CN112661858A - Recombinant human growth hormone Fc fusion protein, application and engineering cell strain thereof - Google Patents

Abstract

The invention relates to the technical field of biomedical engineering, in particular to the technical field of gene recombination long-acting human growth hormone, and specifically relates to development of a recombinant human growth hormone Fc fusion protein. The human growth hormone Fc fusion protein contains human growth hormone (hGH), flexible peptide linker (L)) and human immunoglobulin Fc fragment. The invention also discloses an activity determination method of the fusion protein and a host cell capable of expressing the fusion protein. The recombinant human growth hormone Fc fusion protein constructed by the invention has longer serum half-life than recombinant hGH, greatly reduces the injection administration frequency required in the treatment time, thereby improving the compliance of patients and increasing the convenience of doctors and patients, and simultaneously, the production process is simpler and more efficient, thus being particularly suitable for preparing the gene recombinant long-acting human growth hormone in an industrialized large scale.

Description

Recombinant human growth hormone Fc fusion protein, application and engineering cell strain thereof

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to the technical field of gene recombination long-acting human growth hormone, and specifically relates to development and application of a recombinant human growth hormone Fc fusion protein and an engineering cell strain thereof.

Background

Growth Hormone (HGH) is a peptide hormone which is synthesized, stored and secreted by growth hormone cells in the pituitary. Mature wild-type human GH (hGH identified by SEQ ID NO: 2) is composed of 191 amino acid residues, most of which are present in the form of 22kDa molecular weight, a small amount of growth hormone is present in the form of 20kDa, has an isoelectric point of 4.9, and contains 4 cysteine residues at positions 53, 165, 182 and 189, which stabilize the three-dimensional structure of the protein by forming two intramolecular disulfide bonds linking C53 to C165 and C182 to C189, respectively. Growth hormone and a specific growth hormone receptor (hGHR) thereof are mutually combined on the surface of a target cell to mediate biochemical tandem reaction, and a plurality of definite signal paths such as JAK2-STATs, MAPK, PI3K-GSK3 and the like in cells are activated, so that the proliferation and differentiation of the cells are maintained. Insulin-like growth factor-1 (IGF-1) is expressed in combination with growth hormone receptors and is involved in the growth and regeneration of cells. It is well known that growth hormone is produced in the pituitary gland of normal humans and gradually increases to puberty and gradually decreases with age.

The most important physiological role of growth hormone is to promote human metabolism and growth by promoting protein synthesis and fat decomposition of receptor cells in various tissues, organs and bones of the body. If the human body has insufficient growth hormone secretion, the growth hormone can cause 'dysplasia' in a popular way. Therefore, growth hormone is currently used clinically mainly for the treatment of growth retardation in children and growth hormone deficiency in adults. In addition, the growth hormone has application in the aspects of beauty treatment, anti-aging, severe burns and the like in recent years, and experiments prove that the growth hormone can actually re-secrete fat in vivo, increase mucopolysaccharide in subcutaneous elastic tissues and enable the skin to become firm and elastic.

The domestic growth hormone stock market in 2019 is about 55 million yuan, and the growth hormone stock market is in a rapid growth period. According to the factory caliber calculation, the market scale of the growth hormone is increased year by year, the growth is gradually increased from 11.90 billion in 2014 to 55.19 billion in 2019, and the composite growth rate is 35.91%. At present, a plurality of products of recombinant human growth hormone are available on the market at home and abroad, for example, the indications of Jianhaoning (recombinant human growth hormone for injection) of the feverfew are as follows: growth disorders caused by insufficient endogenous growth hormone secretion or by Turner's syndrome or chronic renal insufficiency; nutropin, Genentech; of norhondride

(somatropin), the saybolt of the golden horse pharmaceutical industry (recombinant human growth hormone injection), and the amsugen of the andrology (recombinant human growth hormone for injection), etc.

However, these drugs are usually short-acting products, and in clinical application, since the half-life of growth hormone in human body is very short, and the half-life is about 2 hours, the therapeutic drug administration requires one injection per day by subcutaneous injection, and the administration period is long (for example, up to about 6 months or more). Frequent injection of growth hormone can cause inconvenience to patients and the cost of administration is expensive. The long-acting PEG (polyethylene glycol) growth hormone developed in the market can obviously improve the half-life period of the medicament and enable the long-acting PEG growth hormone to be long-acting after being modified by polyethylene glycol, but because PEG has potential risk as a foreign body in a human body, pain can be caused in the injection process, the safety is limited, the metabolic pathway of the PEG growth hormone is filtered and removed by glomeruli, and researches show that the PEG component is related to the hollowing of renal tubular epithelial cells, and potential hidden danger exists.

The current long-acting protein drug technology which is the hottest research and the most rapidly developed is the Fc fusion protein technology, the Fc fragment is the main factor for keeping the in vivo longer half-life of IgG, and simultaneously, the Fc fragment can stabilize the protein and is used for developing various fusion proteins. The Fc fragment functions by two mechanisms, the first is binding to Fc receptors on the cell surface, killing the cell by phagocytosis or lysis and digesting the pathogen by the antibody-dependent cellular cytotoxicity (ADCC) pathway. Among the four human IgG subtypes, IgG1 and IgG3 bind efficiently to Fc receptors on cell surfaces, whereas IgG2 and IgG4 bind to receptors with lower affinity. The second is binding to C1q of the first complement component C1, which cleaves the pathogen through the Complement Dependent Cytotoxicity (CDC) pathway. Human IgG1 and IgG3 bind C1q efficiently to mediate the complement cascade, human IgG2 binds C1q poorly, and human IgG4 does not bind C1 q. The Fc fusion protein is mainly used for increasing the half-life of a protein drug in vivo and simplifying the production process so as to reduce the cost, and the Fc mediated IgG effect and the function of cell lysis are not necessary and even generate side effects. The Fc fusion protein technology not only increases the clearance rate of the molecular weight reduction of protein drugs like PEG, but also can prolong the half life in a slow release mode through an FcRn binding domain and pH sensitive binding force. The fusion partner and the effector molecule are connected together at the gene level, no additional chemical modification is needed, the purification and preparation processes are simple, the quality control is relatively easy, and the long-acting method is very promising in comprehensive view.

