CN110256575A

CN110256575A - A kind of long-acting recombinant human growth hormone fusion protein and its engineering cell

Info

Publication number: CN110256575A
Application number: CN201910503065.0A
Authority: CN
Inventors: 刘景会; 刘涵; 俞露; 刘琳琳; 刘玉林; 张宇; 薛毅勇; 郭胜仓; 许佳慧
Original assignee: Changchun Institute of Biological Products
Current assignee: Changchun Institute of Biological Products
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2019-09-20
Anticipated expiration: 2039-06-11
Also published as: CN110256575B

Abstract

The present invention relates to gene engineering technology fields, the especially long-acting human growth hormone (HGH) technical field of genetic recombination, more particularly to a kind of long-acting recombinant human growth hormone fusion protein and its engineering cell, long-acting recombinant human growth hormone fusion protein rhGH-Fc of the invention can extend serum half-life, increase the bioactivity of albumen in vivo, reduce the immunogenicity of albumen, so as to improve drug effect, drug administration by injection frequency required in treatment time is substantially reduced, thus the convenience for improving the compliance of patient, increasing doctor and patient；The engineering cell for the expression rhGH-Fc that the present invention constructs has the characteristics that expressing quantity is high, bioactivity is good, especially suitable for industrializing the long-acting human growth hormone (HGH) of large scale preparation genetic recombination.

Description

A kind of long-acting recombinant human growth hormone fusion protein and its engineering cell

Technical field

The present invention relates to the long-acting human growth hormone (HGH) technical fields of gene engineering technology field, especially genetic recombination, specifically It is related to a kind of long-acting recombinant human growth hormone rhGH-Fc fusion protein, its engineering cell and construction and screening method.

Background technique

Human growth hormone (HGH) (Human Growth Hormone, hGH) is the most important hatching egg of growth promotion after mankind's birth White matter parahormone is secreted by the acidophic cell of human pituitary's frontal lobe with species specificity, the albumen of human growth hormone (HGH) by Body spreads each position of whole body, and generates physiological effect not by target gland effect.Human growth hormone (HGH) contains 191 ammonia Base acid molecule, wherein most be in the form of molecular weight 22KDa exist, isoelectric point 4.9, the 53rd cysteine with There are two disulfide bond between 165 cysteines and the 182nd cysteine and 189 cysteines, A small amount of growth hormone exists in the form of 20KDa.The most important physiological action of growth hormone be by promote body respectively organize, organ And the protein of recipient cell synthesizes the decomposition with fat in bone, and then promotes the metabolism and growth and development of people.

If growth hormone secretion deficiency will lead to growth retardation and each dysfunction of body, if hypersecretion can cause it is bad Reaction, such as there is " acromegaly ", " gigantism ".Human growth hormone (HGH) the most significantly effect be enhance protein synthesis, Enhance the synthesis of collagen, promote the development of bone tissue.It is raw in bone although hGH is particularly significant to the growth for maintaining bone Growth hormone has been not direct effect in length, it can be promoted by sulfatin factor (SM) Lai Shixian, SM in stimulation blood The division of various histocytes and mother cell in into body.Human growth hormone (HGH) can also promote amino acid to enter in cell, reduce The discharge of Ammonia substance, nitrogenous substance keeps the metabolism state of body to balance.Human growth hormone (HGH) can also promote lipolysis to make With inhibiting the oxidation of glucose, slow down the depletion rate of glucose, also have the function of stable glycogen.Growth swashs at present Element is still a kind of extremely complex hormone, and there are also to be studied for many functions.

Human growth hormone (HGH) is clinically mainly used for children growth hormone deficiency, and Tuner syndrome promotes large area to burn Hurt the healing etc. of the surface of a wound.At present in China, the slow incidence of upgrowth and development of children is up to 9.9%, occupies the whole world second.According to China Medical association's statistics, it is growth hormone deficiency by therein 1/3 that there are about 7,000,000 people for 4~15 years old short and small infant sum in the whole nation at present Calculate, the children of domestic demands growth hormone therapy probably have 2,000,000, but can receive rational therapy number at present still less than 30000 people.Patient find when long-term follow-up: more than about 23% meeting child patient every month three needle of no marking；Nearly 13% Patient children understand no marking prescribed dose more than half；Clinical evidence shows not strictly observe therapeutic scheme patient reachable 50% or more.Main reasons is that human growth hormone recombinant common at present needs daily injection primary, to keep blood appropriate Clear level of growth hormone.Patient is very painful, and curative compliance is poor, and expensive price.Those are needed to receive life for a long time The patient of long hormone therapy causes therapeutic effect to reduce often due to cannot inject on time.At present at home, recombinant human growth The approved indication of hormone mainly has 2 kinds, and wherein most is applied to treatment children " dwarfism ", and country children are short and small at present Disease patient's treatment rate is very low, so long-acting, high activity human growth hormone recombinant has biggish future market potential.

Expression system currently used for expressing protein drug is divided into two classes, uses prokaryotic expression system earliest, mainly For Escherichia coli.Another kind of is eukaryotic expression system, and insect, yeast, mammalian cell belong to such expression system.Although big Coli cell have good operability, advantage of lower cost, but tend not to carry out correctly it is glycosylation modified, Inclusion body easy to form intracellular.Currently, domestic company mostly uses greatly E. coli secretion expression technology to produce growth hormone, Albumen contains endotoxin, and cumbersome purification step is needed to remove, and causes final products yield lower, since Escherichia coli are protokaryons Expression vector, so the activity of destination protein is relatively low.Chinese hamster ovary celI (hamster ovary cell, Chinese Hamster Ovary) Belong to eukaryocyte, has the function of to transcribe the posttranslational modification of rhetorical function and protein, the conformation of the destination protein of expression is divided Minor structure, physicochemical property, biological function are all very close with natural protein.Chinese hamster ovary celI is fibroblastic one kind, The intrinsic protein secretory volume of its own is few, highly beneficial for isolating and purifying for later period recombinant protein；In terms of culture, CHO Cell had both had the characteristic of adherent growth, if can also carry out suspension culture through taming, was resistant to the energy of larger shearing force and osmotic pressure Power is preferable；The purity of product can be improved in free serum culture, facilitates isolating and purifying for product, while can reduce due to serum band The pollution come and anaphylactogen, can also extend the G1 phase or G0 phase of cell, so that destination protein expression quantity is improved, it is also big for the later period Technical scale production provides convenience.

