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

Abstract

The present invention relates to the technical field of genetic engineering, in particular to the technical field of gene recombinant long-acting human growth hormone, in particular to a long-acting recombinant human growth hormone fusion protein and engineering cells thereof. The long-acting recombinant human growth hormone fusion protein rhGH- Fc can prolong the serum half-life, increase the biological activity of the protein in the body, reduce the immunogenicity of the protein, thereby improving the efficacy of the drug, greatly reducing the frequency of injections required during the treatment period, thereby improving patient compliance and increasing the number of doctors and patients. convenience; the engineered cells expressing rhGH-Fc constructed by the present invention have the characteristics of high protein expression and good biological activity, and are especially suitable for large-scale industrial production of recombinant long-acting human growth hormone.

Description

A long-acting recombinant human growth hormone fusion protein and its engineered cells

technical field

The invention relates to the technical field of genetic engineering, in particular to the technical field of gene recombinant long-acting human growth hormone, in particular to a long-acting recombinant human growth hormone rhGH-Fc fusion protein, its engineering cells, and its construction and screening methods.

Background technique

Human Growth Hormone (hGH) is the most important protein hormone for human growth after birth. It has species specificity and is secreted by eosinophils in the anterior lobe of the human pituitary. The body spreads all over the body, rather than producing physiological effects through the action of target glands. Human growth hormone contains 191 amino acid molecules, most of which exist in the form of a molecular weight of 22KDa, an isoelectric point of 4.9, between the cysteine at the 53rd position and the cysteine at the 165th position and at the 182nd position There are two disulfide bonds between cysteine and cysteine 189, and a small amount of growth hormone exists in the form of 20KDa. The main physiological function of growth hormone is to promote human metabolism, growth and development by promoting protein synthesis and fat decomposition of recipient cells in various tissues, organs and bones of the body.

Insufficient secretion of growth hormone will lead to growth stagnation and various body dysfunctions, and excessive secretion will cause adverse reactions, such as "acromegaly" and "giantism". The most obvious effect of human growth hormone is to enhance protein synthesis, enhance collagen synthesis, and promote bone tissue development. Although hGH is very important to maintain bone growth, growth hormone does not play a direct role in bone growth. It is achieved by stimulating the sulfur factor (SM) in the blood. SM can promote the growth of various tissue cells and mother cells in the body. split. Human growth hormone can also promote the entry of amino acids into cells, reduce the discharge of ammonia and nitrogen-containing substances, and maintain the balance of the body's metabolic state. Human growth hormone can also promote the decomposition of fat, inhibit the oxidation of glucose, slow down the consumption of glucose, and stabilize glycogen. At present, growth hormone is still a very complex hormone, and many functions have yet to be studied.

Human growth hormone is mainly used clinically for children with growth hormone deficiency, Tuner syndrome, and to promote the healing of large burn wounds. Currently in China, the incidence of stunted growth and development among children is as high as 9.9%, ranking second in the world. According to the statistics of the Chinese Medical Association, the total number of short children aged 4 to 15 in the country is about 7 million at present. According to the calculation that 1/3 of them are growth hormone deficiency, there are about 2 million children who need growth hormone treatment in China, but they can The number of people receiving reasonable treatment is still less than 30,000. During the long-term follow-up of the patients, it was found that about 23% of children missed more than three injections every month; nearly 13% of children missed more than half of the prescribed dose; clinical evidence showed that patients who did not strictly abide by the treatment plan Up to 50% or more. The main reason is that the current generic recombinant human growth hormone requires a daily injection to maintain proper serum growth hormone levels. Patients are very painful, treatment compliance is poor, and the price is expensive. For those patients who need to receive growth hormone therapy for a long time, the therapeutic effect is often reduced due to failure to inject on time. At present, in China, there are mainly 2 approved indications for recombinant human growth hormone, most of which are used to treat children's "short stature". Somatropin has great potential in the future market.

At present, the expression systems used to express protein drugs are divided into two categories. The earliest use is the prokaryotic expression system, mainly Escherichia coli. The other is the eukaryotic expression system, and insect, yeast, and mammalian cells all belong to this type of expression system. Although Escherichia coli cells have good operability and relatively low cost, they often cannot carry out correct glycosylation modification, and inclusion bodies are easily formed in the cells. At present, most domestic companies use E. coli secretion expression technology to produce growth hormone. The protein contains endotoxin, which requires cumbersome purification steps to remove, resulting in a low yield of the final product. Since E. coli is a prokaryotic expression vector, the activity of the target protein Also lower. CHO cells (hamster ovary cells, Chinese Hamster Ovary) are eukaryotic cells that have functions of transcriptional modification and post-translational modification of proteins. The conformation, molecular structure, physical and chemical properties, and biological functions of the expressed target protein are very similar to natural proteins. near. CHO cells are a type of fibroblasts, and their own endogenous protein secretion is very small, which is very beneficial for the separation and purification of recombinant proteins in the later stage; in terms of culture, CHO cells not only have the characteristics of adherent growth, but if domesticated, they can also Suspension culture can be carried out, and the ability to withstand large shear force and osmotic pressure is better; serum-free culture can improve the purity of the product, facilitate the separation and purification of the product, and at the same time reduce the pollution and allergens caused by serum, and also It can prolong the G1 phase or G0 phase of the cells, thereby increasing the expression of the target protein, and also facilitates the large-scale industrial production in the later stage.

