CN103897064B

CN103897064B - Long-acting recombinant human chorionic gonadotrophin fusion

Info

Publication number: CN103897064B
Application number: CN201310617181.8A
Authority: CN
Inventors: 侯永敏; 李强; 徐桢琦; 吴茂柏; 张玉杰; 王安新
Original assignee: GUANGZHOU YOULIANKANG PHARMACEUTICAL TECHNOLOGY Co Ltd
Current assignee: Guangzhou best Biotechnology Co., Ltd.
Priority date: 2013-11-29
Filing date: 2013-11-29
Publication date: 2016-05-11
Anticipated expiration: 2033-11-29
Also published as: CN103897064A

Abstract

Do you the invention discloses recombined human chorionic gonadotrophin fusion (Human? chorionic? gonadotrophin-Fc? fusion? protein, be called for short HCG-Fc) and preparation method thereof, this HCG-Fc albumen is dimerization fusion, does its amino acid sequence hold C end to comprise successively HCG β subunit, HCG α subunit, flexible peptide linker and human IgG from N? Fc variant, compared with existing chorionic gonadotrophin, its Half-life in vivo is longer, bioavilability is higher, side effect is less. The invention still further relates to restructuring HCG-Fc fusion protein compositions and treat and/or prevent the purposes in the medicine of infertility in preparation.

Description

Long-acting recombinant human chorionic gonadotrophin fusion

Technical field

The present invention relates to molecular biology and field of medicaments. More specifically, the present invention relates to long-acting recombinant human chorionic gonadotrophinFusion, its preparation method and application. This fusion Half-life in vivo significant prolongation, bioavilability significantly increases, largeLarge medication number of times and the dosage of reducing, reduces side effect.

Background technology

Physex (Humanchorionicgonadotrophin is called for short HCG) is that human reproduction field is conventionalMedicine main component, is widely used in gynemetrics and sterility treatment, and main application comprises brings out ovulation, promotes sperm development,Treatment functional uterine bleeding and habitual abortion, control premature labor, alleviates production pain etc. HCG master in China market at presentWill be the product extracting in pregnant woman urine, existence virus be polluted, raw material sources are limited, collection is difficult, content is low and purge processThe defects such as complexity, the developed countries such as America and Europe generally do not accept to urinate product-derived listing and sell.

Comparatively speaking, restructuring HCG is the product of producing by genetic engineering and mammaliancellculture, and it is pure at itThe aspects such as degree, antigenicity, security, virus-free infection have advantages of that urine source HCG product is incomparable. HCG is a kind of glycosylChange albumen, it is 50KD that SDS-PAGE detects its molecular weight. In addition, HCG is the glycosylation egg with two strands (α chain and β chain)In vain, interchain connects with non-covalent bond, the biologically active of the correct folding guarantee HCG of two chains. How at protein expression andThe normal combination that keeps two chains in purge process is a challenge. As a kind of medicine, ensure its biologically active, necessityCondition be to there is correct three-dimensional structure and glycosylation modified. Having the posttranslational modification of improving function is mammalian cell quiltBe elected to be most of biological medicament protein expression hosts' main cause. Wherein, Chinese hamster ovary cell (ChineseHamsterOvaryCell, is called for short CHO) be for the most successfully host cell of eucaryote exogenous gene expression, existing more and morePharmaceutical protein obtained therein high efficient expression, a lot of people are gone on the market with recombinant protein medicine. Compared with other expression system,This system has many advantages, as has process after complete translation, comprises glycosylation, hydroxylating, makes the external source of expressingEukaryotic gene product can keep its natural structure and activity, and expression product is secreted into outside born of the same parents, is conducive to foreign proteinSeparation and purification.

Although the existing reconstituted drug HCG listing that utilizes Chinese hamster ovary celI to produce at present, but still there are the following problems: first,Its half-life is shorter, in order to reach effective result for the treatment of, needs frequent drug administration clinically, brings very big misery to patient. ExampleAs, when for ovulation induction, 1-2 HCG of patient's palpus intramuscular injection every day, needs injection continuously 5 days conventionally. This therapeutic schemeNormal with bad side effect, comprise nerve, endocrine and immune stimulation and toxic and side effect, and cause also commonSend out disease as OHSS, show as ovary increase, the increase of whole body capillary permeability and ascites and form, severe oneCan jeopardize patient's life. Secondly, the HCG expression of recombinating is at present low, and preparation process is complicated and production cost is huge. In addition, HCG isThe glycosylated protein with two strands (α chain and β chain), interchain connects with non-covalent bond, correct folding could the guarantor of two chainsThe biologically active of card HCG. The normal combination that how to keep two chains in protein expression and purge process is a challenge. ForThe defect of existing restructuring HCG and like product, the present invention utilizes modern molecular biology technique, cells produce and protein purification workThe restructuring HCG product of skill development of new, with prolong half-life, raising bioavilability and raising restructuring HCG protein expression level,Thereby reduce administration number of times and dosage, reduce side effect.

Summary of the invention

The present invention aims to provide a kind of recombined human chorionic gonadotrophin fusion (HumanchorionicGonadotrophin-Fcfusionprotein, be called for short HCG-Fc) and preparation method thereof and application, to solve prior artThe problem that middle restructuring HCG expression is low, purifying is difficult, Half-life in vivo is short.

An object of the present invention is to provide restructuring HCG-Fc fusion, this fusion is dimerization fusion,Its amino acid sequence holds C end to comprise successively HCG β subunit, HCG α subunit, peptide linker (Linker is called for short L) and human IgG from NFc variant (vIgG2Fc, vIgG4Fc and vIgG1Fc), (HCG β-HCG α-vIgG2Fc, HCG as shown in SEQIDNO:2,4 and 6β-HCG α-vIgG4Fc, HCG β-HCG α-vIgG1Fc amino acid sequence), preferred amino acid sequence is as SEQIDNO:2 (HCGβ-HCG α-vIgG2Fc), above-mentioned fusion is referred to as HCG-Fc.

The amino acid sequence of described HCG β subunit has been removed the 1-20 amino acids residue in complete HCG β subunit,The amino acid sequence of ripe HCG β subunit is as shown in SEQIDNO:8.

The amino acid residue sequence of described HCG α subunit has been removed the 1-24 amino acids residue in complete HCG α subunit,Be that the amino acid sequence of ripe HCG α subunit is as shown in SEQIDNO:7.