Currently, the long lasting growth hormone of domestic goods is PEGylated growth hormone, PEGylated its is capable of increasing albumen point Son amount, not easily passs through glomerulus, reduces clearance rate, prevents the enzymolysis of protease, to extend half of drug in vivo It declines the phase, but protein PEGylation process can lose bioactivity.

Research is most hot at present, and developing most rapid long acting protein drug technique is Fc fusion protein technology, and Fc segment is The principal element of longer half-life in IgG keeping body, while albumen can be stablized, it has been used for the exploitation of a variety of fusion proteins. For Fc segment by two kinds of mechanisms play functions, the first is to pass through phagocytosis or splitting action in conjunction with the Fc receptor of cell surface It kills cell and pathogen is digested by antibody-dependent cytotoxicity (ADCC) approach.In four kinds of human IgG hypotypes IgG1 with IgG3 can be effectively combined the Fc receptor of cell surface, and the affinity of IgG2 and IgG4 in conjunction with receptor is lower.It is for second In conjunction with the C1q of the first complement component C1, pathogen is cracked by cell toxicant (CDC) approach of Complement Dependent.Human IgG1 and IgG3 can be effectively combined C1q so that the binding ability of mediate complement cascade reaction, human IgG2 and C1q are weaker, and human IgG 4 is not In conjunction with C1q.Fc fusion protein primarily to increase protein drug half-life period in vivo and simplify production technology reduce at This, the function of IgG effect and lytic cell that Fc is mediated is not necessary, or even can generate side effect, based on the above reasons Selection IgG4 hypotype simultaneously carries out corresponding amino acid mutation to weaken the function of its effector molecule.Fc fusion protein technology is not only such as Increasing protein drug molecular mass as PEGylated reduces clearance rate, can more also be combined by FcRn binding domain by pH sensibility Power extends half-life period in a sustained manner.Current many researchs show that Fc fusion protein is longer with having than PEGylated drug Half-life period.

Specific to human growth hormone (HGH), have the several researchs that Fc segment is merged to it, by taking CN 102875683B as an example, A kind of long-acting recombinant human growth hormone Fc fusion protein hGH-L-vFc fusion protein is disclosed, human growth hormone (HGH), about 2- are contained The flexible peptide linker and human IgG Fc variant of 20 amino acid, the human IgG Fc variant can selected from containing S228P and 4 hinge area of human IgG of L235A mutation, the region CH2 and CH3.But in above-mentioned several researchs, human growth hormone (HGH) half-life period is prolonged Long effect is unsatisfactory.

Therefore, more long-acting, high activity the human growth hormone derivatives of exploitation are still needed further exist for.

Summary of the invention

To solve the deficiencies in the prior art, the present invention provides a kind of long-acting recombinant human growth hormone rhGH-Fc to merge egg White, the fusion protein is made of human growth hormone (HGH), link peptide and Fc segment；Successively by N-terminal to C-terminal are as follows: human growth hormone (HGH), company Connect peptide, Fc segment, amino acid sequence totally 431 amino acid, wherein preceding 191 amino acid sequences are human growth hormone sequence, 192nd to the 206th is connection peptide sequence, the 207th to the 431st Fc segment for human IgG.

Further, the Fc segment is selected from the Fc segment of human IgG, preferably the Fc segment of human IgG 4.

Further, the Fc segment of the human IgG 4 includes four amino acid mutations, the amino acid mutation are as follows: S228P, F234A, L235A and V308P, wherein the number of the residue in the area IgG Fc is numbered according to Kabat Eu Index.Wherein, S228P site mutation is the stability in order to maintain albumen；F234A and L235A site mutation is to lower Immune Fusion egg The white affinity with Fc receptor is to have the function that the ADCC activity for reducing immune fusion protein；And V308P site mutation Rely on the combination of fusion protein and FcRn in pH, thus can by improve molecular weight of albumen to not by glomerular filtration and The mutation in the site V308P makes long-acting recombinant human growth hormone rhGH-Fc Immune Fusion to make albumen rely on two aspects in pH The half-life period of albumen is higher than common Fc fusion protein drug.

Further, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:1, the coding of the fusion protein Nucleotide sequence is as shown in SEQ ID NO:2.

The present invention also provides a kind of engineering cell, the engineering cell expresses foregoing fusion albumen, it is highly preferred that the work Journey cell is CHO-k1 cell, can stable transfection have the expression vector of foregoing fusion albumen.