At present, the long-acting growth hormone commercialized in China is PEGylated growth hormone. PEGylated growth hormone can increase the molecular weight of the protein, make it difficult to pass through the glomerulus, reduce the clearance rate, and prevent the enzymatic hydrolysis of proteases, thereby prolonging the half-life of the drug in the body , but the protein PEGylation process will lose biological activity.

At present, the hottest and fastest-growing long-acting protein drug technology is Fc fusion protein technology. The Fc fragment is the main factor for IgG to maintain a long half-life in vivo, and can stabilize the protein at the same time. It has been used in the development of various fusion proteins. Fc fragments function through two mechanisms, the first is binding to Fc receptors on the cell surface, killing cells through phagocytosis or lysis and digesting pathogens through the antibody-dependent cellular cytotoxicity (ADCC) pathway. Among the four subtypes of human IgG, IgG1 and IgG3 can effectively bind to Fc receptors on the cell surface, while IgG2 and IgG4 bind to the receptors with lower affinity. The second is binding to C1q of the first complement component C1, which lyses pathogens through the complement-dependent cytotoxicity (CDC) pathway. Human IgG1 and IgG3 can effectively bind C1q to mediate the complement cascade reaction, human IgG2 has a weak binding ability to C1q, and human IgG4 does not bind to C1q. The main purpose of Fc fusion protein is to increase the half-life of protein drugs in the body and simplify the production process to reduce costs. The Fc-mediated IgG effect and the function of lysing cells are not necessary, and may even cause side effects. Based on the above reasons, the IgG4 subtype was selected and carried out. Corresponding amino acid mutations to attenuate the function of its effector molecules. Fc fusion protein technology not only increases the molecular weight of protein drugs and reduces the clearance rate like PEGylation, but also prolongs the half-life in a slow-release manner through the FcRn binding domain and pH-sensitive binding force. Many current studies have shown that Fc fusion proteins have a longer half-life than PEGylated drugs.

Specific to human growth hormone, there have been several studies on its fusion Fc fragment. Taking CN 102875683B as an example, it discloses a long-acting recombinant human growth hormone Fc fusion protein hGH-L-vFc fusion protein, which contains human growth hormone , a flexible peptide linker of about 2-20 amino acids, and a human IgG Fc variant, which may be selected from the human IgG4 hinge region, CH2 and CH3 regions containing S228P and L235A mutations. However, in the above several studies, the effect of prolonging the half-life of human growth hormone is not satisfactory.

Therefore, there is still a further need to develop more long-acting, highly active human growth hormone derivatives.

Contents of the invention

In order to solve the deficiencies in the prior art, the present invention provides a long-acting recombinant human growth hormone rhGH-Fc fusion protein, the fusion protein is composed of human growth hormone, connecting peptide and Fc fragment; from N-terminal to C-terminal are : Human growth hormone, connecting peptide, Fc fragment, its amino acid sequence has a total of 431 amino acids, of which the first 191 amino acid sequences are human growth hormone sequences, the 192nd to 206th are connecting peptide sequences, and the 207th to 431st Fc fragment of human IgG.

Further, the Fc fragment is selected from the Fc fragment of human IgG, preferably the Fc fragment of human IgG4.

Further, the Fc fragment of human IgG4 includes four amino acid mutations, the amino acid mutations are: S228P, F234A, L235A and V308P, wherein the numbering of the residues in the IgG Fc region is based on Kabat Eu Index numbering. Among them, the S228P site mutation is to maintain the stability of the protein; the F234A and L235A site mutations are to reduce the affinity between the immune fusion protein and the Fc receptor so as to reduce the ADCC activity of the immune fusion protein; and the V308P site mutation makes The combination of the fusion protein and FcRn is pH-dependent, so the protein can be pH-dependent by increasing the molecular weight of the protein so as not to be filtered by the glomerulus and the mutation of the V308P site, so that the long-acting recombinant human growth hormone rhGH-Fc The half-life of the immune fusion protein is higher than that of common Fc fusion protein drugs.

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

The present invention also provides an engineered cell expressing the aforementioned fusion protein, more preferably, the engineered cell is a CHO-k1 cell capable of stably transfecting the expression vector of the aforementioned fusion protein.

Further, the engineered cells are constructed by the following steps:

(1) First, the human antibody IgG4 Fc sequence (accession number: DI488112) was mutated at a specific site and connected to the hGH gene (accession number: NM_000515) with a flexible linker peptide, and the growth hormone signal peptide sequence (accession number : CAI94177), the whole gene synthetic rhGH-Fc sequence;

(2) Digest the double expression plasmid p327.8GS twice with restriction endonucleases HindⅢ, EcoRI and XbaI, XhoI, and mix the recovered large fragment with the rhGH-Fc sequence synthesized in step (1) with T4DNA ligase Evenly, connect overnight at 15-16°C;

(3) Prepare competent Escherichia coli DH5α by the CaCl ₂ method, and then carry out plasmid transformation, culture and screen on LB plates containing ampicillin, pick positive colonies and inoculate them into LB liquid medium, and extract the plasmid after 14-16 hours of low-speed shaking culture ; The plasmid was identified and sequenced by HindⅢ, EcoRI, XbaⅠ, and XhoⅠ, and the recombinant plasmid with the correct gene sequence identification was named rhGH-Fc-1;