Disclosure of Invention

The invention aims at providing a recombinant human growth hormone Fc fusion protein, a vector of nucleotide for coding the fusion protein, a host cell and application thereof. The fusion protein is used as a medicine for treating growth retardation of children and growth hormone deficiency of adults, severe burns and the like, can prolong the half-life of serum, reduce the injection administration frequency required in the treatment time, and improve the compliance of patients and the convenience of doctors and patients.

The invention provides a long-acting recombinant human growth hormone Fc fusion protein, which preferably comprises the following components from the N end to the C end in sequence: human growth hormone, linker peptide, Fc fragment (abbreviated as rGH-Fc), less preferably Fc fragment, linker peptide, human growth hormone (abbreviated as Fc-rGH).

Wherein the amino acid sequence of the growth hormone is the same as the amino acid sequence in GenBank: M13438.1, and the total amino acid number is 191.

Further preferred flexible peptide linker amino acids are of the general structural formula (GGGGS) nA, wherein n is an integer greater than or equal to 2, in some embodiments n-3, in other embodiments n-2 or 4.

The value of n in the protection range does not influence the construction, the implementation and the test result of the specific embodiment.

In some embodiments, the Fc variant of human IgG (numbering of residues is according to the Kabat Eu Index) is selected from the group consisting of:

1) IgG1 Fc containing at least the following two mutations: leu234Ala, Leu235Ala, Asn297Ala, Val308Pro and deletion of C-terminal Lys, the nucleotide sequence of the IgG1 Fc is as set forth in SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4 is shown in the specification;

2) IgG2 Fc containing at least the following two mutations: asn297Ala, Val308Pro, Pro331Ser, and deletion of C-terminal Lys, the nucleotide sequence of IgG2 Fc is set forth in SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6 is shown in the specification;

3) IgG4 Fc containing at least the following two mutations: at least the hinge region, CH2 and CH3 region of human IgG4 containing Ser228Pro, Glu233Pro, Leu234Ala or Leu234Val, Leu235Ala, Asn297Ala, Val308Pro and Arg409Lys and deleting C-terminal Lys, the nucleotide sequence of IgG4 Fc is as shown in SEQ ID NO: 7, and the amino acid sequence is shown as SEQ ID NO: 8 is shown in the specification;

in order to achieve the purpose, the invention adopts the technical scheme that:

firstly, determining an amino acid sequence of a recombinant human growth hormone Fc fusion protein, adding a proper enzyme cutting site, a Koaza sequence and a signal peptide for guiding secretion at two ends, referring to a website or a database for translating each codon utilization rate of Chinese hamster protein, referring to some literature data, performing gene coding region codon optimization on the premise of not changing the amino acid sequence, adopting rat species to bias codons, removing sequences (such as TATA box, AATAA Transcription termination site, Crytic splice sites and the like) which possibly influence antibody expression, finally reversely converting the sequences into corresponding gene sequences, and sending the gene sequences to gene synthesis.

The DNA coding the fusion Protein is constructed into pCDNA3.1 vector (from Invitrogen), transiently transfected into Expi293 eukaryotic cells, and after transfection for 5 days, cell culture supernatant is collected and purified by Protein A to obtain the target fusion Protein. The fusion protein of interest was analyzed for purity, isoelectric point, stability, plasma half-life in rat, response to rat serum IGF-1, and its effect on the STAT5 signaling pathway upon binding to Growth Hormone Receptor (GHR).

After confirming that the target fusion protein can stimulate an IGF-1 reaction of rats, the plasma half-life of the rats in vivo is obviously prolonged, and the STAT5 signal channel can be effectively activated after the target fusion protein is combined with a Growth Hormone Receptor (GHR), DNA for coding the fusion protein is constructed into a D2M vector, CHO-S cells are transfected, and a stable cell strain for highly expressing rGH-Fc/Fc-rGH is obtained by MTX pressure screening.

The beneficial effect that above-mentioned technical scheme produced lies in: the recombinant human growth hormone fusion protein provided by the invention has the characteristics of specifically binding to a Growth Hormone Receptor (GHR), activating a downstream STAT5 signal pathway, maintaining the proliferation and differentiation of cells and having longer half-life, and is used for treating growth hormone deficiency of children and adults, severe burns and the like.

Drawings

FIG. 1 shows the rGH-Fc/Fc-rGH protein after SDS-PAGE (polyacrylamide gel electrophoresis) detection and purification of proteinA by one-step affinity chromatography, wherein the first lane from the left is Fc-rGH protein, the second lane is rGH-Fc protein, and the third lane is protein Ladder;

FIG. 2 shows SEC detection of rGH-Fc protein purity;

FIG. 3 shows the purity of Fc-rGH protein measured by SEC;

FIG. 4 shows the detection of the binding of rGH-Fc/Fc-rGH protein to the growth hormone receptor GHR by ELISA;

FIG. 5 is an ELISA for detection of rGH-Fc/Fc-rGH protein binding to complement C1 q;

FIG. 6 shows the luciferase reporter gene method for detecting the biological activity of rGH-Fc;

FIG. 7 is a graph showing the change in serum IGF-1 levels in rats after intravenous injection of equimolar amounts of rGH-Fc and GH;

FIG. 8 is the change in GH content in rat serum after intravenous injection of equimolar amounts of rGH-Fc and GH;

FIG. 9 shows the growth curves of rGH-Fc engineered cell lines, with the numbers of cells (LCC) and the cell viability rates (VIA) on the ordinate.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1 construction of rGH-Fc/Fc-rGH expression vectors

1.1 Synthesis of GH Gene

The NCBI database searches an amino acid sequence (GenBank Accession: M13438.1) of GH, analyzes the nucleotide sequence, carries out modification such as decorrelation enzyme cutting site or reduction of stem-loop structure aiming at the nucleotide sequence, entrusts the whole gene to synthesize the rGH-Fc full-length gene, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

1.2PCR amplification of XbaI-rGH-Fc-HindIII and XbaI-Fc-rGH-XhoI

The following primers were designed:

GH-Fc amplification: XbaI-GH fragment was amplified by PCR using a synthetic plasmid containing GH gene (pUC57-GH) as a template and P1 and P2 as primers and Prime STAR HS polymerase. The Fc-HindIII fragment was obtained by PCR using the existing IgG-Fc gene plasmid as a template, P3 and P4 as primers, and Prime STARHS polymerase. Then XbaI-GH-Fc-HindIII obtained by the PCR amplification is used as a template, P1 and P4 are used as primers, and XbaI-GH-Fc-HindIII is obtained by the SOE-PCR amplification.