Described peptide linker contains 2-20 amino acid residue, and peptide linker contains two or more and is selected from glycine, silkThe amino acid residue of propylhomoserin, alanine and threonine, preferred peptide linker amino acid sequence isGlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。

Described human IgG Fc variant comprises: human IgG2's hinge region, CH2He CH3 district that (1) contains Pro331Ser sudden changeTerritory; (2) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change; (3) containHuman IgG1's hinge region, CH2 and the CH3 region of Leu234Val, Leu235Ala and Pro331Ser sudden change. Preferred human IgG FcVariant is to contain human IgG2's hinge region, CH2 and CH3 region that Pro331Ser suddenlys change.

Below specifically introduce human IgG Fc variant of the present invention, peptide linker function:

IgGFc variant

The immunoglobulin (Ig) of IgG class is rich in protein in human blood, and their half-life can be up to 21 days, andFc fragment be in IgG holder compared with long half-lift main cause, there is the effect of stabilize proteins simultaneously.

Fc is from the Fc region of immunoglobulin (Ig), and Fc has important function in the immune defense of eliminating pathogen. IgG'sEffector function is mediated by Fc, by two kinds of main mechanisms: the combination of (1) and cell surface Fc acceptor (Fc γ Rs), by anti-Body dependent cellular cytotoxicity (ADCC) approach digestion pathogen, or the combination of the Clq of (2) and the first complement component Cl part, drawSend out cytotoxicity (CDC) approach that depends on complement, thus cracking pathogen. In four kinds of human IgG isotypes, IgG1 and IgG3Can be effectively in conjunction with Fc γ Rs. IgG4 with the binding affinity of Fc γ Rs than a low order of magnitude of IgG1 and IgG3, and IgG2So low that to be difficult to measure with the combination of Fc γ Rs. Human IgG1 and IgG3 can also be effectively in conjunction with Clq, and activating complement cascade reaction.Human IgG2 is fixing very weak to complement, and the extreme ability that lacks activating complement cascade of IgG4 performance. For medical application,The Fc region of restructuring dimerization albumen must not can mediate effector function and cracking or remove these cells. Therefore, HCG-FcFc region must be non-cracking performance, in conjunction with Fc γ Rs and Clq and aspect trigger effect subfunction, Fc is preferably without livingProperty. Obviously, do not have a kind of natural IgG isotype to be applicable to producing HCG-L-Fc restructuring dimerization albumen. In order to obtain non-splittingThe Fc of solution property, must make some amino acid mutations in natural Fc region, to reduce its effector function.

By analyzing the amino acid sequence of IgG isotype of different genera, find near the Fc portion amino terminal of CH2 regionPoint be presented in the combination of IgGFc and Fc γ Rs and work, and be bonded to pass part and parcel and be positioned at the CH2 of human IgG with ClqNear the c-terminus of region. The effector function causing in order to reduce the combination of Fc and Fc γ Rs and Clq, the present invention uses followingHuman IgG Fc variant (Fig. 1): (1) IgG2Fc variant: human IgG2's hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change;(2) IgG4Fc variant: human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change; (3)IgG1Fc variant: human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.Preferred human IgG Fc variant is to contain human IgG2's hinge region, CH2 and CH3 region that Pro331Ser suddenlys change.

Above-mentioned Fc variant shows much lower effector function than natural IgG2Fc, IgG4Fc and IgG1Fc, is more suitable forPreparation restructuring HCG-Fc fusion.

Peptide linker

The length that connects peptide is extremely important to the activity of restructuring dimerization albumen. Existing people has reported hematopoietin(EPO) derivative (as dimer), compared with EPO monomer, (3 to 7 amino acid peptides of being separated by connect to contain 2 complete EPO regionsHead) restructuring dimerization albumen show the activity that weakens (referring to QiuH etc., JBiolChem, 273:11173-11176,1998). But, in the time that the length of the interregional peptide linker of these two EPO is 17 amino acid, dimer EPO molecule externalObviously improve (SytkowskiAJ etc., JBiolChem, 274:24773-24778,1999 with in vivo bioactivity; The U.S. is specialProfit No.6,187,564). This possible explanation is the connection peptide increasing between restructuring dimerization albumen two parts, makes two of this moleculePoint can exercise respectively its function (referring to AshkenaziA etc., CurrOpininImmunol, 9:195-200,1997).

The inventor is through long-term and deep research, and the hinge region peptide linker that has designed first a kind of uniqueness reduces skyBetween steric effect, can make the C end of HCG α chain and the restructuring dimerization albumen of Fc variant coupling, centre has soft peptide to connectHead. This restructuring dimerization albumen not only can not cause the afunction of HCG, can maintain on the contrary, even improve HCG-Fc restructuringThe biologically active of dimerization albumen. The amino acid residue sequence of preferred peptide joint isGlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。

Restructuring HCG-Fc fusion of the present invention has following characteristics: this recombination fusion protein is that dimerization merges eggIn vain, its amino acid sequence holds C end to comprise successively HCG β subunit, HCG α subunit, peptide linker and human IgG Fc variant from N. Human IgGFc variant has the effect that extends Half-life in vivo and stabilize proteins, and Fc variant is non-cracking performance, can reduce with Fc γ Rs andClq in conjunction with and trigger effector function. Carry out the coupling of C end with the Fc variant of HCG α chain by soft peptide linker, canMaintain, even improve the biologically active of restructuring HCG-Fc fusion.

The present invention is connected peptide linker with human IgG Fc variant (vIgG2Fc, vIgG4Fc or vIgG1Fc) first in an orderly mannerIn HCG and form novel restructuring HCG-Fc fusion. The position of human IgG Fc variant and peptide linker in this fusionArrangement can maintain the steric configuration that HCG is correct, and does not affect its biologically active, and can the significant prolongation half-life, improves biological profitExpenditure, can reduce frequency of injection when clinical practice, reduces the toxic and side effect producing in existing therapeutic scheme.

Another object of the present invention is to provide a kind of preparation method of the HCG-Fc of restructuring fusion, this preparation method's bagDraw together:

(1) expression vector of structure coding restructuring HCG-Fc fusion;

(2) stably express of restructuring HCG-Fc fusion in mammalian host cell;

(3) high-density cells is cultivated restructuring HCG-Fc fusion;

(4) purification of restructuring HCG-Fc fusion.