(4) The cells are inoculated in the retort flask of complete medium with serum, and when the cell density reaches 90-95%, the liposome transfection method is adopted, and under the mediation of Lipofectamin 2000, the step (3) The prepared recombinant plasmid rhGH-Fc-1 was stably transfected into CHO-k1 cells;

(5) Transfer the cells transfected in step (4) into a 96-well plate, add SMM-CHO GSI medium (containing 10-12% FBS+25-30 μmol MSX) and mix well, and place in a CO ₂ incubator for 35- After 10-12 days in a constant temperature incubator at 37°C, mark the single clone under a microscope;

(6) Gradually increase the MSX concentration and use the ELISA method to gradually screen high-expression strains, from 96-plate (25-30 μmol MSX) → 24-well plate (50-55 μmol MSX) → 6-well plate (50-55 μmol MSX) → 75cm2 culture flask (70-75 μmol MSX) → 125ml shake flask (70-75 μmol MSX), select the cell line with higher expression level and better proliferation state for subcloning screening, and name the final screened cell line as rhGH-Fc cell line, expressing The target protein is rhGH-Fc immune fusion protein.

The present invention also provides an expression vector, the expression vector is preferably a double expression vector, more preferably, the expression vector is a double expression plasmid p327. After two double digestions, mix the recovered large fragment with the nucleic acid encoding the fusion protein according to claim 1 with T4 DNA ligase, and connect overnight at 15-16°C, so that the optimized target gene is cloned into the double expression vector and get.

The present invention also provides a screening method for positive clones and subclones of fusion proteins. The method is as follows: gradually increase the concentration of methionine sulfonamide (MSX), from 96 plates (25-30 μmol MSX) → 24-well plates (50-30 μmol MSX). 55μmol MSX) → 6-well plate (50-55μmol MSX) → 75cm2 culture flask (70-75μmol MSX) → 125ml shake flask (70-75μmol MSX), use ELISA method to gradually screen out cells with higher expression and better proliferation strain, the final screened cell strain was named rhGH-Fc cell strain, and the target protein expressed was rhGH-Fc immune fusion protein.

Further, the fusion protein can prolong the serum half-life, greatly reduce the frequency of injections required during treatment, thereby improving patient compliance and convenience for both doctors and patients; the CHO cells can be used to prepare The recombinant human long-acting growth hormone has high protein expression, good biological activity, reduces protein immunogenicity, and can improve drug efficacy.

Beneficial effect

The rhGH-Fc fusion protein of the present invention increases the molecular weight of human growth hormone so as not to be filtered by the glomerulus by constructing a fusion protein of human growth hormone and Fc fragment, and improves the stability of the Fc fragment through the amino acid mutation of the Fc fragment, Reducing the ADCC activity of the fusion protein and improving the pH dependence of the binding of the fusion protein to FcRn, combined with the above effects, further increases the half-life of the fusion protein compared to common Fc fusion protein drugs.

The rhGH-Fc fusion protein of the present invention has significantly prolonged serum half-life and high biological activity, can greatly reduce the frequency of injection and administration required within the treatment period, and can improve patient compliance and convenience for both doctors and patients.

The engineered cells of the present invention use CHO-k1 cells as host cells, and through stable transfection with GS double expression vectors, the obtained engineered cells have high protein expression, good biological activity, reduced protein immunogenicity, and can significantly improve drug efficacy .

Description of drawings

Figure 1: Plasmid map of the p327.8GS dual expression vector.

Figure 2: Sequencing map of the plasmid.

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

Figure 4: ELISA results of culture supernatants in 125ml shake flasks.

Figure 5: ELISA results of culture supernatant in cell line D 75cm2.

Figure 6: ELISA results of culture supernatant in cell line E 75cm2.

Figure 7: ELISA results of culture supernatant in cell line H 75cm2.

Figure 8: ELISA results of culture supernatants of D1, E2, and H2 in 125ml shake flasks of subclones.

Figure 9: Growth curves of D1, E2, H2 cells in subcloned 125ml shake flasks.

Figure 10: SDS-PAGE profile of the target protein, 1: non-reduced rhGH-Fc protein; 2: protein marker; M: reduced rhGH-Fc protein.

Figure 11: Isofocus electrophoresis of target protein, 1: rhGH standard; M1: HighpIKit, pH 5-10.5; M2: BroadpIKit, pH 3-10; 2: rhGH-Fc protein.

Figure 12: HPLC profile of the supernatant of the cell culture fluid of the rhGH-Fc immunofusion protein.

Figure 13: HPLC profile of rhGH-Fc immunofusion protein.

Fig. 14: Trend chart of body weight growth of rats in each group after administration.

Detailed ways

Hereinafter, the present invention is described in more detail to facilitate understanding of the present invention.

It should be understood that the terms or words used in the specification and claims should not be understood as having the meaning defined in the dictionary, but should be understood as having the meaning consistent with the meaning in the context of the present invention on the basis of the following principles Meaning: The concepts of the terms may be appropriately defined by the inventor for the best description of the invention.

For the experimental methods that do not indicate specific conditions in the following examples, generally follow conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual, or according to the conditions suggested by the manufacturer.