Fc-GH amplification: the plasmid of the existing IgG-Fc gene is used as a template, P6 and P7 are used as primers, PrimeSTARHS polymerase is used for PCR amplification to obtain an Fc-pre fragment, and then the Fc-pre fragment is used as a template, and P5 and P8 are used as primers for amplification to obtain XbaI-Fc. The GH-XhoI fragment was obtained by PCR using a synthetic plasmid containing the GH gene (pUC57-GH) as a template and P9 and P10 as primers and Prime STAR HS polymerase. And then XbaI-Fc-GH-XhoI is obtained by amplifying XbaI-Fc-GH-XhoI through SOE-PCR by using XbaI-Fc and GH-XhoI obtained by the PCR amplification as templates and P5 and P10 as primers.

The amplification system is as follows:

the PCR reaction program is as follows:

1.3 double digestion

XbaI/HindIII enzyme digestion vector pCD-cFc (modified from pCDNA3.1+) and target fragment XbaI-GH-Fc-HindIII, XbaI/XhoI double-enzyme digestion target fragment XbaI-Fc-GH-XhoI and pCD-cFc vector, wherein the vector and enzyme digestion products are recovered by DNA gel and then used for connecting and transforming, wherein the enzyme digestion system is as follows:

1.4 ligation transformation and sequencing

Taking 3 mu l of enzyme-linked product, transforming into DH-5a competent cells, coating an ampicillin plate, picking out clone and sequencing to obtain eukaryotic expression plasmids pCD-rGH-Fc and pCD-Fc-rGH, and sequencing to identify the correct vector sequence.

Example 2rGH-Fc/Fc-rGH protein expression and purification

2.1 expression and characterization of rGH-Fc/Fc-rGH

Inoculating Expi293 cells into a 24-well culture plate at an inoculation density of 2.5-3 × 10⁶cells/ml, inoculum size 1 ml/well, cell viability greater than 90% at transfection. Mu.g of DNA (pCD-rGH-Fc/Fc-rGH) was diluted in 50. mu.l of OptiMEM, gently mixed, 2.7. mu.l was diluted in 50. mu.l of OptiMEM, gently homogenized, incubated at room temperature for 5 minutes, then the resulting DNA dilution and the resulting Expifeacylamine dilution were mixed, gently homogenized, incubated at room temperature for 20 minutes, the resulting DNA-Expifeacylamine complex (total volume: about 100. mu.l) was added to the culture well plate, and the plate was shaken back and forth to distribute uniformly. And (3) putting the cells into an incubator for continuous culture for 16h, adding transfection Enhancer1 according to the addition ratio of 5 mul/ml, adding transfection Enhancer2 according to the addition ratio of 50 mul/ml, adding antibiotics according to the addition ratio of 1 mul/ml, gently mixing, continuously culturing for 4 days, and collecting all cells for centrifugation when the cell viability is lower than 50%. After confirming the expression of rGH-Fc/Fc-rGH fusion protein by ELISA, the transfection system was expanded to 30ml (same procedure as above), and the supernatant purified protein was collected 5 days after transfection.

2.2 purification and purity characterization of rGH-Fc/Fc-rGH protein

The culture supernatant was collected, purified with proteinA resin, and then concentrated by ultrafiltration with an ultrafiltration centrifuge tube having a molecular weight cut-off of 30kDa, and characterized by SDS-PAGE as shown in fig. 1, wherein lane 1 is Fc-rGH, lane 2 is rGH-Fc, lane 3 is protein Ladder, indicating that the molecular weight of rGH-Fc/Fc-rGH is consistent with the theory (97KD) and the purity is high. FIG. 2 and FIG. 3 are SEC identification plots for rGH-Fc and Fc-rGH proteins, respectively, and integration results show that the purity of the main peak is greater than 95%.

Example 3 binding of rGH-Fc/Fc-rGH protein to GHR

Growth hormone receptor protein (GHR) was diluted to 1. mu.g/ml with PBS and plated overnight at 4 ℃. TPBS was washed 3 times, oven-blocked at 37 ℃ for 1h, TPBS was washed 3 times, and gradient dilutions of rGH-Fc/Fc-rGH fusion protein (50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19nM) were added, 100. mu.l per well, incubated with shaking at room temperature for 1 h. After 3 TPBS washes, 100. mu.l of sheep anti-human Fc-HRP (1:4000 dilution) was added to each well, incubated at room temperature with shaking for 1 hour, and after 3 TPBS washes, OPD was added to develop color, reacted at room temperature for 10min, and 1MH2SO4 was added to terminate the reaction, 100. mu.l of each well, and the absorbance was measured at 490 nm. The detection result is shown in FIG. 4, and the result shows that both rGH-Fc and Fc-rGH can bind with GHR and present a concentration gradient-dependent trend.

Example 4 binding of rGH-Fc/Fc-rGH protein to C1q

The rGH-Fc/Fc-rGH protein and Herceptin antibody antigens were diluted with PBS to 8, 4, 2.000, 1, 0.5, 0.25, 0.125, 0. mu.g/mL, added to a 96-well plate at 100. mu.l/well and plated overnight at 4 ℃. TPBS is washed for 3 times, an oven is sealed for 1h at 37 ℃, 2 mu g/mL human C1q protein is added after TPBS is washed for 3 times, 50 mu l of each hole is added, and the incubation is performed for 1 hour at room temperature with shaking. After 3 TPBS washes, 100. mu.l of sheep anti-human C1q-HRP (1:500 dilution) was added to each well, incubated with shaking at room temperature for 1 hour, washed 3 TPBS, and then colored by addition of OPD, reacted at room temperature for 10min and then stopped by addition of 1MH2SO4, 100. mu.l of each well, and the absorbance was measured at 490 nm. The detection results are shown in FIG. 5, and the results show that the binding of Herceptin of the IgG1 subtype and the C1q protein is in a concentration gradient-dependent trend, while the rGH-Fc/Fc-rGH protein is not bound with the C1 q.