Further, the engineering cell is constructed to obtain by following steps:

(1) first by human antibody IgG4Fc sequence (accession number: DI488112) after specific site is mutated with hGH gene (accession number: NM_000515) is connected with flexible peptide linker, and growth hormone signal peptide sequence is added before hGH sequence and (logs in Number: CAI94177), full genome synthesizes rhGH-Fc sequence；

(2) by double expression plasmid p327.8GS through restriction enzyme Hind III, EcoR I and Xba I, Xho I double enzymes twice It cuts, the rhGH-Fc sequence that the large fragment of recycling is synthesized with step (1) is mixed through T4DNA ligase, 15~16 DEG C connected Night；

(3) CaCl is used₂Method prepares competence bacillus coli DH 5 alpha, then carries out plasmid conversion, through containing ampicillin LB plate culture screening, picking positive bacterium colony is inoculated into LB liquid medium, extracts matter after 14~16h of low speed shake culture Grain；Plasmid through Hind III, EcoR I and Xba I, Xho I, identify and be sequenced twice by double digestion, and gene order is identified correctly weight Group plasmid is named as rhGH-Fc-1；

(4) cell inoculation is cultivated in the retorts bottle of the complete medium with serum, when cell density reaches 90~ When 95%, using lipofection, under the mediation of Lipofectamin 2000, by the recombinant plasmid of step (3) preparation RhGH-Fc-1 stable transfection is to CHO-k1 cell；

(5) cell after step (4) transfection is transferred in 96 orifice plates, be added SMM-CHO GSI culture medium (containing 10~ 12%FBS+25~30 μm ol MSX) it mixes, in CO₂After 35~37 DEG C of incubator constant incubator 10~12 days, in microscope Under, mark monoclonal；

(6) MSX concentration is gradually increased, strain is expressed using ELISA method Stepwise Screening height, from 96 plates (25~30 μm of ol MSX) → 24 orifice plates (50~55 μm of ol MSX) → 6 orifice plates (50~55 μm of ol MSX) → 75cm2 culture bottle (70~75 μm of ol MSX) → 125ml shaking flask (70~75 μm of ol MSX), selection expression quantity is higher and the preferable cell strain of vegetative state carries out subclone sieve Choosing will finally screen cell strain and be named as rhGH-Fc cell strain, and the destination protein of expression is rhGH-Fc immune fusion protein.

The present invention also provides a kind of expression vector, the expression vector is preferably dual-expression vector, it is highly preferred that the table It is with double expression plasmid p327.8GS through restriction enzyme Hind III, EcoR I and Xba I, Xho I double enzymes twice up to carrier After cutting, the large fragment of recycling and the code nucleic acid of fusion protein described in claim 1 are mixed through T4DNA ligase, 15~16 DEG C connection overnight, so that the target gene optimized is cloned on dual-expression vector and is obtained.

The present invention also provides a kind of positive colony of fusion protein and the screening technique of subclone, the methods are as follows: pass through Methionine sulfonamide (MSX) concentration is gradually increased, from 96 plates (25~30 μm of ol MSX) → 24 orifice plates (50~55 μm of ol MSX) → 6 orifice plates (50~55 μm of ol MSX) → 75cm2 culture bottle (70~75 μm of ol MSX) → 125ml shaking flask (70~75 μm of ol MSX), go out that expression quantity is higher and the preferable cell strain of vegetative state using ELISA method Stepwise Screening, cell strain life will be finally screened Entitled rhGH-Fc cell strain, the destination protein of expression are rhGH-Fc immune fusion protein.

Further, fusion protein described in the fusion protein can extend serum half-life, substantially reduce treatment time Interior required drug administration by injection frequency, to improve the compliance of patient and the convenience of doctor and patient；The Chinese hamster ovary celI can be used for Preparation and reorganization people's long lasting growth hormone, expressing quantity is high, bioactivity is good, reduces the immunogenicity of albumen, can improve Drug effect.

Beneficial effect

RhGH-Fc fusion protein of the invention is by improving life for human growth hormone (HGH) and Fc segment construction of fusion protein The molecular weight of long hormone is not to by glomerular filtration, and by the amino acid mutation of Fc segment so as to improve the steady of Fc segment ADCC activity that is qualitative, reducing fusion protein and improve pH dependence of the fusion protein in conjunction with FcRn, in summary effect, To further improve the half-life period of fusion protein relative to common Fc fusion protein drug.

RhGH-Fc fusion protein of the invention has significant extended serum half-life, and has high bioactivity, energy Drug administration by injection frequency required in treatment time is enough substantially reduced, the compliance of patient and the convenience of doctor and patient can be improved.

Engineering cell of the invention selects CHO-k1 cell as host cell, carries out by using GS dual-expression vector steady Fixed transfection, the engineering cell expressing quantity of acquisition is high, bioactivity is good, reduces the immunogenicity of albumen, can significantly improve Drug effect.

Detailed description of the invention

The plasmid map of Fig. 1: p327.8GS dual-expression vector.

Fig. 2: the Sequencing chromatogram of plasmid.

Fig. 3: the identification of recombinant plasmid rhGH-Fc-1 double digestion, 1: plasmid rhGH-Fc-1 HindIII, EcoRI double digestion Product；2: plasmid rhGH-Fc-1 XhoI, XbaI double enzyme digestion product；M:DNA marker DL 10000.

Culture supernatant ELISA result in Fig. 4: 125ml shaking flask.

Culture supernatant ELISA result in Fig. 5: cell strain D 75cm2.

Culture supernatant ELISA result in Fig. 6: cell strain E 75cm2.

Culture supernatant ELISA result in Fig. 7: cell strain H 75cm2.

Fig. 8: D1, E2, H2 culture supernatant ELISA result in subclone 125ml shaking flask.

Fig. 9: D1, E2, H2 cell growth curve in subclone 125ml shaking flask.

Figure 10: destination protein SDS-PAGE map, 1:rhGH-Fc albumen non-reduced；2: protein marker；M: RhGH-Fc albumen reduced form.

Figure 11: the point focusing such as destination protein electricity point electrophoretogram, 1:rhGH standard items；M1:HighpIKit, pH5~10.5； M2:BroadpIKit, pH3~10；2:rhGH-Fc albumen.

The HPLC map of the supernatant of the cell culture fluid of Figure 12: rhGH-Fc immune fusion protein.

The HPLC map of Figure 13: rhGH-Fc immune fusion protein.

Figure 14: body weight increase tendency chart after the administration of each group rat.

Specific embodiment

The present invention is described below in more detail to facilitate the understanding of the present invention.