Example 1: Design and synthesis of rhGH-Fc fusion protein

The human antibody IgG4 Fc sequence (DI488112) was mutated at a specific site and then connected to the hGH gene (NM_000515) with a flexible Linker to obtain a mature rhGH-Fc, and the growth hormone signal peptide sequence (CAI94177) was added in front of the hGH sequence to obtain a rhGH-Fc carrying a signal Peptide rhGH-Fc. Whole gene synthesis of mature rhGH-Fc and rhGH-Fc carrying signal peptide, its amino acid sequence is shown in SEQ ID NO: 1; whole gene synthesis of mature rhGH-Fc coding gene, its nucleotide sequence is shown in SEQ ID NO: 2 shown.

SEQ ID NO 1:

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

FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQ NPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLR SVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFK QTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSV EGSCGFSSASTKGPSVFPLAPGPPCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTPLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK

SEQ ID NO 2:

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

TTCCCCACCATTCCTCTGTCCAGGCTGTTCGACAACGCCATGCT GAGGGCCCACAGGCTGCATCAACTGGCCTTCGACACCTACCAG GAGTTCGAGGAAGCCTACATCCCTAAGGAGCAGAAATACTCCTT CCTGCAGAACCCCCAGACTTCTCTGTGCTTCTCCGAGTCCATCC CAACCCCCTCCAACAGGGAGGAAACCCAACAGAAGTCCAACCT GGAACTGCTGAGGATCTCTCTGCTGCTGATTCAGTCCTGGCTGG AGCCCGTGCAATTCCTGAGGTCTGTGTTCGCAAACTCCCTGGTG TACGGCGCCTCCGACTCCAACGTGTACGATCTGCTGAAGGACCT GGAGGAGGGAATCCAGACACTGATGGGCAGGCTGGAAGACGG CTCCCCAAGGACCGGCCAAATCTTCAAGCAGACCTACTCCAAGT TTGACACCAACTCCCACAACGATGACGCCCTGCTGAAAAACTAC GGACTGCTGTACTGCTTTAGGAAAGATATGGACAAAGTGGAGAC CTTTCTGAGGATTGTGCAGTGCAGGTCCGTGGAGGGCTCCTGC GGCTTCTCCTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCT GGCCCCTGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGgccgcc GGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACAC CCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTG GACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGA GGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACC CCCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCA AGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCAT CTCCAAGGCCAAGGGCCAG CCTCGCGAGCCTCAGGTGTACACC CTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCGGTGTCCCT GACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTG GAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCA CCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCC CGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGT TCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGTCCCTGTCCCTGGGCAAGT

According to the amino acid sequence shown in SEQ ID NO: 1, the mature rhGH-Fc amino acid sequence has a total of 431 amino acids, of which the first 191 amino acids are the human growth hormone sequence, "SSASTKGPSVFPLAP" is the connecting peptide Linker, and G207-K431 is the human IgG4 The Fc fragment has four amino acid mutations of S228P, F234A, L235A and V308P (according to the Eu code of Kabat).

The mutation of S228P is to maintain the stability of the protein, IgG4 sometimes dissociates the disulfide bond between the heavy chains in the hinge region to form a half-antibody molecule, which usually does not happen in the other three IgG isotype molecules . Position 228 in IgG1 and IgG2 is Pro, while position 228 in IgG4 is Ser. Substituting the Ser residue of IgG4 with Pro can keep IgG4 an intact antibody molecule. F234A, L235A mutation is to reduce the affinity of immune fusion protein and Fc receptor so as to reduce the ADCC activity of immune fusion protein. The V308P mutation in the Fc fragment makes the binding of the fusion protein to FcRn pH-dependent. When biological endocytosis occurs, that is, when the pH is about 6, the fusion protein has a higher affinity with FcRn, which can avoid the degradation of lysosome. It is normally combined with FcRn at normal pH7.4, so that the half-life of the long-acting growth hormone is higher than that of ordinary Fc fusion protein drugs.

Example 2: Construction of recombinant expression plasmid containing rhGH-Fc fusion protein coding gene

(1) Connection of target gene sequence and GS double expression vector

Take 8.0 μl double expression plasmid p327.8GS (its plasmid map is shown in Figure 1) and mix with 0.5 μl HindⅢ, 0.5 μl EcoRI, 2.0 μl Cut Smart buffer, 9.0 μl ddH ₂ O, 0.5 μl XbaⅠ, 0.5 μl XhoⅠ, 2.0 μl Cut Smart buffer, 9.0 μl ddH ₂ O and two enzyme digestion systems were mixed in an EP tube, and placed in a 37°C incubator for 2 hours of reaction for double enzyme digestion. Then add 5 μl of plasmid GS, 5 μl of rhGH-Fc gene sequence carrying signal peptide, 2.0 μl of Buffer, and 7.0 μl of ddH ₂ O into the EP tube and mix well, add 1.0 μl of melted T4 DNA ligase to the mixed system, mix well, and keep at 16°C Connect overnight.