Example 5 determination of the biological Activity of rGH-Fc proteins

Luciferase reporter gene containing STAT5 promoter and GHR receptor plasmid are transferred into HEK293 cells to obtain HEK293-GHR-Luc cell strain. HEK293-GHR-Luc cells in logarithmic growth phase were taken and trypsinized before being resuspended in DMEM + 10% FBS + 1% PS + 2. mu.g/ml of Puromycin medium to a cell concentration of 6 x 105 cells/ml. 50 μ l of the cell suspension, 30000cells/well, was added to the 96-well plate and 100 μ l of PBS buffer, CO2, was added around the 96-well plate for 16-24 h. The rGH-Fc protein was diluted to 200. mu.g/ml with phenol red-free DMEM + 10% FBS medium, and 8-fold gradient dilution was performed for 8 gradients, and 50. mu.l of the diluted protein was added to a 96-well plate (i.e., 100 final concentration, 14.2857, 2.0408, 0.2915, 0.0416, 0.0059, 0.0008, 0.0001. mu.g/ml), and then placed in a CO2 incubator for 5 hours, 100. mu.l of Promega Bio-GloTM luciferase assay System which had reached room temperature was added to each well, and placed at room temperature and shaken with a shaker for 10min and then tested on the machine. The test results are shown in FIG. 5. FIG. 5 shows: after the HEK293-GHR-Luc is stimulated by GH-Fc, the GH-Fc is combined with a receptor GHR to activate a JAK2-STATs signal path, so that the transcription of a luciferase gene downstream of the STAT5 reaction element is activated, and the expression level of the luciferase gene is positively correlated with the content of the GH-Fc of the GHR combined on a target cell membrane.

Example 6 rGH-Fc protein and Effect on rat IGF-1 levels

Male mature Sprague Dawley rats (about 7 weeks old, body weight 130-180g) were divided into 3 groups of 5, and blood samples were taken after a single administration of 0, 1, 4, 12, 24, 48, 96h by intravenous injection of equal volumes of physiological saline, 15nmol of growth hormone water needle and 15nmol of recombinant human growth hormone protein (rGH-Fc), and the IGF-1 content in the serum of the rats was determined using Rat IGF-1 ELISAKit (manufacturer: Elapscience, cat # E-EL-R0010 c). The specific operation is as follows: first, the enzyme label plate coated with anti-IGF-1 antibody is taken out and balanced to room temperature, 100. mu.L of rat IGF-1 standard substance protein (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0ng/mL) which is diluted in a gradient manner and a sample to be detected are added, the liquid is discarded after incubation for 90 minutes at 37 ℃, 100. mu.L of biotinylated antibody working solution is added after drying, and the mixture is incubated for 1 hour at 37 ℃. After washing the plate 3 times, 100. mu.L of the enzyme conjugate working solution was added to each well and incubated at 37 ℃ for 30 minutes. And washing the plate for 5 times, adding 90 mu L of substrate solution TMB into each hole, incubating for 15 minutes at 37 ℃ in the dark, adding 50 mu L of stop solution into each hole, immediately measuring the optical density (OD value) of each hole at the wavelength of 450nm by using an enzyme labeling instrument, and calculating the IGF-1 concentration of the sample according to the light absorption value of the standard GH protein. The detection results are shown in figure 6, and the results show that intravenous injection of GH and rGH-Fc can cause transient increase of IGF-1 level in rat serum.

Example 7 plasma half-life of rGH-Fc protein in rats

Male mature Sprague Dawley rats (about 7 weeks old, 130-g) were divided into 3 groups of 5 rats, and blood samples were taken after intravenous injection of equal volumes of physiological saline, 15nmol growth hormone water needle and 15nmol recombinant human growth hormone protein (rGH-Fc) for 0, 0.25, 1, 2, 4, 8, 12, 24, 32, 48, 72, 96, 144, 192h of single administration, and the GH content in rat sera were measured by ELISA, as follows: anti-GH antibody 4D9(1:1000) was first diluted with PBS and added to a 96-well plate at 100. mu.l per well and plated overnight at 4 ℃. TPBS is washed for 3 times, an oven is sealed for 1h at 37 ℃, after TPBS is washed for 3 times, the standard GH protein and the sample to be tested (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625ng/mL) which are diluted in a gradient manner are added, each well is 100 mu l, and the incubation is performed for 1h at room temperature with shaking. TPBS was washed 3 times, then 6A4-HRP (1:1000) was added at 100. mu.l per well, incubated for 1 hour at room temperature with shaking, TPBS was washed 3 times, OPD was added for color development, 1MH2SO4 was added after reaction for 10min at room temperature to stop the reaction, 100. mu.l per well, absorbance was measured at 490nm, and GH and rGH-Fc concentrations of the samples were calculated from the absorbance of the standard GH protein. The detection result is shown in figure 7, and the result shows that the frontal plasma half-life of the rGH-Fc in rats is longer than that of the GH.

Example 8 construction and screening of engineered cell lines highly expressing rGH-Fc protein

Constructing rGH-Fc optimized by nucleotides into a DN2 vector, transfecting the rGH-Fc into CHO cells after PvuI enzyme digestion linearization, pressurizing by using 50-100-200-500 nM MTX, paving by using 0.5cell/well, detecting the content of fusion protein in culture supernatant by using an ELISA method, screening to obtain 5 preferred primary subclones with relatively high expression quantity and good cell growth state, screening secondary subclones by using 0.3cell/wel as the primary subclones, and finally obtaining 1 preferred clone and 2 suboptimal clones after screening. The cell growth curve of the preferred clone at 14 days of culture is shown in FIG. 7, with rGH-Fc content in the supernatant > 2 g/L.

The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts of the present invention. The foregoing is only a preferred embodiment of the present invention, and it should be noted that there are objectively infinite specific structures due to the limited character expressions, and it will be apparent to those skilled in the art that a plurality of modifications, decorations or changes may be made without departing from the principle of the present invention, and the technical features described above may be combined in a suitable manner; such modifications, variations, combinations, or adaptations of the invention using its spirit and scope, as defined by the claims, may be directed to other uses and embodiments.

