt ccccccaaaa 900

cccaaggaca ccctcatgat ctcccggaca cctgaggtca catgcgtggt ggtggacgtg 960

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 1020

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgggtggt cagcgtcctc 1080

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1140

gccctcccag cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1200

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1260

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1320

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1380

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1440

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1500

aaatga 1506

<210> 10

<211> 501

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

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 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser

130 135 140

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

145 150 155 160

Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys

165 170 175

Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr

180 185 190

Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg

195 200 205

Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr

210 215 220

Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala

225 230 235 240

Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His

245 250 255

Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

290 295 300

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

305 310 315 320

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

325 330 335

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

340 345 350

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

355 360 365

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

370 375 380

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

385 390 395 400

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

405 410 415

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

420 425 430

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

435 440 445

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

450 455 460

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

465 470 475 480

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

485 490 495

Leu Ser Pro Gly Lys

500

<210> 11

<211> 492

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60

tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120

agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180

ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240

agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300

tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360

tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420

gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480

ctgccacagt ga 492

<210> 12

<211> 163

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala

1 5 10 15

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

20 25 30

Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp

100 105 110

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

115 120 125

Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro

130 135 140

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

145 150 155 160

Leu Pro Gln

<210> 13

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctgcc 60

cccgatgtgc aggactgccc tgaatgtact ctgcaggaga accccttctt ttctcagccc 120

ggcgctccta tcctgcagtg tatgggatgc tgttttagta gagcatatcc taccccactg 180

cgctcaaaga aaacaatgct ggtccagaag aatgtgacaa gcgaatctac ttgctgtgtg 240

gctaaatcct acaaccgcgt gaccgtgatg ggcggcttca aggtggagaa tcacacagca 300

tgccattgtt ctacttgcta ctaccataag agttag 336

<210> 14

<211> 111

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala

1 5 10 15

Val Ser Ala Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln

20 25 30

Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met

35 40 45

Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys

50 55 60

Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val

65 70 75 80

Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu

85 90 95

Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

100 105 110

<210> 15

<211> 489

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60

tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120

attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180

gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240

gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300

cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360

tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420

ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480

ccacagtga 489

<210> 16

<211> 162

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

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 Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser

130 135 140

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

145 150 155 160

Pro Gln

<210> 17

<211> 363

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

atggactact atcggaagta tgctgcagtg atcctggcta ttctgtccgt cttcctgcag 60

attctgcata gctttcctga tggggagttc accatgcagg gttgcccaga gtgtaaactg 120

aaggaaaaca aatacttctc caagctgggg gcccccatct atcagtgtat gggttgctgt 180

ttctccagag cctaccccac acctgctcgc agtaagaaaa ctatgctggt gcctaagaat 240

attactagcg aggctacctg ctgtgtcgct aaagccttca ccaaggccac agtgatggga 300

aacgcccgag tcgagaatca caccgaatgc cattgtagta catgctacta tcacaaatca 360

tag 363

<210> 18

<211> 120

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 18

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> 19

<211> 99

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

ccccgtttcc aggattccag ctctagtaaa gctccccctc cttccctgcc ctcaccctca 60

agactgcctg gaccttccga cactcccatc ctgccacag 99

<210> 20

<211> 33

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 20

Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

1 5 10 15

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

20 25 30

Gln

Claims

1. a kind of reorganization FSH fusion protein, which is characterized in that the fusion protein is optional certainly:

(1) recombination hFSH-CTP fusion protein is made of hFSH α subunit and hFSH β-CTP subunit, wherein hFSH β-CTP subunit Amino acid sequence includes hFSH β subunit and CTP from N-terminal to C-terminal；Or,

(2) recombination pFSH-CTP fusion protein is made of pFSH α subunit and pFSH β-CTP subunit, wherein pFSH β-CTP subunit Amino acid sequence successively includes pFSH β subunit and CTP from N-terminal to C-terminal.

2. reorganization FSH fusion protein according to claim 1, wherein the amino acid sequence of CTP described in (1) and (2) For 33 amino acid residues from hCG β chain carboxyl terminal, as shown in SEQ ID NO:20.

3. reorganization FSH fusion protein described according to claim 1~any one of 2, wherein being wrapped in fusion protein described in (1) The amino acid sequence of the hFSH β-CTP subunit contained is as shown in SEQ ID NO:12, the hFSH α subunit that includes in the fusion protein Amino acid sequence as shown in SEQ ID NO:14；The ammonia for the pFSH β-CTP subunit for wherein including in fusion protein described in (2) Base acid sequence is as shown in SEQ ID NO:16, the amino acid sequence for the pFSH α subunit for including in the fusion protein such as SEQ ID Shown in NO:18.

4. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes any in a effective amount of claims 1 to 3 Reorganization FSH fusion protein and pharmaceutically acceptable carrier described in.

5. the preparation method of reorganization FSH fusion protein, step described in a kind of claim 1 include:

The gene that coding hFSH-CTP or pFSH-CTP fusion protein is obtained using artificial synthesis, it is thin to be inserted into mammal Cellular expression carrier obtains the expression plasmid containing hFSH-CTP or pFSH-CTP antigen-4 fusion protein gene；

Expression vector containing hFSH-CTP or pFSH-CTP fusion protein is transfected into mammalian host cell, screening is stablized Express the cell strain of hFSH-CTP or pFSH-CTP fusion；

D) recombinate hFSH-CTP or pFSH-CTP fusion protein purifying: including use segmentation ammonium sulfate precipitation, precipitating redissolve and Ultrafiltration is changed liquid, Anionic column chromatography and hydrophobic chromatography and is purified.

6. preparation method according to claim 5, wherein the expression vector be pCDNA3, pCMV/ZEO, PIRES, pDR, pBK, pSPORT or pCMV-DHFR, preferably pCDNA3, the pCDNA3 more preferably after genetic modification； Wherein the cell transfecting method includes electroporation transfection method, calcium phosphate transfection, liposome transfection and Protoplast fusion, excellent It is selected as electroporation transfection method；Wherein the mammalian host cell includes CHO, HEK293, BHK, NS0 and Sp2/0 thin Born of the same parents, preferably Chinese hamster ovary celI, more preferably DHFR deficient CHO suspension cell (CHO DHFR^-)。

7. reorganization FSH fusion protein according to any one of claims 1 to 3 is preparing answering in animal reproduction field of medicament With.

8. application of the reorganization FSH fusion protein according to any one of claims 1 to 3 in sow timing semen deposition.

9. application according to claim 8, wherein the sow is Suprapubic arch sling or replacement gilt.