It should be understood that the term or word used in the specification and in the claims is not construed as having The meaning limited in dictionary, and be interpreted as having on the basis of following principle and its meaning one in the context of the present invention The meaning of cause: the concept of term can suitably limit best illustration of the invention by inventor.

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al., Molecular cloning: condition described in laboratory manual, or according to the normal condition proposed by manufacturer.

The design and synthesis of embodiment 1:rhGH-Fc fusion protein

By human antibody IgG4Fc sequence (DI488112) after specific site is mutated with hGH gene (NM_000515) with soft Property Linker connection, obtain mature rhGH-Fc, growth hormone signal peptide sequence (CAI94177) be added before hGH sequence, obtain The rhGH-Fc of signal peptide must be carried.The mature rhGH-Fc of the full genome synthesis and rhGH-Fc for carrying signal peptide, ammonia Base acid sequence is as shown in SEQ ID NO:1；The mature rhGH-Fc encoding gene of full genome synthesis, nucleotide sequence such as SEQ Shown in ID NO:2.

SEQ ID NO 1:

The amino acid sequence of long-acting recombinant human growth hormone rhGH-Fc immune fusion protein:

FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQ NPQTSLCFSESIPTPSNREETQQKS NLELLRISLLLIQSWLEPVQFLR SVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFK QTYSK FDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSV EGSCGFSSASTKGPSVFPLAPGPPCPPCPAPEAAG GPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTPLH QDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK

SEQ ID NO 2:

The coding nucleotide sequence of long-acting recombinant human growth hormone rhGH-Fc immune fusion protein:

TTCCCCACCATTCCTCTGTCCAGGCTGTTCGACAACGCCATGCT GAGGGCCCACAGGCTGCATCAACTGGC CTTCGACACCTACCAG GAGTTCGAGGAAGCCTACATCCCTAAGGAGCAGAAATACTCCTT CCTGCAGAACCCCC AGACTTCTCTGTGCTTCTCCGAGTCCATCC CAACCCCCTCCAACAGGGAGGAAACCCAACAGAAGTCCAACCT G GAACTGCTGAGGATCTCTCTGCTGCTGATTCAGTCCTGGCTGG AGCCCGTGCAATTCCTGAGGTCTGTGTTCGCA AACTCCCTGGTG TACGGCGCCTCCGACTCCAACGTGTACGATCTGCTGAAGGACCT GGAGGAGGGAATCCAGAC ACTGATGGGCAGGCTGGAAGACGG CTCCCCAAGGACCGGCCAAATCTTCAAGCAGACCTACTCCAAGT TTGACA CCAACTCCCACAACGATGACGCCCTGCTGAAAAACTAC GGACTGCTGTACTGCTTTAGGAAAGATATGGACAAAG TGGAGAC CTTTCTGAGGATTGTGCAGTGCAGGTCCGTGGAGGGCTCCTGC GGCTTCTCCTCCGCCTCCACCAAG GGCCCTTCCGTGTTCCCTCT GGCCCCTGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGgccgcc GGCGGCCC TTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACAC CCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGT GGTG GACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCACAACGCCA AGACCAAGCCTCGCGA GGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACC CCCCTGCACCAGGAC TGGCTGAACGGCAAGGAGTACAAGTGCA AGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCAT CTC CAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACC CTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCG GTGTCCCT GACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTG GAGTGGGAGTCCAACGGCCAGCC TGAGAACAACTACAAGACCA CCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCC CGCCTGACCG TGGACAAGTCCCGCTGGCAGGAGGGCAACGTGT TCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACA CC CAGAAGTCCCTGTCCCTGTCCCTGGGCAAGT

The amino acid sequence as shown in SEQ ID NO:1 is it is found that mature rhGH-Fc amino acid sequence totally 431 amino Acid, wherein preceding 191 amino acid is human growth hormone sequence, " SSASTKGPSVFPLAP " is link peptide Linker, G207- K431 is the Fc segment of people IgG4, and has the mutation of tetra- amino acid of S228P, F234A, L235A and V308P (according to Kabat Eu coding).

The mutation of S228P is the stability in order to maintain albumen, and IgG4 is it some times happens that two sulphur between heavy chain in hinge area To form semi-antibody molecule, such case will not usually occur in other three kinds of IgG isotype molecules for the dissociation of key.? 228 sites in IgG1 and IgG2 are Pro, and in IgG4 228 be Ser.The Ser residue Pro of IgG4 is replaced, it can So that IgG4 keeps complete antibody molecule.F234A, L235A mutation are the parents in order to lower immune fusion protein Yu Fc receptor With power to reach the ADCC activity for reducing immune fusion protein.V308P is mutated in Fc segment, makes fusion protein and FcRn It is relied in conjunction in pH.When biological encytosis occurs, i.e. when pH is 6 or so, this fusion protein and FcRn affinity are higher, can To avoid the degradation of lysosome.It is normally combined in normal acidity-basicity ph 7.4 with FcRn, to make the half of the long lasting growth hormone The phase decline higher than common Fc fusion protein drug.

Embodiment 2: the building of the recombinant expression plasmid of the encoding gene of fusion protein containing rhGH-Fc

(1) connection of objective gene sequence and GS dual-expression vector

Take respectively 8.0 μ l double expression plasmid p327.8GS (its plasmid map is as shown in Figure 1) respectively with 0.5 μ l Hind III, 0.5μl EcoRⅠ、2.0μl Cut Smart buffer、9.0μl ddH₂O and 0.5 μ l Xba I, 0.5 μ l Xho I, 2.0 μ l Cut Smart buffer、9.0μl ddH₂Digestion system mixes O in EP pipe twice, is put into 37 DEG C of insulating boxs and reacts 2h Carry out double digestion.Then 5 μ l plasmid GS, 5 μ l are carried to rhGH-Fc gene order, 2.0 μ l Buffer, 7.0 μ l of signal peptide ddH₂O is added in EP pipe and mixes, and the T4DNA ligase after 1.0 μ l melt is added in mixed system, mixes, 16 DEG C connected Night.