Specifically, the expression vector step of structure coding restructuring HCG-Fc albumen is: adopt artificial synthesisObtain coding restructuring HCG-Fc antigen-4 fusion protein gene and carried out codon optimized nucleotide sequence (as SEQIDNO:1,3 and 5Shown in), be inserted into mammalian cell expression vector (as pCDNA3), obtain and contain HCG-Fc destination gene expression plasmidPCDNA3-HCG-Fc (Fig. 6), preferred nucleotide sequence is as SEQIDNO:1. The nucleotide sequence optimization of gene is based on the food in one's mouthThe codon of breast animal reservoir cell preference is selected design.

Described mammalian cell expression vector can adopt commercially available but be not limited to, as: pCDNA3, pCMV/ZEO,PIRES, pDR, pBK, pSPORT etc. can be used for the carrier that eukaryotic cell system is expressed, preferably, and pCDNA3.

The stably express step of restructuring HCG-Fc fusion in mammalian host cell is: will contain HCG-Fc eggWhite expression plasmid is transfected into suitable mammalian host cell, utilizes selection markers screening and amplification selected marker to obtainStablize the cell line of high-expression target proteins.

Described mammalian host cell comprises CHO, HEK293, COS, BHK, NS0 and Sp2/0 cell. Preferably, CHOCell; More preferably, the DHFR (DihydrofolateReductase, two that adapt to suspension growth in serum free medium have been tamedHydrogen folic acid reductase) deficiency Chinese hamster ovary celI (abbreviation CHO-DHFR^-)。

Described transfection method comprises calcium phosphate method, and electricity turns method, liposome transfection, and preferred transfection method is that electricity turns method.

Described screening technique is first to utilize selection markers to screen, and then can improve expression by amplification selected markerMeasure and obtain and stablize overexpression cell line. Selection markers is any one suitable selective resistance mark known in the artNote, as ZEO (Zeocin, bleomycin), G418 (aminoglycoside antibiotics), PUR (puromycin, puromycin), HYP(Hygromycin, hygromycin), preferred resistant gene is ZEO; Selection markers also can be known in the art any oneFluorescence labeling gene, comprises GFP (GreenFluorescentProtein, green fluorescent protein), RFP (RedFluorescentProtein, red fluorescent protein), preferred fluorescence labeling gene is GFP. Amplification selected marker is thisKnown DHFR sequence or GS (Glutaminesynthetase, glutamine synthelase) sequence in field, preferred amplification choosingSelecting property gene is DHFR. Due to CHO-DHFR-cell self disappearance dihyrofolate reductase (DHFR), cannot self tetrahydrobiopterin synthesisFolic acid, so must be in the cultivation of having added hypoxanthine (hypoxanthine), thymidine (Thymidine) and glycineIn liquid, just can survive. And by genes of interest and DHFR gene co-transfection, not only obtain in the not training containing above-mentioned additiveSupport the cell clone that also can grow on base, what is more important due to DHFR can by folacin MTX (Methotrexate,Methotrexate) institute suppresses, and under MTX concentration selection pressure, DHFR gene a lot of copy number that must increase could be survived, fromAnd obtain anti-MTX clone; Dye owing to tending to be incorporated into cell together with it with the genes of interest of DHFR gene co-transfection againThe same area on colour solid, thus the sequence fragment of encoding exogenous recombinant protein also increase along with the amplification of DHFR gene, canObtain the cell clone of mass expressing external albumen.

The step that high-density cells is cultivated restructuring HCG-Fc fusion is: the above-mentioned stable cell line screening is proceeded toShaking flask or biological reactor carry out large-scale culture, and particularly, the present invention, by the optimization to cell culture condition, obtains high tableReach the cell culture fluid of restructuring HCG-Fc fusion. Cell culture processes of the present invention can be realized high-density cells and cultivate, weighsHistone Quality and yield promotes, the degree of glycosylation of recombinant protein increases and sialic acid content improves.

The optimization of described cell culture condition comprises cooling cultivation, specifically, and when cell density reaches 1 × 10⁷Individual/when mL, temperature to be down to 33 DEG C by 37 DEG C, at this temperature, cultivate until express output and no longer increase. The method can be carriedThe activity level of high expressed albumen and the cumulative production of recombinant protein.

The optimization method of described cell culture condition is also included in culture medium and adds special additive, preferably,Basal medium adds 100 μ MCu²⁺, feeding culture medium adds 2mMManNAc (N-acetyl group-D-epichitosamine). ShouldMethod can make the to recombinate degree of glycosylation of HCG-Fc fusion increases, and sialic acid content improves approximately 20%.

The purification step of restructuring HCG-Fc fusion is:

1) ProteinA affinity chromatography: centrifugal, collect supernatant, the characteristic of albumen coupling Fc fragment according to the present invention, utilizesAffine ProteinA column chromatography.

2) hydrophobic chromatography post purifying: adopt hydrophobic chromatography post, according to the hydrophobicity difference of restructuring HCG-Fc fusion, enterOne step is removed the impurity in target protein after above-mentioned ProteinA purifying.

Described drainage column comprises ButylSepharose4FastFlow, OctylSepharose4FastFlow,PhenylSepharose6FastFlow，Butyl-SSepharose6FastFlow，ButylSepharose4B，OctylSepharoseCL-4B, PhenylSepharoseCL-4B. Preferably, PhenylSepharose6FastFlow.

The preparation method of restructuring HCG-Fc fusion disclosed in this invention, it is high that this preparation method can obtain expression outputRestructuring HCG-Fc fusion. Because of with the coupling of IgGFc variant, the recombinant protein forming can be affine by ProteinAChromatography obtains efficiently purifying easily. The fusion purity obtaining after hydrophobic chromatography is further purified reaches more than 98%.In addition, in the restructuring HCG-Fc fusion that the present invention announces, α chain and β chain can correctly fold, and have avoided α-α dimerizationBody, the dimeric appearance of β-β, simplified purification step greatly, reduced production cost.

An also object of the present invention has been to provide a kind of pharmaceutical composition that contains long-acting restructuring HCG-Fc fusion,It is characterized in that, comprise pharmaceutically acceptable carrier or excipient or diluent, and effective dose is of the present invention heavyGroup leader is imitated HCG-Fc fusion.