(2) Transformation and extraction of plasmids

Escherichia coli DH5α and LB medium were inoculated in liquid LB medium (without resistance) at a ratio of 1:100 by volume, and cultured overnight at a constant temperature of 37°C with slight shaking. The next day, inoculate the bacterial solution after shaking overnight in LB medium (without resistance) at a volume ratio of 1:50, and continue to cultivate until the OD600 is 0.3-0.5. Place the cultured Escherichia coli on ice for 10 min, mix well, transfer to a 1.5 ml centrifuge tube, centrifuge at 4000 rpm for 5 min at 4 °C, and discard the supernatant. Accurately draw 100 μl of pre-cooled 0.1 mol CaCl ₂ solution with a pipette gun and transfer it to a centrifuge tube, flick it evenly with your fingers and place it on ice for 10 minutes. Centrifuge at 3000 rpm at 4°C for 5 min at low temperature, and discard the supernatant. Use a pipette gun to accurately draw 50 μl of pre-cooled 0.1mol CaCl ₂ solution into the centrifuge tube and mix well.

Take 100 μl of competent Escherichia coli DH5α and 1 μl of the plasmid obtained in step (1), mix them in an EP tube, and put them in an ice bath for 30 minutes. The remaining competent cells should be stored at -80°C. Then put the EP tube in a warm water bath at 42°C for 90 seconds, take it out immediately, and then ice-bath for 2 minutes. Add 200 μl of LB culture medium without resistance, and incubate at a constant temperature of 37° C. with low-speed shaking for 50 minutes. Centrifuge at 2000 rpm for 1 min, discard 200 μl of supernatant, and mix the remaining solution evenly. Spread the liquid evenly on the LB plate containing ampicillin resistance (Amp), and incubate in a constant temperature incubator at 37°C for about 16 hours. Use an inoculation needle to pick up positive colonies and inoculate them into 5ml LB liquid medium, culture with low-speed shaking at 200rpm for 14h, and prepare to extract plasmids.

Then take 5ml of the bacterial solution into a centrifuge tube, centrifuge at 7000rpm for 3min to collect the bacteria, and discard the supernatant. Add 500 μl Buffer P1 containing RNaseA to the centrifuge tube and resuspend fully. Then add 500 μl Buffer P2 to the centrifuge tube, gently invert it up and down 5-6 times, and let it stand at room temperature for 5 minutes until the solution becomes clear and viscous. Add 500μl Buffer E3 to the centrifuge tube, immediately invert 6-8 times, place at room temperature for 5 minutes, centrifuge at 1300rpm for 10min, add the supernatant to the collection tube that has been added with Endo-Remover Column, centrifuge at 1300rpm for 1min, and remove the filtrate in the collection tube Transfer to a centrifuge tube. Put the adsorption column into the collection tube, add 200 μl Buffer Ps, centrifuge at 1300 rpm for 2 min, balance, discard the filtrate, and put the adsorption column into the collection tube again. Add 450 μl of isopropanol to the centrifuge tube, mix well by inverting up and down, and transfer the mixture into a well-balanced adsorption column. Centrifuge at 1300rpm for 1min, discard the waste liquid, and put the adsorption column back into the collection tube again. Add 750 μl of Buffer PW containing absolute ethanol, centrifuge at 1300 rpm for 1 min, and discard the waste liquid. Centrifuge at 1300rpm for 2min, discard the waste liquid, and dry at room temperature for 5min. Put the adsorption column in a new centrifuge tube. Add 200 μl Endo-Free Buffer EB to the middle of the adsorption membrane, place it at room temperature for 5 minutes, and centrifuge at 300 rpm for 2 minutes. The extracted plasmid will be sequenced and identified, and the recombinant plasmid with correct gene sequence identification will be named rhGH-Fc-1. Figure 2 shows the sequencing map of the plasmid. Figure 3 shows the 1% agarose gel electrophoresis analysis of the recombinant plasmid rhGH-Fc-1 with HindⅢ, EcoRI, XbaI and XhoI twice, and the target band of about 1300bp can be seen in the figure , consistent with the expected 1362bp. Therefore, the sequencing and identification results confirmed that it fully met the design requirements.

Example 3: CHO-k1 cell culture and transfection

First, discard all the cell culture medium (preferably IMDM medium) for culturing CHO-k1 cells, add an appropriate amount of HYQtase (if it is an adherent cell), and then put it in a CO ₂ incubator for accelerated digestion at 37°C. After the digestion is complete, add An appropriate amount of culture medium was used to dilute the digestion solution to terminate the digestion. Pour all the cells into a centrifuge tube, centrifuge at 1500rpm for 5min, and discard the supernatant. Add an appropriate amount of cryopreservation solution (medium + 10% DMSO), mix the cells in the centrifuge tube, and count the cells so that the final concentration is between 3×10 ⁶ and 1×10 ⁷ cells/ml. The above-mentioned cells were transferred to a pre-cooled cryovial, placed in a freezing box with isopropanol, placed at -80°C overnight and then transferred to liquid nitrogen the next day.

Remove the frozen plastic tube from the liquid nitrogen tank. Quickly place the cryovial in a 38°C water bath to allow it to thaw quickly. Add appropriate complete medium with serum to a 75cm ² retort flask, and inoculate the cells in the retort flask for culture.

Transfection was started when the cell density reached 95%. According to the mediation of Lipofectamin 2000, 6 μg of high-purity plasmid DNA was diluted with 250 μl of IMDM medium, and left at room temperature for 5 minutes. Dilute 20 μl of Lipofectamin with 250 μl of IMDM medium, and place at room temperature for 5 minutes. The two were mixed and placed for 20min. Cells were washed three times with IMDM serum-free medium. Put the DNA-liposome mixture into the retort, add about 14ml of IMDM medium, culture at 37°C, 5% CO ₂ , and saturated humidity for 4-6 hours, replace the medium, and discard the original medium , add 16ml IMDM+10% FBS medium to continue culturing.

