CN114507291A - Expression method of Fc fusion protein of recombinant human immunoglobulin epsilon and gamma - Google Patents

Abstract

The invention relates to an expression method and application of Fc fusion protein (antiallergic fusion protein) of recombinant human immunoglobulin epsilon and gamma. The recombinant human immunoglobulin epsilon and gamma Fc fusion protein can be combined with Fc epsilon receptor I (Fc epsilon RI) and inhibitory Fc gamma receptor IIb (Fc gamma RIIb) which mediate anaphylactic reaction on the surfaces of mast cells and basophils. The Fc fusion protein of the recombinant human immunoglobulin epsilon and gamma can be used for allergic diseases, such as allergic asthma, rhinitis and the like.

Description

Expression method of Fc fusion protein of recombinant human immunoglobulin epsilon and gamma

Technical Field

The invention belongs to the field of biological medicine, and relates to an expression method and operation of Fc fusion protein (antiallergic fusion protein) of recombinant human immunoglobulin epsilon and gamma.

Background

Allergy is a series of phenomena of excessive reaction caused by the contact of human body with allergen in the environment, including allergic rhinitis, food allergy, urticaria, asthma and the like.

Immediate hypersensitivity (also called type I hypersensitivity) is an IgE-mediated immune reaction, is a common type of allergy, and can be mainly divided into two major types, namely systemic anaphylaxis and atopic anaphylaxis.

The principle of allergy generation is: when an allergen (antigen) is contacted with an immune B cell for the first time, the B cell and the T cell are contacted to differentiate into plasma cells and produce a large amount of antibody IgE, which is bound to immune cells such as mast cells, and when the allergen is contacted for the next time, the immune cells bound to IgE are activated and release histamine and the like, which causes allergic symptoms and affects immune response.

In 1995, the first report by Daeron et al, a research group in France, on IgE high affinity receptors (Fc) that mediate mast cell activation_εRI) are subject to low affinity IgG receptors (Fc)_γRII) negative regulation, the same polyvalent ligand cross-linking of Fc on mast cell surface_εRI and Fc_γRII inhibits activation of cells and prevents release of mediators [ Daeron M, Malbec O, Latour S, arc M, Fridman W.Regulation of high-affinity IgE receptor-mediated map cell activation by human muscle low-affinity IgG receptors J Clin Invest,95: 577-cell 585,1995.]In the literature at that time, there is no attention paid to different kinds of Fc_γThere are differences in RII. This difference was found in later studies.

WuJ, et al, showed that Fc_γThe RII family of monoclonal antibodies plays an important role in allergic reactions, Fc_γRII can be further subdivided into Fc_γRIIa，Fc_γRIIb，Fc_γRIic. Wherein the receptor having negative IgE regulation is Fc_γRIIb，Fc_γRIIa showed a positive correlation with specific responses, while other receptors were not clearly Associated with specific responses and asthma [ Wu J, Lin R, Huang J, Guan W, Oetting WS, et al (2014) Functional Fcgamma Receptor Associated with Human allergy. PLoS ONE9(2): e89196. doi:10.1371/journal. po. 0089196.]. Wherein Fc_γRIIa is unique to humans. At Fc_γIn RII family, Fc_γThe affinity of RIIb and IgG is higher than that of Fc_γRIIa and Fc_γRIic is low.

As the research proceeds, it is found that different kinds of Fc_γThe differences in the in vivo effects of the RII receptors are quite significant. The studies by Rosales c. et al further demonstrate that the regulatory effects of the different receptors are diametrically opposed. [ Rosales C.Fc. gamma. Receptor selectivity in Leucocyte Functional Responses.Front Immunol.2017；8:280.Published 2017Mar 20. doi:10.3389/fimmu.2017.00280.]In humans, Fc_γThe RIIb receptor can activate an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) on the intracellular chain, which is also the only receptor that can activate ITIM, and FC_γRIIa and FC_γRIIc activates an Immunoreceptor Tyrosine-based Activation Motif (ITAM) in the intracellular chain, and the same Activation effect as IgE leads to Activation of SYK (protein spleen Tyrosine kinase), which aggravates asthma.