Specifically, described pharmaceutical composition contains effective dose (as 0.000001-90wt%; Preferably 0.1-50wt%; Better, 5-40wt%) recombinant long-acting HCG-Fc fusion of the present invention, and pharmaceutically acceptable yearBody. Conventionally, the fusion of the present invention of effective dose can be formulated in to nontoxic, inertia and pharmaceutically acceptable aqueous carrierIn medium, wherein pH is about 5-8 conventionally, and preferably, pH is about 6-8.

Described pharmaceutically acceptable carrier comprises (but being not limited to): sucrose, sweet mellow wine, polysorbas20 (Tween20),Methionine, salt solution, buffer solution, glucose, water, glycerine and composition thereof. Conventionally pharmaceutical preparation should match with administering mode,Pharmaceutical composition of the present invention can be made into injection form, for example, with physiological saline or the water that contains glucose and other assistant agentsSolution is prepared by conventional method. Described pharmaceutical composition should be manufactured under aseptic condition. The dosage of active componentIt is treatment effective dose. Pharmaceutical preparation of the present invention also can be made into sustained release preparation.

The effective dose of fusion of the present invention can be with order of severity of the pattern of administration and disease to be treated etc.And change. The selection of preferred effective dose can be determined and (for example pass through according to various factors by those of ordinary skill in the artClinical testing). Described factor includes but not limited to: such as biological utilisation of the pharmacokinetic parameter of described fusionRate, metabolism, half-life etc.; Animal will be treated the order of severity, the body weight of animal, the immune state of animal, the administration way of diseaseFootpath etc.

A further object of the present invention is restructuring HCG-Fc fusion treating and/or preventing in people's infertilityApplication.

Restructuring HCG-Fc fusion Half-life in vivo significant prolongation of the present invention, bioavilability obviously improves, therebyImprove pharmacokinetics and drug effect, with existing HCG comparison, in clinical practice, can reduce patient infusion number of times and dosage, fallenLow side effect, alleviates patient suffering and financial burden.

The advantage of restructuring HCG-Fc fusion of the present invention and preparation method thereof is summarized as follows:

1. restructuring HCG-Fc fusion of the present invention is by IgGFc variant (vIgG2Fc, vIgG4Fc or vIgGIFc)With the dimer protein that HCG coupling forms, Half-life in vivo that can significant prolongation albumen, greatly improve HCG in Chinese hamster ovary celIExpression, has significant inside and outside activity.

2. the α chain of dimerization strand HCG-Fc fusion and β chain are correctly folding with covalent bond, avoid forming α-α dimerizationBody and β-β dimer, simplified purifying process greatly, reduces production costs.

3. restructuring HCG-Fc fusion Half-life in vivo significant prolongation of the present invention, its half-life is that existing people urinates HCGWith restructuring 10 times of HCG, and absolute bioavailability is 2 times that people urinates HCG and restructuring HCG, can greatly reduce frequency injection andReduce ID, alleviate the side effect that existing therapeutic scheme produces.

Brief description of the drawings

Fig. 1. show the ratio of the hinge region of human IgG1, IgG2, IgG4 and their variants and the amino acid sequence in CH2 regionRight. Relatively this three partial amino-acid series: amino acid region 228,234-237 and 330-331. The amino acid mutation of these variantsShow with bold Italic. Amino acid residue numbering is to demarcate according to EU number system.

Fig. 2. show HCG-Fc strand and dimerization structural representation. A) strand HCG-Fc; B) HCG-Fc of dimerization.

Fig. 3. show the nucleotide sequence of the HCG-Fc of HindIII-EcoRI fragment in PCDNA3 expression vector and pushed awayThe amino acid sequence of leading. The nucleotide sequence of HCG-Fc comprises that coding is containing leader peptide (1-20), ripe HCG β chain, ripe HCG αChain, peptide linker, IgG2Fc variant (vIgG2Fc). Ripe restructuring HCG-Fc fusion contain ripe HCG β chain (21-165),Ripe α chain (166-257), peptide linker (258-273) and IgG2Fc variant (vIgG2Fc) are (274-496).

Fig. 4. show the nucleotide sequence of the HCG-Fc of HindIII-EcoRI fragment in PCDNA3 expression vector and pushed awayThe amino acid sequence of leading. The nucleotide sequence of HCG-Fc comprises that coding is containing leader peptide (1-20), ripe HCG β chain, ripe HCG αChain, peptide linker, IgG4Fc variant (vIgG4Fc). Ripe restructuring HCG-Fc fusion contain ripe HCG β chain (21-165),Ripe HCG α chain (166-257), peptide linker (258-273) and IgG4Fc variant (vIgG4Fc) are (274-502).

Fig. 5. show the nucleotide sequence of the HCG-Fc of HindIII-EcoRI fragment in PCDNA3 expression vector and pushed awayThe amino acid sequence of leading. The nucleotide sequence of HCG-Fc comprises that coding is containing leader peptide (1-20), ripe HCG β chain, ripe HCG αChain, peptide linker, IgG1Fc variant (vIgG1Fc). Ripe restructuring HCG-Fc fusion contain ripe HCG β chain (21-165),Ripe HCG α chain (166-257), peptide linker (258-273) and IgG1Fc variant (vIgG1Fc) are (274-500).

Fig. 6. show the eukaryon expression plasmid collection of illustrative plates of constructed coding HCG-Fc fusion. This expression plasmid total length9063bp, contains 10 oligogene segments, comprises 1.CMV promoter; 2. target gene HCG-Fc; 3.IRES; 4.ZeocinResistance screening gene; 5.BGH terminator; 6.SV40 promoter; 7.DHFR amplification gene; 8.SV40 terminator; 9. ammonia benzyl mouldElement resistant gene (Ampicillin); 10.ColE1 origin of replication (Ori).

Fig. 7. the accumulation that has shown the HCG-Fc cell line expression-secretion HCG-Fc albumen of recombinating in 7L bioreactor becomesPower curve figure.

Fig. 8 .Westembloting result has shown the successful expression of restructuring HCG-Fc fusion in Chinese hamster ovary celI.Non-reduced glue, Lane1: the HCG (50KDa) in urine source; Lane2: restructuring HCG-vIgG2Fc fusion of the present invention(148KDa); Lane3: restructuring HCG-vIgG1Fc fusion of the present invention (148KDa); Lane4: restructuring HCG-of the present inventionVIgG4Fc fusion (148KDa).