(2) conversion and extraction of plasmid

Taking bacillus coli DH 5 alpha and LB culture medium, 1:100 ratio is inoculated in liquid LB culture medium (without anti-by volume Property), it shakes, is incubated overnight by a small margin under 37 DEG C of temperature constant states.Next day is by the bacterium solution 1:50 ratio by volume after shaking a night Example is inoculated in LB culture medium (without resistance), continues culture to OD600 0.3~0.5.Cultured Escherichia coli are placed in 10min on ice is transferred in 1.5ml centrifuge tube after mixing, the 4000rpm low-temperature centrifugation 5min at 4 DEG C, abandons supernatant.Use liquid relief Device rifle accurately draws the 0.1mol CaCl of 100 μ l pre-cooling₂Solution is transferred in centrifuge tube, and with finger, gently bullet is uniformly placed on 10min is stood on ice.4 DEG C of 5 min of 3000rpm low-temperature centrifugation abandon supernatant.50 μ l pre-cooling is accurately drawn with pipettor gun 0.1mol CaCl₂Solution is added in centrifuge tube and mixes.

The plasmid for taking 100 μ l competent E.coli DH5 α and 1 μ l step (1) to obtain mixes in EP is managed, ice bath 30min.Remaining competent cell saves under the conditions of being placed in -80 DEG C.Then EP pipe is put into 42 DEG C of tepidarium 90s, immediately It takes out, then ice bath 2min.The not resistant LB culture solution of 200 μ l, low speed shake culture 50min at 37 DEG C of constant temperature is added. 2000rpm is centrifuged 1min, discards 200 μ l supernatants, resting solution is uniformly mixed.By liquid in (Amp) containing ampicillin resistant LB plate on coating uniformly, cultivated 16 hours or so in 37 DEG C of constant incubators.It is inoculated into transfer needle picking positive bacterium colony In 5ml LB liquid medium, 200rpm low speed shake culture 14h prepares to extract plasmid.

Then take 5ml bacterium solution into centrifuge tube, 7000rpm is centrifuged 3min and collects bacterium, abandons supernatant.It is added into centrifuge tube 500 μ l contain the Buffer P1 of RNaseA, are sufficiently resuspended.500 μ l Buffer P2 are added into centrifuge tube again, gently upper It overturns 5~6 times down, is placed at room temperature for 5min, until solution becomes limpid sticky.500 μ l Buffer E3 are added into centrifuge tube, immediately It is 6~8 times reverse, it is placed at room temperature for 5min, 1300rpm is centrifuged 10min, supernatant is added to, Endo-Remover Column has been added Collecting pipe in, 1300rpm be centrifuged 1min, the filtrate in collecting pipe is transferred in centrifuge tube.Adsorption column is packed into collecting pipe Middle 200 μ l the Buffer Ps, 1300rpm that are added are centrifuged 2 min, and balance discards filtrate, adsorption column is put into collecting pipe again In.450 μ l isopropanols are added into centrifuge tube, mixed liquor is transferred in the adsorption column balanced by mixing of turning upside down. 1300rpm is centrifuged 1min, discards waste liquid, again puts back to adsorption column in collecting pipe.750 μ l are added containing dehydrated alcohol Buffer PW, 1300rpm centrifugation 1min, discards waste liquid.1300rpm sky discards waste liquid from 2min, is placed in drying at room temperature 5min. Adsorption column is put in new centrifuge tube.200 μ l Endo-Free Buffer EB are added among to adsorbed film, are placed at room temperature for 5min, 300rpm are centrifuged 2 min, and the plasmid extracted will be sequenced and be identified, gene order is identified correctly recombination Plasmid is named as rhGH-Fc-1.What Fig. 2 was shown is exactly the Sequencing chromatogram of plasmid.Fig. 3 is shown recombinant plasmid rhGH-Fc- 1 is analyzed with the product after Hind III, EcoR I and Xba I, Xho I twice double digestion through 1% agarose gel electrophoresis, in figure See the about purpose band of 1300bp, it is consistent with expected 1362bp.Therefore sequencing and qualification result confirmation comply fully with design requirement.

Embodiment 3:CHO-k1 cell culture and transfection

The cell culture fluid (preferably IMDM culture medium) for cultivating CHO-k1 cell is all discarded first, is added appropriate HYQtase (if attached cell), is then placed in CO₂37 DEG C of acceleration digestion are carried out in incubator, are added after digesting completely appropriate Culture medium dilutes digestive juice and terminates digestion.Cell is all poured into centrifuge tube, 1500rpm is centrifuged 5min, abandons supernatant.It is added Suitable frozen stock solution (culture medium+10%DMSO), cell in centrifuge tube is mixed, cell count, makes its final concentration 3 × 10⁶ ~1 × 10⁷Between a/ml.Above-mentioned cell is transferred in the cryovial of pre-cooling, is put into the freeze box with isopropanol, sets It is transferred in liquid nitrogen again within second day overnight in -80 DEG C.

Freezing plastic tube is taken out from liquid nitrogen container.Cryovial is put into 38 DEG C of water-baths rapidly, melts it rapidly.? 75cm²The complete medium appropriate with serum is added in retorts bottle, cell inoculation is cultivated in retorts bottle.

Start to be transfected when cell density is up to 95%.Under being mediated according to Lipofectamin 2000, with 250 μ l's IMDM culture medium dilutes 6 μ g high-purity plasmid DNAs, is placed at room temperature for 5min.Dilute 20 μ l's with the IMDM culture medium of 250 μ l Lipofectamin is placed at room temperature for 5min.The two is mixed and places 20min.Cell is washed three times with IMDM serum free medium. DNA- liposomal mixtures are added in retorts bottle, 14ml or so IMDM culture medium is added, is placed in 37 DEG C, 5%CO₂, saturation After cultivating 4~6h under humidity, culture medium is replaced, original culture medium is discarded, 16ml IMDM+10%FBS culture medium is added and continues Culture.