SEQUENCE LISTING

<110> Anhui Anke bioengineering (group) corporation

<120> development and application of recombinant human growth hormone Fc fusion protein and engineering cell strain thereof

<130> 2020

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 651

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of growth hormone

<400> 1

atggccaccg gcagccggac ctctctgctc ctggccttcg gactgctctg cctgccttgg 60

ctgcaggaag gcagcgcttt ccctacaatc cctctgagcc ggctgttcga taacgccatg 120

ctgagagccc acaggctgca ccagctggcc ttcgacacct accaggagtt cgaggaagcc 180

tacatcccta aggagcagaa gtacagcttc ctgcagaacc ctcagaccag cctgtgtttc 240

tctgagtcca tccctacccc aagcaaccgg gaggaaaccc agcagaagag caacctggag 300

ctgctcagaa tctccctgct cctgatccag agctggctgg agcctgtgca gttcctgcgg 360

agcgtgttcg ccaacagcct ggtgtacgga gccagcgaca gtaacgtgta cgacctgctc 420

aaggacctgg aggaaggcat ccagaccctg atgggcagac tggaggacgg cagccctcgg 480

acaggccaga tcttcaagca gacctacagc aagttcgaca caaacagcca caacgatgac 540

gccctgctca agaactacgg cctgctctac tgcttcagaa aggacatgga taaggtggaa 600

acattcctga gaatcgtgca gtgcaggtcc gtggaaggca gctgcggctt c 651

<210> 2

<211> 217

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of growth hormone

<400> 2

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

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

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe

210 215

<210> 3

<211> 699

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG1 Fc nucleotide sequence

<400> 3

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt ctcctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaatga 699

<210> 4

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG1 Fc amino acid sequence

<400> 4

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

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

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

35 40 45

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

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

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

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 5

<211> 687

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG2 Fc nucleotide sequence

<400> 5

gagaggaaat gctgtgtgga gtgccctccc tgtcctgctc ctcccgtggc tggacctagc 60

gtgttcctgt tccctcccaa gcctaaagac accctgatga tcagccggac ccctgaagtg 120

acatgcgtgg tcgtggacgt gtcccacgaa gaccctgagg tgcagttcaa ctggtacgtg 180

gatggcgtgg aagtgcacaa cgccaagaca aaacctcggg aggaacagtt caacagcacc 240

ttcagagtgg tgtccgtgct gaccgtggtc caccaggact ggttgaacgg caaggagtac 300

aaatgcaagg tgagcaacaa gggcctccct gctcccatcg agaagaccat ctccaagacc 360

aagggccagc ctcgggaacc tcaggtgtac actctgcctc caagcaggga ggaaatgacc 420

aagaatcagg tgtctctgac ctgcctcgtg aagggcttct accctagcga catttccgtg 480

gagtgggaat ctaacggaca gcctgagaac aattacaaga ccacacctcc catgcttgac 540

tccgacggca gcttctttct gtactctaag ctcaccgtgg acaaaagcag gtggcagcag 600

ggcaatgtgt tctcttgtag cgtcatgcac gaggccctgc acaaccatta cacccagaag 660

agcctgtccc tctctcctgg caagtga 687

<210> 6

<211> 228

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG2 Fc amino acid sequence

<400> 6

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

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

195 200 205

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

210 215 220

Ser Pro Gly Lys

225

<210> 7

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG4 Fc nucleotide sequence

<400> 7

gaatctaagt acggccctcc ctgccccagc tgtcctgccc ctgaatttct gggcggaccc 60

tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360

gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420

accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt tctgtattct cgcctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtccctgt ccctgtctct gggaaagtga 690