Fig. 9. show that the recombinant single chain of purifying and dimerization HCG-Fc fusion are in reducing condition and non-reduced conditionUnder 10%SDS-PAGE electrophoresis pattern. Lane1: non-reduced glue, restructuring dimerization HCG-vIgG2Fc fusion (148KDa)Lane2: non-reduced glue, restructuring dimerization HCG-vIgG1Fc fusion (148KDa); Lane3: non-reduced glue, restructuring dimerizationChange HCG-vIgG4Fc fusion (148KDa); Lane4: go back virgin rubber, recombinant single chain HCG-vIgG2Fc fusion(74kDa); Lane5: go back virgin rubber, recombinant single chain HCG-vIgG1Fc fusion (74kDa); Lane6: go back virgin rubber, recombinant single chainHCG-vIgG4Fc fusion (74kDa).

Figure 10. the restructuring HCG-Fc fusion, the people that have shown purifying urinate HCG and the metabolism of restructuring HCG in rat bodyCurve. A) restructuring HCG-vIgG2Fc, restructuring HCG-vIgG4Fc and restructuring HCG-vIgG1Fc; B) people urinates HCG and restructuring HCG.

Detailed description of the invention

Below in conjunction with specific embodiment, further set forth the present invention. Should be understood that these embodiment are only for illustrating the present inventionLimit the scope of the invention and be not used in. The experimental technique of unreceipted actual conditions in the following example, according to normal condition asThe people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1. builds the expression vector of coding restructuring HCG-Fc fusion

Gene order design is to be optimized based on Chinese hamster ovary celI preference codon, adopts the synthetic warp of artificial synthetic methodCross the encode signal peptide of HCG albumen β chain and the fusion of mature peptide section and HCG α chain mature peptide section thereof of containing of optimizing, by instituteSynthetic 756bpDNA fragment is inserted transfer vector as between the EcoRV restriction enzyme site in pUC57, has obtained HCG plasmid(pHCG), use the correctness of DNA sequencing method validation insetion sequence.

The artificial synthetic flexible peptide linker of coding that contains BamHI (5 ' end) and EcoRI (3 ' end) restriction enzyme site respectivelyFusion L-vIgG2Fc, the L-of (Linker detects " L ") and Fc variant (vIgG2Fc, vIgG4Fc and vIgG1Fc) fragmentVIgG4Fc and L-vIgG1Fc. The fusion fragment of acquisition is inserted into respectively to transfer vector as the BamHI of PUC19 andBetween EcoRI site, obtain the plasmid containing three kinds of variants, be respectively pL-vIgG2Fc, pL-vIgG4Fc and pL-vIgG1Fc. Pass throughThe gene order of DNA sequencing checking L-vIgG2Fc, L-vIgG4Fc and L-vIgG1Fc. Merge base for preparing two kinds of HCG-L-FcCause, with restriction enzyme SpeI and BamHI double digestion pHCG plasmid, after gel electrophoresis, glue reclaims the letter of coding HCG albumen β chainThe fusion fragment of number peptide and mature peptide section and HCG α chain mature peptide section, purified said gene fragment is inserted into respectively5 of peptide linker '-end in pL-vIgG2Fc, pL-vIgG4Fc and pL-vigG1Fc plasmid, T4 ligase connects and composes pHCG-L-VIgG2Fc, pHCG-L-vIgG4Fc and pHCG-L-vIgG1Fc plasmid. Constructed fusion is by HCG β, HCG α, peptide linkerWith Fc variant gene composition, its nucleotide sequence as shown in SEQIDNO:1,3 and 5, its single-stranded structure as shown in Figure 2 a, dimerizationChange structure as shown in Figure 2 b.

Restriction enzyme SpeI/EcoRI is double digestion pHCG-L-vIgG2Fc, pHCG-L-vIgG4Fc and pHCG-respectivelyL-vIgG1Fc plasmid, DNA gel purifying obtains HCG-L-vIgG2Fc, HCG-L-vIgG4Fc and HCG-L-vIgG1Fc fragment.Two kinds of good purifying HCG-L-Fc fragments are inserted into mammalian cell expression plasmid as the phase of pCDNA3 (Invitrogen)Answer between restriction enzyme site final acquisition containing fusion gene expression plasmid pCDNA3-HCG-vIgG2Fc, pCDNA3-HCG-vIgG1FcAnd pCDNA3-HCG-vIgG4Fc, be referred to as pCDNA3-HCG-Fc plasmid, as shown in Figure 6. This plasmid is high containing mammalian cellThe promoter CMV that efficient expression exogenous gene albumen is required; This plasmid also contains two kinds of selected markers, thereby in bacteriumCan there is amicillin resistance, in mammalian cell, can there is bleomycin (zeocin) resistance. In addition, work as placeWhen chief cell is DHFR gene expression deficiency, the dihyrofolate reductase (DHFR) of the contained mouse of PCDNA3 expression vectorGene, makes it in the time that amethopterin (MTX) exists, can coamplification pHCG-Fc fusion and DHFR gene.

The coupling of carrying out HCG and Fc fragment by peptide linker (preferably for flexible joint) can improve the biology of albumen and liveProperty, for the purpose of the present invention, preferably length is approximately 20 or (but can not be less than 2) amino acid whose peptide linker still less, certainlyBy the peptide linker of 1 Amino acid profile also in protection scope of the present invention, should use contain or by 2 or more multiselect from belowThe peptide linker of Amino acid profile: glycine, serine, alanine and threonine. The peptide linker of the embodiment of the present invention contains Gly-Ser peptide member, its amino acid sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.

Embodiment 2. stably express of HCG-Fc fusion in mammalian cell of recombinating

The expression plasmid pCDNA3-HCG-L-Fc that embodiment 1 is built is transfected into DHFR deficient CHO host cell(CHO-DHFR^-), Fig. 2 b has shown the schematic diagram of restructuring dimerization HCG-Fc fusion. Adopt electroporation method to turnDye, use 960 μ Fd electric capacity GenePulserElectroporator (Bio-RadLaboratories, Hercules,CA), its electric field is set to 250V, in cuvette 2～5 × 10⁷In individual cell, add the linearizing matter of 10 μ g PvuIGrain DNA. In transfection two days later, make culture medium into growth medium containing 100 μ g/mLZeocin resistant maker genes, obtainThrough the transfectant of resistance primary dcreening operation. Adopt the method for westemblotting, with the expression of anti-HCG antibody test HCG-Fc, as figure8. Utilize DHFR amplification selected marker to improve the expression of restructuring dimerization albumen, containing progressive concentration for this reasonIn the growth medium of MTX, with the restructuring dimerization GFP of DHFR gene coamplification transfection. With Method of Limited Dilution method subcloneThe transfectant that can grow in up to 10 μ M/mLMTX culture mediums. The secretion rate of subclonal cell line is done further to analyze. SieveSelect secretion level (preferably approximately 20) the μ g/10 that exceedes approximately 10⁶The cell line of (1,000,000) individual cell/24 hour, obtains stable highExpress the cell line of restructuring HCG-Fc fusion.