Embodiment 4: stability and high efficiency expresses the screening of the engineering cell of rhGH-Fc fusion protein

(1) screening of positive colony

The IMDM culture medium of CHO-k1 cell after embodiment 3 is transfected discards, and 2ml or so HYQtase digestion is added, It is put into insulating box and accelerates digestion.After digestion completely, be added has blood serum medium to terminate digestion in right amount.Cell and culture solution are fallen Enter in centrifuge tube, 1500rpm is centrifuged 5 min, discards supernatant.30ml SMM-CHO GSI culture medium is added in centrifuge tube (to contain 10% FBS+25 μm of ol MSX) it mixes.Required culture medium and cell are mixed, moved into 96 orifice plates with the volley of rifle fire, 100 holes μ l/, The counting of every hole cell should be 5 × 10 in 96 orifice plates³~8 × 10³It is a.It is sealed with preservative film, is put into CO₂It is cultivated in incubator.One 50 μ l SMM-CHO GSI culture mediums is added after week (containing 10%FBS+25 μm of ol MSX).12 days or so after transfection, in microscope Under, it marks with monoclonal hole location.The cell culture fluid for marking hole location is blotted only, SMM-CHO GSI is added in 150 holes μ l/ (25 μm of ol MSX) serum free medium.After being cultivated 3~4 days in CO2 incubator, using ELISA method detection culture supernatant, into Row screens for the first time.

The relatively good monoclonal of ELISA result is scraped and is moved into 24 orifice plates, 1ml SMM-CHO GSI is added in every hole (25 μm of ol MSX) culture medium, is placed in CO₂After cultivating 3~4 days in incubator, the first hole takes 150 μ l supernatants, and excess-three hole is every Hole adds 200 μ l PBS, takes 50 μ l to the second hole mixing successively to dilute from the first hole, takes 4 gradient dilutions of supernatant, uses The detection of ELISA method, carries out programmed screening.

It will test the relatively good hole location piping and druming of result uniformly, move into 6 orifice plates, 3ml SMM-CHO GSI is added in every hole (50 μm of ol MSX), is placed in CO₂After being cultivated 3~4 days in incubator, 4 gradient dilutions of supernatant point are taken, are examined using ELISA method Culture supernatant is surveyed, third time screening is carried out.

Choose the relatively good immigration 75cm of testing result²In retorts bottle, every bottle is added 13ml SMM-CHO GSI (75 again μm ol MSX), it is placed in CO₂It is cultivated in incubator.Continue culture one week or so, takes 75cm²4 gradients of supernatant in retorts bottle Dilution carries out the 4th screening using ELISA method detection.

The relatively good carry out cell cryopreservation of testing result is chosen, is left some to continue to cultivate.Cell grows to logarithmic phase When be transferred in the small shaking flask of 125ml, continue shake culture.By in the small shaking flask of 125ml cell and culture solution 1500rpm centrifugation 5min is added the piping and druming of 10ml SMM-CHO GSI (75 μm of ol MSX) culture medium and mixes progress cell count.In 125ml shaking flask Middle addition 30ml SMM-CHO GSI (75 μm of ol MSX) fresh culture, moves into appropriate cell, makes bottle inner cell sum be 1.2×10⁷A, batch cultivation counts observation daily.It takes cells and supernatant in 125ml bottles within 3~4 days, carries out 6 ladders respectively Degree dilution carries out the 5th screening using ELISA method detection.The selection result is shown in Fig. 4, according in screening 125ml shaking flask Culture solution supernatant ELISA as a result, choose that expression quantity is relatively high and cell growth state tri- plants of cell cryopreservations of preferable D, E, H, It is ready for the subclone screening of the second wheel.

(2) subclone screening

The expression quantity that selecting step (1) obtains is higher and vegetative state tri- plants of cell strains of preferable D, E, H are subcloned Screening.

Cell count is carried out first.Then diluting cells, culture medium are SMM-CHO GSI+10% FBS (25 μm of ol MSX), make altogether containing 500 cells or so in 5ml, each cell strain spreads 5 piece of 96 orifice plate, 100 holes μ l/, 96 orifice plates completed It is sealed with preservative film, is put into 37 DEG C, CO₂It is cultivated in incubator.The screening experiment method phase of subsequent screening technique and positive colony Together, from 96 plates (25 μm of ol MSX) → 24 orifice plates (50 μm of ol MSX) → 6 orifice plates (50 μm of ol MSX) → 75cm²Culture bottle (75 μm of ol MSX) → 125ml shaking flask (75 μm of ol MSX).

Cell strain D is shown in 75cm in Fig. 5²Middle culture supernatant ELISA chooses D1 and continues to cultivate as a result, as shown in figure； Cell strain E is shown in 75cm in Fig. 6²Middle culture supernatant ELISA chooses E2 and continues to cultivate as a result, as shown in figure；Fig. 7 is shown Be cell strain H in 75cm²Culture supernatant ELISA in culture bottle chooses H2 and continues to cultivate as a result, as shown in figure；It will be at sub- gram D1, E2, H2 culture supernatant ELISA method detection in 125ml shaking flask in grand screening under identical cultivation conditions, it is aobvious according to Fig. 8 The ELISA shown is as a result, D1, E2, H2 cell growth curve, selection cell grow shape in the subclone 125ml shaking flask that Fig. 9 is shown State and the preferable H2 of testing result are to finally screen cell strain, and be named as rhGH-Fc cell strain, and the destination protein of expression is RhGH-Fc immune fusion protein.