Example 4: Screening of engineered cells stably and efficiently expressing rhGH-Fc fusion protein

(1) Screening of positive clones

Discard the IMDM medium of the CHO-k1 cells transfected in Example 3, add about 2ml of HYQtase for digestion, and put them in an incubator to accelerate digestion. After the digestion is complete, add an appropriate amount of serum-containing medium to terminate the digestion. Pour the cells and culture medium into a centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Add 30ml of SMM-CHO GSI medium (containing 10% FBS+25μmol MSX) into the centrifuge tube and mix well. Mix the required medium with the cells, transfer it into a 96-well plate with a row gun, 100 μl/well, and the count of cells in each well of the 96-well plate should be 5×10 ³ to 8×10 ³ . Seal it with plastic wrap and put it in a _CO2 incubator for cultivation. One week later, 50 μl of SMM-CHO GSI medium (containing 10% FBS+25 μmol MSX) was added. About 12 days after transfection, under the microscope, the marker grows with monoclonal well sites. Aspirate the cell culture solution in the marked wells, and add 150 μl/well of SMM-CHO GSI (25 μmol MSX) serum-free medium. After culturing in a CO2 incubator for 3 to 4 days, use the ELISA method to detect the culture supernatant for the first screening.

Scrape and transfer the monoclonal with relatively good ELISA results into a 24-well plate, add 1ml of SMM-CHO GSI (25μmol MSX) medium to each well, place it in a _CO2 incubator for 3-4 days, and take 150μl of the first well For the supernatant, add 200 μl PBS to each well of the remaining three wells, take 50 μl from the first well and mix it in the second well to dilute in turn, take the supernatant and dilute in 4 gradients, use ELISA method for detection, and perform the second screening.

Pipette the wells with relatively good test results evenly, transfer them into a 6-well plate, add 3ml SMM-CHO GSI (50μmol MSX) to each well, and place them in a _CO2 incubator for 3 to 4 days, then take the supernatant and divide it into 4 A serial dilution was used to detect the culture supernatant by ELISA for the third screening.

Select relatively good detection results and transfer them into 75cm ² retort flasks, add 13ml SMM-CHO GSI (75μmol MSX) to each bottle, and place them in a CO ₂ incubator for cultivation. Continue to cultivate for about a week, take 4 gradient dilutions of the supernatant in the 75cm ² retort flask, use ELISA method to detect, and carry out the fourth screening.

Cells with relatively good test results were selected for cryopreservation, and the rest continued to be cultured. When the cells grow to the logarithmic phase, they are transferred to a 125ml small shake flask, and the shaking culture is continued. The cells and culture medium in the 125ml small shake flask were centrifuged at 1500rpm for 5min, and 10ml of SMM-CHO GSI (75 μmol MSX) medium was added to mix the cells by blowing and mixing. Add 30ml of SMM-CHO GSI (75μmol MSX) fresh medium to a 125ml shake flask, transfer appropriate amount of cells, so that the total number of cells in the flask is 1.2× ¹⁰⁷ , culture in batches, count and observe every day. Take 125ml of the cell culture supernatant in the bottle on the 3rd to 4th day, carry out 6 serial dilutions respectively, use the ELISA method to detect, and carry out the fifth screening. Figure 4 shows the screening results. According to the ELISA results of screening the supernatant of the culture medium in 125ml shake flasks, three cell lines D, E, and H with relatively high expression levels and good cell growth status were selected and frozen for the second round. Subclonal screening.

(2) Subclone screening

Three cell lines D, E, and H obtained in step (1) with higher expression levels and better proliferation status were selected for subcloning screening.

First perform a cell count. Then dilute the cells, the medium is SMM-CHO GSI+10% FBS (25 μmol MSX), so that 5 ml contains a total of about 500 cells, and each cell line is paved with 5 96-well plates, 100 μl/well, and the paved 96-well plates are used Seal it with plastic wrap and place it in a 37°C, CO ₂ incubator for cultivation. The follow-up screening method is the same as the screening experiment method for positive clones, from 96 plates (25 μmol MSX) → 24-well plates (50 μmol MSX) → 6-well plates (50 μmol MSX) → 75cm ² culture flasks (75 μmol MSX) → 125ml shake flasks (75 μmol MSX).

What Fig. ⁵ shows is cell line D culture supernatant ELISA result in 75cm , as shown in the figure, select D1 to continue cultivating; What Fig. ⁶ shows is cell line E culture supernatant ELISA result in 75cm , as shown in the figure , select E2 to continue culturing; Figure 7 shows the ELISA results of the culture supernatant of cell line H in a 75cm ² culture flask, as shown in the figure, select H2 to continue culturing; the same culture state in the 125ml shake flask in subcloning screening The culture supernatants of D1, E2, and H2 were detected by ELISA method. According to the ELISA results shown in Figure 8, the growth curves of D1, E2, and H2 cells in the subcloned 125ml shake flasks shown in Figure 9, the growth state of the selected cells and the detection results were selected. The better H2 is the final screened cell line, named as rhGH-Fc cell line, and the target protein expressed is rhGH-Fc immune fusion protein.