Embodiment 3. recombinate production and the purifying of HCG-Fc fusion

The high yield cell line that adopts embodiment 2 to obtain first carries out serum-free domestication and cultivates in culture dish, then turnsMove on in shaking flask and suspend and tame cultivation, in domestication process, carry out the screening of culture medium simultaneously, add different compositions to observeThe growth conditions of cell, growth tendency, and the biochemical indicator such as activity and sialic acid of expression product, preferred cell is cultivated barPart is: basal medium adds 100 μ MCu²⁺, feeding culture medium adds 2mMManNAc (the amino sweet dew of N-acetyl group-D-Sugar), the method can make to recombinate degree of glycosylation of HCG-Fc fusion increases, and sialic acid content improves approximately 20%. Tame intoAfter merit, carry out cell amplification, amplification, to q.s, is carried out the monitoring of 7L bioreactor and is cultivated, and exceedes 1 × 10 at cell density⁷Individual/to start to be cooled to 33 DEG C of cultivations when mL, batch growth cycle is 20 days. Recombinant protein is entered with 1mlProteinA column chromatographyRow preliminary purification, the expression of mensuration restructuring HCG-Fc fusion, result shows, restructuring HCG-vIgG2Fc, restructuring HCG-The cumulative production that vIgG4Fc and restructuring HCG-L-vIgG1Fc cell line are expressed is respectively 1.20g/L, 0.89g/L, 0.82g/L(Fig. 7).

The purifying of restructuring HCG fusion comprises the following steps:

1) ProteinA affinity chromatography: centrifugal, collect supernatant, the characteristic of albumen coupling Fc fragment according to the present invention, utilizesAffinity chromatography method, is loaded onto supernatant on the ProteinA post of phosphate buffer saline (PBS) balance; Fusion to be reorganizedProtein combination, after ProteinA, is washed this post with PBS, until OD280 value is lower than 0.01, then the acetic acid that is 4.0 with 20mMpHThe restructuring HCG-Fc fusion of sodium buffer solution elution combination, finally uses in the 1MTris-HCl buffer solution of pH10.0 and activeCollect liquid. The HCG-Fc purity of protein of purifying can reach more than 95%.

2) hydrophobic chromatography: with hyperfiltration process, above-mentioned proteinA activity being collected to fluid exchange is 20mMTris-HCl-1.5MNaCl (pH8.0) buffer solution, by this sample pipetting volume to crossing by 20mMTris-HCl-1.5MNaCl (pH8.0) balancePhenyl-6FastFlow post, first uses identical equilibrium liquid drip washing, then uses 20mMTris-HCl-1.35MNaCl (pH8.0)Drip washing, finally uses 20mMTris-HCl-0.5MNaCl (pH8.O) buffer solution elution.

Westernbloting result has shown the successful expression of restructuring HCG-Fc fusion in Chinese hamster ovary celI, as figureShown in 8, SDS-PAGE gel electrophoresis collection of illustrative plates under non-reduced condition, people urinates HCG (commercial product) and restructuring HCG-Fc of the present inventionFusion shows corresponding target protein hybridization band at 50KDa and 148KDa respectively, has verified the weight that the present invention obtainsIn group HCG fusion, contain HCG albumen. Fig. 9 be purifying HCG-Fc fusion reduction and non-reduced condition underSDS-PAGE gel electrophoresis collection of illustrative plates. Result demonstration, the HCG-Fc purity of protein of purifying can reach more than 98%, and HCG-Fc is at reduction barMolecular weight under part be under non-reduced condition molecular weight 50%. Confirm its amino acid order through 15 determined amino acid sequences of N endRow and SEQIDNO:2,4 and 6 identical.

The recombinate inside and outside determination of activity of HCG-Fc fusion of embodiment 4.

Restructuring HCG-Fc fusion restructuring HCG-vIgG2Fc of the present invention, restructuring HCG-vIgG4Fc and restructuring HCG-The external activity (immunogen activity) of vIgG1Fc adopts the HCG enzyme immunoassay that BIOCHECK (U.S.) company produces to detect examinationAgent box detects, method reference reagent box description. Activity in vivo adopts the ovary weightening finish method in 2010 editions " British Pharmacopoeias " to carry outDetect. Protein content determination uses LOWRY sizing technique. Urinate HCG, restructuring HCG and restructuring HCG-according to standard items labelled amount, peopleFc fusion (restructuring HCG-vIgG2Fc, restructuring HCG-vIgG4Fc and restructuring HCG-vIgG1Fc) is estimated to tire, and uses dilution(containing 0.1%BSA, the phosphate buffer of pH7.2 ± 0.2) standard items, people are urinated to HCG, restructuring HCG and restructuring HCG-Fc mergesAlbumen is mixed with the working solution containing HCG0.5IU/ml, 1IU/ml, high, normal, basic three dosage of 2IU/ml. Select 19～28 ages in days, yearMust not differ age and exceed 3, body weight must not differ and exceeded the male mouse of SD of 10 grams. Standard items, people urinate HCG group, restructuring HCG and HCG-Fc group is divided into high, normal, basic three dosage, every group of 6 animals. Hypodermic injection after rat neck, injects twice every day, per injection bodyAmass as 0.5ml, inject continuously four days, every day is in same time administration. Last drug administration by injection is after 24 hours, suitable according to administrationOrder adopts dislocation method to put to death animal, gets seminal vesicle, peels off after blotting surface moisture and weighs, and records organ weight. According to standard itemsThe activity that seminal vesicle weightening finish adopts parallel line analysis method calculating people to urinate HCG, restructuring HCG and HCG-Fc. Record restructuring HCG-The external activity that vIgG2Fc, restructuring HCG-vIgG4Fc, restructuring HCG-vIgG1Fc, restructuring HCG and people urinate HCG is respectively9961IU/ml, 9305IU/ml, 9521IU/ml, 8966IU/ml and 8369IU/ml, its activity in vivo be respectively 9120IU/ml,8990IU/ml, 8780IU/ml, 8253IU/ml and 8011IU/ml. Result shows, restructuring HCG-Fc fusion of the present inventionThere is inside and outside BA.