The preliminary purification of embodiment 5:rhGH-Fc immune fusion protein

Culture rhGH-Fc cell strain is cultivated about 10~15 days with producing rhGH-Fc immune fusion protein, close to cell When all dead, supernatant is collected, 4000rpm is centrifuged 20min, and supernatant is collected and prepares purifying rhGH-Fc immune fusion protein. By the supernatant of albumen medium centrifugal and diatomite in starting to carry out affinitive layer purification mesh after 3g/100ml ratio hybrid filtering Albumen.Membrane filtration first by cell culture supernatant through 0.22 μm of aperture.By protein purification column (prepacked column Hitrap Mabselect Sure 5*1ml) it is connected on protein purification instrument.With 50mM Tris+150 mM NaCl (HCl tune pH 7.3~ 7.5) equilibrium liquid balances 3~5 volumes of pillar with 5ml/min flow velocity, shows 0 or so numerical value on Ultraviolet Detector.With 5ml/min flow velocity loading may show hundred values of series on Ultraviolet Detector.3~5, pillar are cleaned with equilibrium liquid after end of the sample Volume shows 0 or so numerical value on Ultraviolet Detector again.With the eluent of 0.1M glycine (HCl tune pH 3.5~3.8) With the flushing of 2~3ml/min flow velocity, eluting peak is collected.3~5 column volumes of purification column are cleaned with 0.1M NaOH cleaning solution, 20% Ethyl alcohol saves.

The identification of embodiment 6:rhGH-Fc immune fusion protein

RhGH-Fc immune fusion protein after preliminary purification is analyzed into phase through non-reduced and 10% SDS-PAGE of reduced form To molecular mass, isoelectric point is measured through isoelectric focusing electropho- resis.The SDS- of rhGH-Fc immune fusion protein is shown in Figure 10 PAGE identifies map, as seen from the figure, non-reduced electrophoresis showed visible differential protein band at relative molecular weight about 95kDa, It is consistent with theoretical value 97.3kDa, reduced form electrophoresis showed is about visible differential protein band at 42kDa in relative molecular weight, with Theoretical value 43.7kDa is consistent.

The isoelectric focusing electropho- resis figure of rhGH-Fc immune fusion protein is shown in Figure 11, and rhGH-Fc is immune as the result is shown The isoelectric point of fusion protein is consistent with theoretical expectation values 5.35 between 5.2~5.85.

The expression of embodiment 7:rhGH-Fc immune fusion protein

The rhGH-Fc cell strain obtained of embodiment 4 is cultivated to produce rhGH-Fc immune fusion protein, condition of culture are as follows: SMM-CHO GSI culture medium (contains 75 μm of ol MSX), and 37 DEG C, CO²Shaken cultivation in incubator, culture measured it after about 12 days RhGH-Fc immune fusion protein expression.

The supernatant of the cell culture fluid of rhGH-Fc immune fusion protein is detected using high performance liquid chromatography (HPLC) And the rhGH-Fc immune fusion protein after affinity chromatography preliminary purification.Chromatographic column uses G3000WS, and Detection wavelength is set as 280nm, flow velocity 1ml/min, applied sample amount are 20 μ l.Mesh is determined by the HPLC testing result of known concentration albumen after purification Albumen retention time, pass through peak area compare determine cells and supernatant in destination protein expression quantity.

The HPLC map of the supernatant of the cell culture fluid of rhGH-Fc immune fusion protein, Tu13Xian is shown in Figure 12 What is shown is the HPLC map of rhGH-Fc immune fusion protein.It can be seen that by HPLC testing result, rhGH-Fc immune fusion protein Main peak retention time be about 10.6 min, in the supernatant of cell culture fluid the peak area of destination protein be 17804703, it is pure The peak area of rhGH-Fc immune fusion protein after change is 21131703, purity 95.21%.Estimate cell conditioned medium expression quantity For 1.4g/L or more.

The biological activity determination of embodiment 8:rhGH-Fc immune fusion protein

The active measurement of rhGH-Fc immune fusion protein is gone to hang down by four 1219 rats of general rule of " Chinese Pharmacopoeia " version in 2015 Body growth hormone bioassary method carries out determination of activity.

It is specific as follows: SD rat, half male and half female, 3~4 week old, the abdominal cavity yellow Jackets (concentration 15mg/ml) 45mg/kg Injecting anesthetic (administration volume 3ml/kg), is fixed on stereotaxis hypophysectomy instrument (Stoelting, USA), hangs down after anesthesia Body removal operation, records the weight and tail length (distance from tail end to anus) of every animal after operation.Operation finishes, postoperative In 3 days, daily single subcutaneous injection penicillin (40 × 10⁴IU/ml) 0.1ml/, and animal is allowed freely to drink 5% glucose Water (is fitted into in water bottle)；Free water and feed.Postoperative 3 weeks, the weight of every surviving animals is measured, postoperative 2~3 weeks bodies are taken It is qualified animal pattern that variation, which is less than operation consent ± 10% to go hypophysis rat, again, and is divided into model control group by weight Stochastic Equilibrium (physiological saline), human growth hormone (HGH) international activity standard items low dose group (0.4IU/kg), high dose group (1.6IU/kg), Low dose group (0.4IU/kg), the high dose group (1.6IU/kg) of rhGH-Fc immune fusion protein, every group 10；Medication: Subcutaneous injection；Administered volume: 2.5ml/kg/d；Administration frequency and time limit: according to grouping be administered, standard item group once a day, continuous 6 Day；Model control group, test sample group interval are injected 1 time, co-injection 2 times for 3 days；Injection dosage and grouping setting are shown in Table 1 in detail.