Example 5: Preliminary purification of rhGH-Fc immune fusion protein

Cultivate rhGH-Fc cell lines to produce rhGH-Fc immune fusion protein, and culture for about 10-15 days. When the cells are close to death, collect the supernatant, centrifuge at 4000rpm for 20min, and collect the supernatant for purification of rhGH-Fc immune fusion protein . The centrifuged supernatant of the protein culture solution was mixed with diatomaceous earth at a ratio of 3g/100ml and filtered, and then affinity chromatography was used to purify the target protein. First, the cell culture supernatant was filtered through a filter membrane with a pore size of 0.22 μm. Connect the protein purification column (prepacked column HitrapMabselect Sure 5*1ml) to the protein purification instrument. Use 50mM Tris+150mM NaCl (HCl to adjust pH 7.3~7.5) equilibrium solution to equilibrate 3~5 volumes of the column at a flow rate of 5ml/min, and the value of about 0 can be displayed on the ultraviolet detector. Load the sample at a flow rate of 5ml/min, and the UV detector may display hundreds of values. After loading the sample, wash the column with equilibration solution for 3 to 5 volumes, and the UV detector will display a value of about 0 again. Rinse with 0.1M glycine (adjust pH 3.5-3.8 with HCl) at a flow rate of 2-3 ml/min, and collect the elution peaks. Wash the purification column with 0.1M NaOH cleaning solution for 3 to 5 column volumes, and store in 20% ethanol.

Example 6: Identification of rhGH-Fc immune fusion protein

The relative molecular mass of the rhGH-Fc immune fusion protein after preliminary purification was analyzed by non-reduced and reduced 10% SDS-PAGE, and the isoelectric point was determined by isoelectric focusing electrophoresis. Figure 10 shows the SDS-PAGE identification spectrum of the rhGH-Fc immune fusion protein. It can be seen from the figure that the non-reduced electrophoresis shows that a specific protein band can be seen at a relative molecular weight of about 95kDa, which is consistent with the theoretical value of 97.3kDa. The reduced electrophoresis shows that Specific protein bands can be seen at the relative molecular weight of about 42kDa, which is consistent with the theoretical value of 43.7kDa.

Figure 11 shows the isoelectric focusing electrophoresis of the rhGH-Fc immunofusion protein, and the results show that the isoelectric point of the rhGH-Fc immunofusion protein is between 5.2 and 5.85, which is consistent with the theoretical prediction value of 5.35.

Example 7: Expression level of rhGH-Fc immune fusion protein

Cultivate the rhGH-Fc cell line obtained in Example 4 to produce rhGH-Fc immune fusion protein, the culture conditions are: SMM-CHO GSI medium (containing 75 μmol MSX), 37 ° C, CO ² Shaking culture in the incubator, culture about After 12 days, the expression level of rhGH-Fc immune fusion protein was measured.

High-performance liquid chromatography (HPLC) is used to detect the supernatant of the cell culture fluid of the rhGH-Fc immune fusion protein and the rhGH-Fc immune fusion protein after preliminary purification by affinity chromatography. The chromatographic column was G3000WS, the detection wavelength was set at 280nm, the flow rate was 1ml/min, and the sample volume was 20μl. The retention time of the target protein was determined by the HPLC detection results of the purified protein of known concentration, and the expression of the target protein in the cell culture supernatant was determined by peak area comparison.

Figure 12 shows the HPLC spectrum of the supernatant of the cell culture fluid of the rhGH-Fc immune fusion protein, and Figure 13 shows the HPLC spectrum of the rhGH-Fc immune fusion protein. It can be seen from the HPLC detection results that the main peak retention time of the rhGH-Fc immune fusion protein is about 10.6 min, the peak area of the target protein in the supernatant of the cell culture medium is 17804703, and the peak area of the purified rhGH-Fc immune fusion protein The area is 21131703 and the purity is 95.21%. It is estimated that the expression level of the cell supernatant is above 1.4g/L.

Example 8: Determination of biological activity of rhGH-Fc immune fusion protein

The activity of rhGH-Fc immune fusion protein was determined according to the "Chinese Pharmacopoeia" 2015 edition four general rules 1219 rat pituitary growth hormone bioassay.

The details are as follows: SD rats, half male and half female, aged 3 to 4 weeks, were anesthetized by intraperitoneal injection of pentobarbital sodium (concentration 15 mg/ml) 45 mg/kg (administration volume 3 ml/kg), and fixed in a stereotaxic pituitary gland after anesthesia. A resection instrument (Stoelting, USA) was used to remove the pituitary gland, and the body weight and tail length (distance from the end of the tail to the anus) of each animal were recorded after the operation. After the operation, within 3 days after the operation, a single subcutaneous injection of penicillin (40×10 ⁴ IU/ml) 0.1ml per animal per day was given, and the animals were allowed to drink 5% glucose water (into a water bottle) freely; free to drink and eat . Three weeks after the operation, the body weight of each surviving animal was measured, and the hypophysectomy rats whose body weight change was less than ±10% before the operation at 2 to 3 weeks after the operation were selected as qualified model animals, and were randomly and evenly divided into model control groups (normal saline) according to body weight. ), low dose group (0.4IU/kg) and high dose group (1.6IU/kg) of human growth hormone international active standard substance, low dose group (0.4IU/kg) and high dose group (0.4IU/kg) of rhGH-Fc immune fusion protein ( 1.6IU/kg), 10 rats in each group; administration method: subcutaneous injection; administration volume: 2.5ml/kg/d; dosing frequency and duration: dosing according to groups, standard group once a day, for 6 consecutive days ; The model control group and the test product group were injected once at intervals of 3 days, for a total of 2 injections; the injection dose and grouping settings are detailed in Table 1.