The research of Wangjingzuo proves the intervention effect and mechanism research of the protein of mouse-origin AAFP (Anti-allergic fusion protein for short) on IgE-mediated anaphylactic reaction of mice [ D]Guiyang-Guiyang medical college, 2014.]By crosslinking Fc_εRI and Fc_γRIIb receptor, activating Fc_γThe ITIM inhibitory motif on the intracellular chain of the RIIb receptor further activates phosphorylation of 5' -inositol phosphatase (SHIP), which phosphorylates Fc_εThe key signal protein spleen tyrosine kinase (Syk) in the RI signaling pathway dephosphorylates, blocking the cellular degranulation initiated by Syk. The AAFP protein acts on the activation of allergic cells and the release of inflammatory mediators, thereby inhibiting the generation of IgE-mediated allergic reaction.

The antiallergic fusion protein AAFP is formed by human Fc_εAnd Fc_γFormation of Fc capable of cross-linking mast cell and basophil surface-mediated allergic reactions_εReceptor I (Fc)_εRI) and inhibitory Fc_γReceptor IIb (Fc)_γRIIb), inhibits activation of mast cells and basophils and release of allergic inflammatory mediators, thereby effectively blocking the development of allergy.

In recent years, the research and development of IgE-mediated allergic disease biological medicines in the market are mainly focused on anti-IgE or anti-Fc_εRI monoclonal antibodies and inhibition of their signaling. However, clinical studies have found that a decrease in IgE levels in vivo results in an increase in the incidence of tumors. AAFP does not simply bind IgE antibodies, so AAFP does not reduce the IgE antibody content in vivo, and does not cause IgE antibody to be in vivoThe content is reduced to cause adverse effects, and compared with the anti-IgE monoclonal antibody, the content is relatively safe, so that a new way for treating IgE-mediated allergic diseases is opened up.

In biology, an expression vector is a vector that can express a target gene by adding expression elements (such as a promoter, an enhancer, a terminator, and the like) to the basic backbone of a cloning vector. Expression vectors are mainly divided into four parts: target gene, promoter, terminator and marker gene.

The expression vector is usually constructed by bacterial plasmid, the ends (mostly viscous ends and flat ends) which are matched with the target gene are cut by using restriction endonuclease in the construction process, and then the ends are connected by adopting DNA ligase and are introduced into organisms to realize expression. Marker genes can help identify plasmids and detect successful integration into chromosomal DNA.

CHO cells (Chinese Hamster Ovary, Chinese Hamster Ovary cells) are a common system for the production of recombinant protein drugs at present, and have many advantages over other expression systems: (1) has the extracellular secretion function of the product, and is convenient for separating and purifying downstream products; (2) has the high-efficiency amplification and expression capacity of the recombinant gene; (3) the culture medium has the characteristics of adherent growth, higher shear stress and osmotic pressure resistance, and higher expression level, and can also be used for suspension culture; (4) CHO cells belong to fibroblasts, rarely secrete self endogenous protein, and are beneficial to starting separation of exogenous protein; (5) CHO cells can grow in a manner of attaching to the wall or in a manner of suspending, and are easy to generate gene mutation and gene transfection; (6) the suspension grows rapidly and stably in serum-free and chemically defined medium; (7) has accurate post-transcriptional modification function, and the expressed protein has molecular structure, physical and chemical properties and biological function close to natural protein molecules; (8) has the high-efficiency amplification and expression capacity of recombinant genes and can stably fuse foreign proteins.

Selectable markers and gene amplification CHO cell expression vectors have two main types of selectable markers. One is non-amplified gene such as neo (neomycin resistance gene) and the like, has no influence on the copy number of a target gene, and is used for constructing a transient expression vector. Another type of gene amplification function is also called co-amplification gene, such as dihydrofolate reductase (DHFR) gene and Glutamine Synthetase (GS) gene.