<210> 8

<211> 229

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 Fc amino acid sequence

<400> 8

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly Lys

225

<210> 9

<211> 1917

<212> DNA

<213> Artificial Sequence

<220>

<223> growth hormone receptor nucleotide sequence

<400> 9

atggatctct ggcagctgct gttgaccttg gcactggcag gatcaagtga tgctttttct 60

ggaagtgagg ccacagcagc tatccttagc agagcaccct ggagtctgca aagtgttaat 120

ccaggcctaa agacaaattc ttctaaggag cctaaattca ccaagtgccg ttcacctgag 180

cgagagactt tttcatgcca ctggacagat gaggttcatc atggtacaaa gaacctagga 240

cccatacagc tgttctatac cagaaggaac actcaagaat ggactcaaga atggaaagaa 300

tgccctgatt atgtttctgc tggggaaaac agctgttact ttaattcatc gtttacctcc 360

atctggatac cttattgtat caagctaact agcaatggtg gtacagtgga tgaaaagtgt 420

ttctctgttg atgaaatagt gcaaccagat ccacccattg ccctcaactg gactttactg 480

aacgtcagtt taactgggat tcatgcagat atccaagtga gatgggaagc accacgcaat 540

gcagatattc agaaaggatg gatggttctg gagtatgaac ttcaatacaa agaagtaaat 600

gaaactaaat ggaaaatgat ggaccctata ttgacaacat cagttccagt gtactcattg 660

aaagtggata aggaatatga agtgcgtgtg agatccaaac aacgaaactc tggaaattat 720

ggcgagttca gtgaggtgct ctatgtaaca cttcctcaga tgagccaatt tacatgtgaa 780

gaagatttct actttccatg gctcttaatt attatctttg gaatatttgg gctaacagtg 840

atgctatttg tattcttatt ttctaaacag caaaggatta aaatgctgat tctgccccca 900

gttccagttc caaagattaa aggaatcgat ccagatctcc tcaaggaagg aaaattagag 960

gaggtgaaca caatcttagc cattcatgat agctataaac ccgaattcca cagtgatgac 1020

tcttgggttg aatttattga gctagatatt gatgagccag atgaaaagac tgaggaatca 1080

gacacagaca gacttctaag cagtgaccat gagaaatcac atagtaacct aggggtgaag 1140

gatggcgact ctggacgtac cagctgttgt gaacctgaca ttctggagac tgatttcaat 1200

gccaatgaca tacatgaggg tacctcagag gttgctcagc cacagaggtt aaaaggggaa 1260

gcagatctct tatgccttga ccagaagaat caaaataact caccttatca tgatgcttgc 1320

cctgctactc agcagcccag tgttatccaa gcagagaaaa acaaaccaca accacttcct 1380

actgaaggag ctgagtcaac tcaccaagct gcccatattc agctaagcaa tccaagttca 1440

ctgtcaaaca tcgactttta tgcccaggtg agcgacatta caccagcagg tagtgtggtc 1500

ctttccccgg gccaaaagaa taaggcaggg atgtcccaat gtgacatgca cccggaaatg 1560

gtctcactct gccaagaaaa cttccttatg gacaatgcct acttctgtga ggcagatgcc 1620

aaaaagtgca tccctgtggc tcctcacatc aaggttgaat cacacataca gccaagctta 1680

aaccaagagg acatttacat caccacagaa agccttacca ctgctgctgg gaggcctggg 1740

acaggagaac atgttccagg ttctgagatg cctgtcccag actatacctc cattcatata 1800

gtacagtccc cacagggcct catactcaat gcgactgcct tgcccttgcc tgacaaagag 1860

tttctctcat catgtggcta tgtgagcaca gaccaactga acaaaatcat gccttag 1917

<210> 10

<211> 638

<212> PRT

<213> Artificial Sequence

<220>

<223> growth hormone receptor amino acid sequence

<400> 10

Met Asp Leu Trp Gln Leu Leu Leu Thr Leu Ala Leu Ala Gly Ser Ser

1 5 10 15

Asp Ala Phe Ser Gly Ser Glu Ala Thr Ala Ala Ile Leu Ser Arg Ala

20 25 30

Pro Trp Ser Leu Gln Ser Val Asn Pro Gly Leu Lys Thr Asn Ser Ser

35 40 45

Lys Glu Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe

50 55 60

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

65 70 75 80

Pro Ile Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln

85 90 95

Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys

100 105 110

Tyr Phe Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys

115 120 125

Leu Thr Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val Asp

130 135 140

Glu Ile Val Gln Pro Asp Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu

145 150 155 160

Asn Val Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu

165 170 175

Ala Pro Arg Asn Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr

180 185 190

Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp

195 200 205

Pro Ile Leu Thr Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys

210 215 220

Glu Tyr Glu Val Arg Val Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr

225 230 235 240

Gly Glu Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Gln

245 250 255

Phe Thr Cys Glu Glu Asp Phe Tyr Phe Pro Trp Leu Leu Ile Ile Ile

260 265 270

Phe Gly Ile Phe Gly Leu Thr Val Met Leu Phe Val Phe Leu Phe Ser

275 280 285

Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro

290 295 300

Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu

305 310 315 320

Glu Val Asn Thr Ile Leu Ala Ile His Asp Ser Tyr Lys Pro Glu Phe

325 330 335

His Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Glu

340 345 350

Pro Asp Glu Lys Thr Glu Glu Ser Asp Thr Asp Arg Leu Leu Ser Ser

355 360 365

Asp His Glu Lys Ser His Ser Asn Leu Gly Val Lys Asp Gly Asp Ser

370 375 380

Gly Arg Thr Ser Cys Cys Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn

385 390 395 400

Ala Asn Asp Ile His Glu Gly Thr Ser Glu Val Ala Gln Pro Gln Arg

405 410 415

Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn

420 425 430

Asn Ser Pro Tyr His Asp Ala Cys Pro Ala Thr Gln Gln Pro Ser Val

435 440 445

Ile Gln Ala Glu Lys Asn Lys Pro Gln Pro Leu Pro Thr Glu Gly Ala

450 455 460

Glu Ser Thr His Gln Ala Ala His Ile Gln Leu Ser Asn Pro Ser Ser

465 470 475 480

Leu Ser Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala

485 490 495

Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Met Ser

500 505 510

Gln Cys Asp Met His Pro Glu Met Val Ser Leu Cys Gln Glu Asn Phe

515 520 525

Leu Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile

530 535 540

Pro Val Ala Pro His Ile Lys Val Glu Ser His Ile Gln Pro Ser Leu

545 550 555 560

Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Ala Ala

565 570 575

Gly Arg Pro Gly Thr Gly Glu His Val Pro Gly Ser Glu Met Pro Val

580 585 590

Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Ile

595 600 605

Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser

610 615 620

Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro

625 630 635

<110> Anhui Anke bioengineering (group) corporation

<120> development and application of recombinant human growth hormone Fc fusion protein and engineering cell strain thereof

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 651

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of growth hormone

<400> 1

atggccaccg gcagccggac ctctctgctc ctggccttcg gactgctctg cctgccttgg 60

ctgcaggaag gcagcgcttt ccctacaatc cctctgagcc ggctgttcga taacgccatg 120

ctgagagccc acaggctgca ccagctggcc ttcgacacct accaggagtt cgaggaagcc 180

tacatcccta aggagcagaa gtacagcttc ctgcagaacc ctcagaccag cctgtgtttc 240

tctgagtcca tccctacccc aagcaaccgg gaggaaaccc agcagaagag caacctggag 300

ctgctcagaa tctccctgct cctgatccag agctggctgg agcctgtgca gttcctgcgg 360

agcgtgttcg ccaacagcct ggtgtacgga gccagcgaca gtaacgtgta cgacctgctc 420

aaggacctgg aggaaggcat ccagaccctg atgggcagac tggaggacgg cagccctcgg 480

acaggccaga tcttcaagca gacctacagc aagttcgaca caaacagcca caacgatgac 540

gccctgctca agaactacgg cctgctctac tgcttcagaa aggacatgga taaggtggaa 600

acattcctga gaatcgtgca gtgcaggtcc gtggaaggca gctgcggctt c 651

<210> 2

<211> 217

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of growth hormone

<400> 2

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu

20 25 30

Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln

35 40 45

Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp

100 105 110

Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val

115 120 125

Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu

130 135 140

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

145 150 155 160

Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser

165 170 175

His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe

180 185 190

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys

195 200 205

Arg Ser Val Glu Gly Ser Cys Gly Phe

210 215

<210> 3

<211> 687

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG4 Fc variant 1 nucleotide sequence

<400> 3

gaatctaagt acggccctcc ctgcccccct tgtcctgccc ctgaagccgc tggcggaccc 60

tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360

gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420

accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt tctgtattct aagctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtccctgt ccctgtctct gggatag 687

<210> 4

<211> 228

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 Fc variant 1 amino acid sequence

<400> 4

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 5

<211> 687

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG4 Fc variant 2 nucleotide sequence