The recombinate pharmacokinetics test of HCG-Fc fusion of embodiment 5.

Be divided into restructuring HCG-vIgG2Fc, restructuring HCG-vIgG4Fc, restructuring HCG-vIgG1Fc, restructuring HCG administration group and peopleUrine HCG administration group, 3 of every group of male SD rats of selecting body weight 200-250g, give after neck subcutaneous by 10000IU/kg respectivelyInjection. Restructuring HCG combination people urinates HCG group 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 56h, 72h after administration and entersRow eye socket venous blood collection, restructuring HCG-Fc fusion respectively after administration 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h,48h, 56h, 72h, 96h, 144h, 264h, 312h, 384h, 432h, 528h carry out eye socket venous blood collection. In addition, every group of difference is anotherGet 3 rats and carry out the dosage intravenous administrations such as relative medicine, same blood sampling time point is taken a blood sample. Blood sample warpAfter the centrifugal 5min of 3000rpm, draw serum ,-20 DEG C of preservations. , adopt ELISA kit (BIOCHECK, the U.S.) to measure when eachBetween put HCG immunocompetence in blood plasma. Use kinetica4.4 software, calculate each group of main pharmacokinetics by statistics moments methodParameter. As shown in figure 10, the half-life, result was as table 1 for each group pharmacokinetic curve. Result demonstration, people urinates HCG in rat bodyThe elimination half-life be about 15.3h, and the present invention recombinate HCG-vIgG2Fc, restructuring HCG-vIgG4Fc, restructuring HCG-vIgG1FcThe elimination half-life of fusion is respectively 168.33h, 166.82h and 156.63h, be people urinate HCG and restructuring 10 times of HCG withOn. The AUC that hypodermic injection medicine is calculated calculates the AUC of gained divided by intravenous injection, be absolute bioavailability, whereinThe absolute bioavailability of restructuring HCG-vIgG2Fc, restructuring HCG-vIgG4Fc, restructuring HCG-vIgG1Fc fusion is restructuringHCG and people urinate the more than 2 times of HCG, and results suggest is urinated HCG with restructuring HCG with people and compared, and reach same curative effect restructuring HCG-FcThe required frequency injection of fusion greatly reduces, dosage significantly reduces.

Table 1 recombinate half-life and the bioavilability of HCG-Fc fusion

Embodiment 6. ovulation effect of HCG-Fc fusion to female rats of recombinating

105 of Healthy female SD rats, body weight 200-250g, under experimental situation, normally feed support quarantine observe 7 days withOn. Be divided at random 7 groups: negative control group (physiological saline 0.2ml/ only), restructuring HCG-vIgG2Fc administration group (6IU/ only), heavyGroup HCG-vIgG1Fc administration group (6IU/ is only), restructuring HCG-vIgG4Fc administration group (6IU/ is only), restructuring HCG control group (10IU/Only), people urinates HCG control group (10IU/ only), he every group of 15 rats, respectively organizes rat body weight and distribute similar. First pass through vaginal smearMethod is observed animal oestrous cycle one-period. Afterwards, start hypodermic injection recombined human in dioestrus first day 10:00 in the morningFSH (fruit receive sweet smell), dosage is 15IU/kg/d, successive administration 3 days, same time administration every day. (proestrum) on the 4th differenceBy grouping hypodermic injection medicine, 10IU/ only. After (oestrus) morning on the 5th, 10:00 anaesthetized rat, carry out ovary and divideFrom, and weigh, liquid-solid fixed with BouinShi to the ovary separating, FFPE, does serial section to fixing ovary every 6 μ m,Under microscope, carry out histological observation, and record follicle number.

Each group Rat Ovulation number of animals, ovulation rate (ovulation number of animals/animal number) and the count results of large follicle number are shown inTable 2, result shows, recombinate HCG fusion, restructuring HCG and people of the present invention urinates HCG and all female rats had to ovulation effect,Ovulation number of animals and ovulation rate and negative control group more all have significant difference (P < 0.01), and large follicle number is with negative rightMore all there is significant difference (P < 0.01) according to group. And three kinds of restructuring HCG-Fc fusions of the present invention only need be injected lowerDosage (6IU/ only) just can reach to restructuring HCG (10IU/ only) and people and urinate the similar curative effect of HCG (10IU/ only).

Table 2 ovulation effect of HCG-Fc fusion to female rats of recombinating

Note: X²Inspection, with negative control group comparison,^*p＜0.05，^**p＜0.01。

The embodiment 8. HCG-Fc fusion of recombinating causes the safety testing of rat ovary hyperstimulation syndrome

60 22 age in days female Wistar rats are divided at random and are divided into Normal group, restructuring HCG-vIgG2Fc administrationGroup, restructuring HCG-vIgG1Fc administration group, restructuring HCG-vIgG4Fc administration group, people urinate HCG control group and restructuring HCG control group,Every group 10. Restructuring HCG-vIgG2Fc administration group, restructuring HCG-vIgG1Fc administration group and restructuring HCG-vIgG4Fc administration groupRat 22-25 age in days all in the morning 9:00 according to 10IU/ only/d dosage lumbar injection rhFSH (fruit receive sweet smell), the 26thAge in days 9:00 in the morning presses only (3 times of drug effect dosage) corresponding restructuring of hypodermic injection HCG-Fc of 18IU/; People urinates HCG and restructuring HCG administrationGroup 22-25 age in days all in the morning 9:00 according to 10IU/ only/d dosage lumbar injection rhFSH (fruit receive sweet smell), the 26th dayThe morning in age, 9:00 pressed only (3 times of drug effect dosage) corresponding HCG of the corresponding injection of hypodermic injection of 30IU/; Normal group injection equal-volumePhysiological saline. All groups all at the 28th age in days 9:00 in the morning through tail vein injection 1%EB dyestuff 0.1ml, de-neck method place after 30minDead rat, carries out ascites scoring immediately. Without ascites or a small amount of ascites person, to intraperitoneal perfusion 5ml physiological saline, perfusion liquid is collectedIn 10ml test tube, be all diluted to 10ml, then add 0.05ml0.1MNaOH solution, with the speed of 3000r/min at room temperature fromHeart 10min, in the row colorimetric estimation of wavelength 600nm place, records OD value with spectrophotometer. Finally, winning both sides ovary weighs. SeeExamining index is: 1, abdominal cavity capillary permeability: according to normal concentration and OD value drawing standard curve thereof, calculate peritoneal fluidEB content. 2, ascites scoring: 1 point: without ascites; 2 points: a small amount of ascites exists; 3 points: middle amount ascites exists; 4 points: ascites is obviously deposited; 5 points: a large amount of ascites exists or ascites is overflowed from abdominal incision. 3, ovary weight.