Table 1 Injection dose and group settings

Daily weighing records (the day of administration of the experimental group was recorded as D1). Figure 14 shows the trend chart of body weight growth of rats in each group after administration. The results of body weight measurement showed that the rhGH-Fc immune fusion protein in the test product group was growing within 3 days after injection, while the standard product group stopped growing immediately after no administration. It can be preliminarily judged that the half-life of rhGH-Fc immune fusion protein is longer than that of growth hormone. Standard. On the 10th day, the animals were killed and the tibias of the hind legs were planed out, and the width of the epiphyseal plate of the tibia was measured according to the method in the appendix of the Pharmacopoeia (10 values were measured on the tibias of each mouse's legs, the highest and lowest values were removed, and the average value was taken). The response value experimental results are arranged according to the format of the quantity-response parallel line determination random design method list in "Chinese Pharmacopoeia" 2015 edition four general rules 1431 biological assay statistics method, and the random design processing results according to the quantity-response parallel line determination method are shown in Table 2. It is the measurement result of the tibial epiphyseal plate width of each group of rats. The data were statistically processed. Table 3 showed the statistical analysis of the measurement data of the epiphyseal plate width of the rats in the low dose group, and Table 4 showed the statistical analysis of the measurement data of the epiphyseal plate width of the rats in the high dose group. As can be seen from the table, the data of the test product and the standard product in the low-dose and high-dose groups prove that the half-life of the immune fusion protein developed by the present invention is significantly longer than the standard product. The theoretical estimated potency of the test product is 1.24IU/mg after conversion by dose, and the potency result calculated by the bioassay statistical method stipulated in Pharmacopoeia is 1.1IU/mg.

Table 2 Measurement results of tibial epiphyseal plate width of rats in each group

Table 3 Statistical analysis of the measurement data of epiphyseal plate width in low-dose group rats

Table 4 Statistical Analysis of Epiphyseal Plate Width Measurement Data in High-dose Group Rats

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.

sequence listing

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

<120> A long-acting recombinant human growth hormone fusion protein and its engineered cells

<141> 2019-05-16

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Claims (10)

1. A long-acting recombinant human growth hormone rhGH-Fc fusion protein, characterized in that: the fusion protein is composed of human growth hormone, connecting peptide and Fc fragment; the fusion protein is followed by the following sequence from the N terminal to the C terminal: Growth hormone, connecting peptide, and Fc fragment, its amino acid sequence has a total of 431 amino acids, of which the first 191 amino acid sequences are human growth hormone sequences, the 192nd to 206th are connecting peptide sequences, and the 207th to 431st are human IgG Fc fragment. 2. The fusion protein according to claim 1, wherein the Fc fragment is selected from the Fc fragment of human IgG, preferably the Fc fragment of human IgG4. 3. The fusion protein according to claim 1, characterized in that: the Fc fragment of the fusion protein comprises four amino acid mutations, the amino acid mutations are: S228P, F234A, L235A and V308P, wherein the residues in the IgG Fc region The numbering is based on the Kabat Eu Index numbering. 4. The fusion protein according to claim 1, characterized in that: the amino acid sequence of the fusion protein is shown in SEQ ID NO:1. 5. The gene encoding the fusion protein according to any one of claims 1 to 4, characterized in that: the nucleotide sequence of the gene encoding is shown in SEQ ID NO:2. 6. An engineered cell, characterized in that: said engineered cell expresses the fusion protein according to any one of claims 1-4. 7. An expression vector, characterized in that: the expression vector is preferably a double expression vector; more preferably, the expression vector is a double expression plasmid p327.8GS through restriction endonucleases Hind III, EcoR I and Xba 1. After Xho I double digestion twice, the recovered large fragment and the nucleic acid encoding the fusion protein according to claim 1 were mixed with T4 DNA ligase, and ligated overnight at 15-16°C, so that the optimized target gene was cloned into on a double expression vector. 8. A CHO cell, characterized in that: the CHO cell is a CHO-k1 cell, which is stably transfected with the expression vector according to claim 7. 9. The screening method of positive clones and subclones expressing the fusion protein described in any one of claims 1 to 4, is characterized in that: by gradually increasing the concentration of methionine sulfonamide (MSX), from 96 plates (25～30 μ mol MSX)→ 24-well plate (50-55 μmol MSX) → 6-well plate (50-55 μmol MSX) → 75cm2 culture flask (70-75 μmol MSX) → 125ml shaker flask (70-75 μmol MSX), the higher expression level was gradually screened by ELISA And the cell line with better proliferative state, the final screened cell line is named as rhGH-Fc cell line, and the target protein expressed is rhGH-Fc immune fusion protein. 10. The fusion protein according to any one of claims 1 to 4 or the CHO cell according to claim 8, characterized in that: the fusion protein can prolong the serum half-life, greatly reducing the required injections during treatment. Drug frequency, thereby improving patient compliance and convenience for both doctors and patients; the CHO cells can be used to prepare recombinant human long-acting growth hormone, which has high protein expression, good biological activity, and reduced protein immunogenicity, which can improve efficacy.