<400> 5

gaatctaagt acggccctcc ctgcccccct tgtcctgccc ctcctgtggc tggcggaccc 60

tctgtgtttc tgttcccccc aaagcctaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtgtccaa caagggcctg cccagctcca tcgaaaagac catcagcaag 360

gccaagggcc agccccggga accccaggtg tacacactgc ctccaagcca ggaagagatg 420

accaagaatc aggtgtccct gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt tctgtattct cgcctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tgttctcctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtccctgt ccctgtctct gggatag 687

<210> 6

<211> 228

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 Fc variant 2 amino acid sequence

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 7

<211> 1917

<212> DNA

<213> Artificial Sequence

<220>

<223> growth hormone receptor nucleotide sequence

<400> 7

atggatctct ggcagctgct gttgaccttg gcactggcag gatcaagtga tgctttttct 60

ggaagtgagg ccacagcagc tatccttagc agagcaccct ggagtctgca aagtgttaat 120

ccaggcctaa agacaaattc ttctaaggag cctaaattca ccaagtgccg ttcacctgag 180

cgagagactt tttcatgcca ctggacagat gaggttcatc atggtacaaa gaacctagga 240

cccatacagc tgttctatac cagaaggaac actcaagaat ggactcaaga atggaaagaa 300

tgccctgatt atgtttctgc tggggaaaac agctgttact ttaattcatc gtttacctcc 360

atctggatac cttattgtat caagctaact agcaatggtg gtacagtgga tgaaaagtgt 420

ttctctgttg atgaaatagt gcaaccagat ccacccattg ccctcaactg gactttactg 480

aacgtcagtt taactgggat tcatgcagat atccaagtga gatgggaagc accacgcaat 540

gcagatattc agaaaggatg gatggttctg gagtatgaac ttcaatacaa agaagtaaat 600

gaaactaaat ggaaaatgat ggaccctata ttgacaacat cagttccagt gtactcattg 660

aaagtggata aggaatatga agtgcgtgtg agatccaaac aacgaaactc tggaaattat 720

ggcgagttca gtgaggtgct ctatgtaaca cttcctcaga tgagccaatt tacatgtgaa 780

gaagatttct actttccatg gctcttaatt attatctttg gaatatttgg gctaacagtg 840

atgctatttg tattcttatt ttctaaacag caaaggatta aaatgctgat tctgccccca 900

gttccagttc caaagattaa aggaatcgat ccagatctcc tcaaggaagg aaaattagag 960

gaggtgaaca caatcttagc cattcatgat agctataaac ccgaattcca cagtgatgac 1020

tcttgggttg aatttattga gctagatatt gatgagccag atgaaaagac tgaggaatca 1080

gacacagaca gacttctaag cagtgaccat gagaaatcac atagtaacct aggggtgaag 1140

gatggcgact ctggacgtac cagctgttgt gaacctgaca ttctggagac tgatttcaat 1200

gccaatgaca tacatgaggg tacctcagag gttgctcagc cacagaggtt aaaaggggaa 1260

gcagatctct tatgccttga ccagaagaat caaaataact caccttatca tgatgcttgc 1320

cctgctactc agcagcccag tgttatccaa gcagagaaaa acaaaccaca accacttcct 1380

actgaaggag ctgagtcaac tcaccaagct gcccatattc agctaagcaa tccaagttca 1440

ctgtcaaaca tcgactttta tgcccaggtg agcgacatta caccagcagg tagtgtggtc 1500

ctttccccgg gccaaaagaa taaggcaggg atgtcccaat gtgacatgca cccggaaatg 1560

gtctcactct gccaagaaaa cttccttatg gacaatgcct acttctgtga ggcagatgcc 1620

aaaaagtgca tccctgtggc tcctcacatc aaggttgaat cacacataca gccaagctta 1680

aaccaagagg acatttacat caccacagaa agccttacca ctgctgctgg gaggcctggg 1740

acaggagaac atgttccagg ttctgagatg cctgtcccag actatacctc cattcatata 1800

gtacagtccc cacagggcct catactcaat gcgactgcct tgcccttgcc tgacaaagag 1860

tttctctcat catgtggcta tgtgagcaca gaccaactga acaaaatcat gccttag 1917

<210> 8

<211> 638

<212> PRT

<213> Artificial Sequence

<220>

<223> growth hormone receptor amino acid sequence

<400> 8

Met Asp Leu Trp Gln Leu Leu Leu Thr Leu Ala Leu Ala Gly Ser Ser

1 5 10 15

Asp Ala Phe Ser Gly Ser Glu Ala Thr Ala Ala Ile Leu Ser Arg Ala

20 25 30

Pro Trp Ser Leu Gln Ser Val Asn Pro Gly Leu Lys Thr Asn Ser Ser

35 40 45

Lys Glu Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe

50 55 60

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

65 70 75 80

Pro Ile Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln

85 90 95

Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys

100 105 110

Tyr Phe Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys

115 120 125

Leu Thr Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser Val Asp

130 135 140

Glu Ile Val Gln Pro Asp Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu

145 150 155 160

Asn Val Ser Leu Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu

165 170 175

Ala Pro Arg Asn Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr

180 185 190

Glu Leu Gln Tyr Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp

195 200 205

Pro Ile Leu Thr Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys

210 215 220

Glu Tyr Glu Val Arg Val Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr

225 230 235 240

Gly Glu Phe Ser Glu Val Leu Tyr Val Thr Leu Pro Gln Met Ser Gln

245 250 255

Phe Thr Cys Glu Glu Asp Phe Tyr Phe Pro Trp Leu Leu Ile Ile Ile

260 265 270

Phe Gly Ile Phe Gly Leu Thr Val Met Leu Phe Val Phe Leu Phe Ser

275 280 285

Lys Gln Gln Arg Ile Lys Met Leu Ile Leu Pro Pro Val Pro Val Pro

290 295 300

Lys Ile Lys Gly Ile Asp Pro Asp Leu Leu Lys Glu Gly Lys Leu Glu

305 310 315 320

Glu Val Asn Thr Ile Leu Ala Ile His Asp Ser Tyr Lys Pro Glu Phe

325 330 335

His Ser Asp Asp Ser Trp Val Glu Phe Ile Glu Leu Asp Ile Asp Glu

340 345 350

Pro Asp Glu Lys Thr Glu Glu Ser Asp Thr Asp Arg Leu Leu Ser Ser

355 360 365

Asp His Glu Lys Ser His Ser Asn Leu Gly Val Lys Asp Gly Asp Ser

370 375 380

Gly Arg Thr Ser Cys Cys Glu Pro Asp Ile Leu Glu Thr Asp Phe Asn

385 390 395 400

Ala Asn Asp Ile His Glu Gly Thr Ser Glu Val Ala Gln Pro Gln Arg

405 410 415

Leu Lys Gly Glu Ala Asp Leu Leu Cys Leu Asp Gln Lys Asn Gln Asn

420 425 430

Asn Ser Pro Tyr His Asp Ala Cys Pro Ala Thr Gln Gln Pro Ser Val

435 440 445

Ile Gln Ala Glu Lys Asn Lys Pro Gln Pro Leu Pro Thr Glu Gly Ala