1 injection dosage of table and grouping setting

Daily weighing record (the experimental group administration same day is denoted as D1).Weight increases after the administration of each group rat is shown in Figure 14 Long tendency chart.Body weight determination after the injection of test sample group rhGH-Fc immune fusion protein in 3 days the results show that all growing, and mark It after quasi- product group is not administered, stops growing immediately, can tentatively judge that the long half time of rhGH-Fc immune fusion protein swashs in growth Plain standard items.10th day execution animal simultaneously digs rear two leg shin bones, (every by pharmacopeia annex method measurement shin bone epiphyseal plate width Two leg shin bone of mouse measures 10 numerical value, removes peak and minimum, takes its average value).By response value experimental result by " China Pharmacopeia " parallel line analysis measurement Random Design method list in 2015 years four 1431 Bioassay-statistical methods of general rule of version lattice Formula arranges, and by quantitative response parallel line assay method Random Design processing result, what table 2 was shown is exactly that each group rat tibia epiphyseal plate is wide Spend measurement result.Data are subjected to statistical procedures, the width measurement data statistics of low dose group rat epiphyseal plate is shown in table 3 The analysis of high dose group rat epiphyseal plate width measurement statistical data is shown in credit analysis, table 4.As can be seen from the table, low The data of test sample and standard items prove that the immune fusion protein half-life period that the present invention develops is significantly long in dosage and high dose group In standard items.Test sample theoretical estimation potency after according to dosage converting are as follows: 1.24IU/mg is counted by the biological standardization of States Pharmacopoeia specifications The potency result that method calculates is 1.1IU/mg.

2 each group rat tibia epiphyseal plate width measurements of table

The analysis of 3 low dose group rat epiphyseal plate width measurement statistical data of table

The analysis of 4 high dose group rat epiphyseal plate width measurement statistical data of table

The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Changchun Institute of Biological Products Co., Ltd.

<120>a kind of long-acting recombinant human growth hormone fusion protein and its engineering cell

<141> 2019-05-16

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1 5 10 15

Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

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

35 40 45

Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

65 70 75 80

Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val

85 90 95

Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu

115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

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Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Ser

180 185 190

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

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

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

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

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

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

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

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

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

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

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

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

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ctggtgtacg gcgcctccga ctccaacgtg tacgatctgc tgaaggacct ggaggaggga 360

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cctgaggtga cctgcgtggt ggtggacgtg tcccaggagg accctgaggt gcagttcaac 780

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tcgccgtgga gtgggagtcc aacggccagc ctgagaacaa ctacaagacc acccctcctg 1140

tgctggactc cgacggctcc ttcttcctgt actcccgcct gaccgtggac aagtcccgct 1200

ggcaggaggg caacgtgttc tcctgctccg tgatgcacga ggccctgcac aaccactaca 1260

cccagaagtc cctgtccctg tccctgggca agt 1293

Claims

1. a kind of long-acting recombinant human growth hormone rhGH-Fc fusion protein, it is characterised in that: the fusion protein is swashed by life is long Element, link peptide and Fc segment composition；The fusion protein by N-terminal to C-terminal successively are as follows: human growth hormone (HGH), link peptide, Fc segment, Its amino acid sequence totally 431 amino acid, wherein preceding 191 amino acid sequences are human growth hormone sequence, the 192nd to the 206 are connection peptide sequence, the 207th to the 431st Fc segment for human IgG.

2. fusion protein according to claim 1, it is characterised in that: the Fc segment is selected from the Fc segment of human IgG, preferably For the Fc segment of people IgG4.

3. fusion protein according to claim 1, it is characterised in that: the fusion protein F c segment includes four amino acid Mutation, the amino acid mutation are as follows: S228P, F234A, L235A and V308P, wherein the number of the residue in the area IgG Fc be according to It is numbered according to Kabat Eu Index.

4. fusion protein according to claim 1, it is characterised in that: the amino acid sequence of the fusion protein such as SEQ ID Shown in NO:1.

5. the encoding gene of any one of Claims 1-4 fusion protein, it is characterised in that: the nucleosides of the encoding gene Acid sequence is as shown in SEQ ID NO:2.

6. a kind of engineering cell, it is characterised in that: the engineering cell expresses the described in any item fusion eggs of Claims 1-4 It is white.

7. a kind of expression vector, it is characterised in that: the expression vector is preferably dual-expression vector；It is highly preferred that the expression Carrier is with double expression plasmid p327.8GS through restriction enzyme Hind III, EcoR I and Xba I, Xho I double enzymes twice After cutting, the large fragment of recycling and the code nucleic acid of fusion protein described in claim 1 are mixed through T4 DNA ligase, 15~16 DEG C connection overnight, be cloned into the target gene optimized on dual-expression vector.

8. a kind of Chinese hamster ovary celI, it is characterised in that: the Chinese hamster ovary celI is CHO-k1 cell, and stable transfection has the right to require 7 institutes The expression vector stated.

9. any one of expression the Claims 1-4 positive colony of the fusion protein and the screening technique of subclone, feature exist In: by the way that methionine sulfonamide (MSX) concentration is gradually increased, from 96 plates (25~30 μm of ol MSX) → 24 orifice plates (50~55 μ Mol MSX) → 6 orifice plates (50~55 μm of ol MSX) → 75cm2 culture bottle (70~75 μm of ol MSX) → 125ml shaking flask (70~ 75 μm of ol MSX), go out that expression quantity is higher and the preferable cell strain of vegetative state using ELISA method Stepwise Screening, will finally screen Cell strain is named as rhGH-Fc cell strain, and the destination protein of expression is rhGH-Fc immune fusion protein.

10. fusion protein according to any one of claims 1 to 4 or Chinese hamster ovary celI according to claim 8, special Sign is: the fusion protein can extend serum half-life, substantially reduce drug administration by injection frequency required in treatment time, from And improve the compliance of patient and the convenience of doctor and patient；The Chinese hamster ovary celI can be used for preparation and reorganization people's long lasting growth hormone, Its expressing quantity is high, bioactivity is good, reduces the immunogenicity of albumen, can improve drug effect.