Experimental result is in table 3, and result shows, although under 3 times of drug effect dosage, restructuring HCG-Fc fusion is female age to childrenProperty Wistar rat ovary have effect of gain, but in ascites scoring and two indexs of abdominal cavity capillary permeability and justNormal control group does not have significant difference, does not cause OHSS. And the people of same dosage urinate HCG control group andRestructuring HCG control group is compared with Normal group, all bright aspect ascites scoring, abdominal cavity capillary permeability and ovary weightAobvious higher, and have significant difference (P < 0.05), illustrate and caused OHSS. Meanwhile, people urinates HCGControl group and restructuring HCG control group indices are apparently higher than three restructuring HCG-Fc administration groups (P < 0.05). Result shows,Under equal higher dosage, the present invention recombinate HCG-vIgG2Fc, restructuring HCG-vIgG1Fc and restructuring HCG-vIgG4Fc phase counterweightIt is less that group HCG and people urinate HCG side effect, and clinical practice is safer.

EB content and ovary weight comparison in the scoring of the each group of table 3 ascites, peritoneal fluid

Note: t inspection, with Normal group this,^aP < 0.05; Compared with restructuring HCG group,^bP < 0.05; Urinate HCG with peopleGroup mutually this,^cp＜0.05。

Claims

1. restructuring HCG-Fc fusion, is characterized in that, described fusion is dimerization fusion, its amino acid orderRow hold C end to comprise successively HCG β subunit, HCG α subunit, peptide linker and human IgG Fc variant, wherein said HCG β subunit from NAmino acid sequence be shown in the HCG β SEQIDNO:8 having removed after 1-18 amino acids residue in conventional H CG β subunitAmino acid sequence; The amino acid residue sequence of wherein said HCG α subunit is the 1-24 position of having removed in conventional H CG α subunitAmino acid sequence shown in HCG α SEQIDNO:7 after amino acid residue; Wherein said peptide linker sequence isGlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer, described peptide linker is present in HCG α subunit and peopleBetween IgGFc variant; Wherein said human IgG Fc variant is selected from lower group:

(i) human IgG2's hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change;

(ii) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change;

(iii) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.

2. restructuring HCG-Fc fusion according to claim 1, is characterized in that the amino acid order of described fusionRow are as shown in SEQIDNO:2,4 and 6.

3. coding, according to the nucleotide sequence of the restructuring HCG-Fc fusion of claim 2, is characterised in that its sequence is as SEQShown in IDNO:1,3 and 5.

4. a preparation method for the restructuring HCG-Fc fusion of claim 1, its step comprises:

1) expression vector of structure coding restructuring HCG-Fc fusion:

Adopt artificial synthesis to obtain the gene of the restructuring HCG-Fc fusion of coding claim 1, be inserted into lactation movingThing fibrocyte expression vector, obtains the expression plasmid that contains restructuring HCG-Fc antigen-4 fusion protein gene;

2) stably express of restructuring HCG-Fc fusion in mammalian host cell:

The expression vector of the restructuring HCG-Fc fusion that contains claim 1 is transfected into mammalian host cell, screeningThe cell line of stably express HCG-Fc fusion;

3) high-density cells is cultivated restructuring HCG-Fc fusion:

The above-mentioned stable cell line screening is proceeded to shaking flask or cell effect tank carries out large-scale culture, when cell density reaches1×10⁷Individual/when mL, temperature to be down to 33 DEG C by 37 DEG C, at this temperature, cultivate until express output and no longer increase;

4) purification of restructuring HCG-Fc fusion:

A) ProteinA affinity chromatography: centrifugal, collect supernatant, according to the present invention, the characteristic of albumen coupling Fc fragment, utilizes affineProteinA column chromatography;

B) hydrophobic chromatography post purifying: after the above-mentioned ProteinA purifying of the further removal of hydrophobic chromatography purifying in target proteinImpurity;

Described drainage column comprises ButylSepharose4FastFlow, OctylSepharose4FastFlow,PhenylSepharose6FastFlow，Butyl-SSepharose6FastFlow，ButylSepharose4B，OctylSepharoseCL-4B，PhenylSepharoseCL-4B。

5. the preparation method of restructuring according to claim 4 HCG-Fc fusion, wherein step 1) described in geneExpression vector is pCDNA3, pCMV/ZEO, pIRES, pDR, pBK, pSPORT or pCMV-DHFR, wherein step 2) described inCell transfecting method comprises electroporation transfection method, calcium phosphate transfection, liposome transfection and Protoplast fusion; Described lactation is movingThing host cell is CHO, HEK293, BHK, NS0 or Sp2/0 cell.

6. the preparation method of restructuring according to claim 5 HCG-Fc fusion, wherein step 1) described in geneExpression vector is pCDNA3; Wherein step 2) described in cell transfecting method be electroporation transfection method; Described lactation is movingThing host cell is Chinese hamster ovary celI.

7. the preparation method of restructuring HCG-Fc fusion according to claim 6, described mammalian host cellFor DHFR deficient CHO-suspension cell.

8. the preparation method of restructuring according to claim 7 HCG-Fc fusion, wherein step 3) in be also included in baseBasal culture medium adds 100 μ MCu²⁺, feeding culture medium adds 2mMManNAc (N-acetyl group-D-epichitosamine).

9. a pharmaceutical composition, is characterized in that, comprises pharmaceutically acceptable carrier or excipient or diluent, Yi JiyouRestructuring HCG-Fc fusion described in the claim 1-3 any one claim of effect amount.

10. the restructuring HCG-Fc fusion described in claim 1-2 any one claim is infertile in preparation treatmentApplication in disease drug.