450 455 460

Glu Ser Thr His Gln Ala Ala His Ile Gln Leu Ser Asn Pro Ser Ser

465 470 475 480

Leu Ser Asn Ile Asp Phe Tyr Ala Gln Val Ser Asp Ile Thr Pro Ala

485 490 495

Gly Ser Val Val Leu Ser Pro Gly Gln Lys Asn Lys Ala Gly Met Ser

500 505 510

Gln Cys Asp Met His Pro Glu Met Val Ser Leu Cys Gln Glu Asn Phe

515 520 525

Leu Met Asp Asn Ala Tyr Phe Cys Glu Ala Asp Ala Lys Lys Cys Ile

530 535 540

Pro Val Ala Pro His Ile Lys Val Glu Ser His Ile Gln Pro Ser Leu

545 550 555 560

Asn Gln Glu Asp Ile Tyr Ile Thr Thr Glu Ser Leu Thr Thr Ala Ala

565 570 575

Gly Arg Pro Gly Thr Gly Glu His Val Pro Gly Ser Glu Met Pro Val

580 585 590

Pro Asp Tyr Thr Ser Ile His Ile Val Gln Ser Pro Gln Gly Leu Ile

595 600 605

Leu Asn Ala Thr Ala Leu Pro Leu Pro Asp Lys Glu Phe Leu Ser Ser

610 615 620

Cys Gly Tyr Val Ser Thr Asp Gln Leu Asn Lys Ile Met Pro

Claims (15)

1. The recombinant human growth hormone Fc fusion protein is characterized by consisting of human growth hormone, a connecting peptide and an Fc fragment, wherein the fusion protein sequentially comprises from N end to C end: the fusion protein comprises human growth hormone, connecting peptide and Fc fragment, or the Fc fragment, the connecting peptide and the human growth hormone are sequentially arranged from the N end to the C end of the fusion protein.

2. The recombinant human growth hormone Fc fusion protein according to claim 1, wherein the nucleotide sequence encoding the human growth hormone gene is as set forth in SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2, respectively.

3. The recombinant human growth hormone Fc fusion protein of claim 1, wherein the linker peptide is a flexible linker peptide consisting of amino acids having the general structural formula (GGGGS) nA, wherein n is an integer greater than or equal to 2.

4. A recombinant human growth hormone Fc fusion protein according to claim 1, wherein the Fc fragment is a human IgG Fc variant.

5. A recombinant human growth hormone Fc fusion protein according to claim 4, wherein the human IgG Fc variant is selected from one or more of the group consisting of:

1) IgG1 Fc containing at least the following two mutations: leu234Ala, Leu235Ala, Asn297Ala, Val308Pro and deletion of C-terminal Lys, the nucleotide sequence of the IgG1 Fc is as set forth in SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4 is shown in the specification;

2) IgG2 Fc containing at least the following two mutations: asn297Ala, Val308Pro, Pro331Ser, and deletion of C-terminal Lys, the nucleotide sequence of IgG2 Fc is set forth in SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6 is shown in the specification;

3) IgG4 Fc containing at least the following two mutations: at least the hinge region, CH2 and CH3 region of human IgG4 containing Ser228Pro, Glu233Pro, Leu234Ala or Leu234Val, Leu235Ala, Asn297Ala, Val308Pro and Arg409Lys and deleting C-terminal Lys, the nucleotide sequence of IgG4 Fc is as shown in SEQ ID NO: 7, and the amino acid sequence is shown as SEQ ID NO: shown in fig. 8.

6. A polynucleotide encoding the amino acid of the recombinant human growth hormone Fc fusion protein according to any one of claims 1 to 5.

7. A vector or set of vectors comprising the polynucleotide of claim 6.

8. A host cell comprising the vector or set of vectors of claim 7, wherein the host cell is prokaryotic or eukaryotic and is one or more of a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof.

9. A method for screening positive clones and subclones expressing the fusion protein of any one of claims 1 to 5, wherein the concentration of MTX is gradually increased from 96 plates → 24 plates → 6 plates → 25cm²And (3) a culture flask → 125ml shake flask, gradually screening cell strains by adopting an ELISA method, and naming the finally screened cell strains as rhGH-Fc/Fc-rGH cell strains, wherein the expressed target protein is rhGH-Fc/Fc-rGH recombinant protein.

10. A pharmaceutical composition comprising the recombinant human growth hormone Fc fusion protein of any one of claims 1 to 5, or comprising the vector or set of vectors of claim 7, and a pharmaceutically acceptable carrier.

11. A kit comprising a recombinant human growth hormone Fc fusion protein according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 10.

12. A method of producing a recombinant human growth hormone Fc fusion protein, comprising culturing the host cell of claim 8 under conditions in which the polynucleotide is expressed.

13. Use of a recombinant human growth hormone Fc fusion protein according to any one of claims 1 to 5, wherein the fusion protein induces a prolonged IGF-1 response.

14. A growth hormone receptor capable of binding to the recombinant human growth hormone Fc fusion protein of any one of claims 1 to 5 and activating the JAK2-STATs signaling pathway, wherein the nucleotide sequence encoding the growth hormone receptor is as set forth in SEQ ID NO: 9, and the amino acid sequence is shown as SEQ ID NO: shown at 10.

15. Use of the recombinant human growth hormone Fc fusion protein according to any one of claims 1 to 5 or the host cell according to claim 8 or a population of strains comprising the host cell according to claim 8 for increasing serum half-life, reducing the frequency of injections required over a treatment period, increasing patient compliance and convenience for both the physician and the patient.

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