The stable cell strain has long screening period of DHFR gene, more than half a year, low yield and less use. The stable cell strain has short screening period of GS gene, generally less than 3 months, stable yield, and can utilize ammonia generated by biological metabolism, and the like, and can avoid or reduce the damage and inhibition effect of ammonia accumulated by glutamine degradation on cells in the cell metabolism process.

The loaded pGS plasmid was transferred into E.coli competent cells DH 5. alpha. and colony PCR was performed, and the plasmid harbored ampicillin resistance gene, so that E.coli containing the plasmid grew on ampicillin-containing medium, while E.coli not containing the plasmid died, and thus it was used for screening colonies into which the plasmid was not introduced.

In addition, the pGS plasmid carries a GS marker (i.e., carries a glutamine synthetase gene), and the CHO-K1 cell is a GS-deficient cell and cannot synthesize glutamine, and in the screening of glutamine analogue MSX (methionine iminosulfone), the CHO-K1 cell into which no plasmid has been transferred dies because glutamine cannot be synthesized, and the surviving cell is a positive cell containing the plasmid, so that the use of the plasmid can also be used for screening a cell into which the plasmid has not been introduced. Therefore, when a vector containing a GS marker is used, a GS-deficient cell is selected as a host cell, and MSX is used as a condition for screening a positive clone.

Lipofectation can be used for transient transfection and stable transfection of DNA. Neutral liposomes encapsulate DNA with a lipid membrane, and the DNA is introduced into the cell membrane via the lipid membrane. For the positively charged cationic liposome, the negatively charged DNA phosphate group is automatically bonded to the positively charged liposome to form a DNA-cationic liposome complex, so that the DNA-cationic liposome complex is adsorbed to the surface of a negatively charged cell membrane and is introduced into cells through endocytosis. This method results in a great improvement in the efficiency, stability and reproducibility of transfection.

Therefore, the linearized pGS-AAFP plasmid is introduced into a CHO-K1 cell by a lipofection method to obtain a CHO cell strain capable of stably expressing the AAFP gene.

Moderate to severe persistent allergic asthma with symptoms that cannot be effectively controlled by inhaled glucocorticoids and long-acting inhaled beta 2-adrenoceptor agonists in patients with IgE (immunoglobulin E) -mediated asthma, AAFP cross-linkable basophilic granulocytes or receptor Fc on the surface of mast cells_γRIIb and Fc_εRI, in which IgE Fc end on AAFP can be linked with Fc of cell surface_εRI binding, IgG Fc end can be connected with Fc_γRIIb is combined. Once crosslinked, Fc_γThe signal path of RIIb is activated to inhibit Fc_εThe conduction of RI, thereby preventing the release of inflammatory mediators by the cells, effectively reducing the incidence of asthma in the patient.

Disclosure of Invention

The invention provides an anti-allergic fusion protein AAFP and expression thereof.

In a first aspect, the present invention provides a nucleotide sequence encoding an Fc fusion protein of recombinant human immunoglobulins epsilon and gamma.

Specifically, the AAFP gene fragment is formed by connecting an IgE Fc fragment and an IgG Fc fragment, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1:

wherein 1-63 is a secretory signal peptide gene sequence of an Ig kappa chain, 64-1192 is a gene of IgE Fc, and 1193-2106 is a gene of IgG Fc.

In order to remove non-human sequences doped in the AAFP gene sequence in the pSecTaq-AAFP vector, the AAFP gene is amplified by using an overlapPCR method; the IgE Fc and IgG Fc gene fragments were first obtained from the pSecTaq-AAFP plasmid using PCR, after which the two fragments were ligated by the overlap PCR method.

In a second aspect, the present invention provides a recombinant human immunoglobulin epsilon and gamma Fc fusion protein expressed by the above gene sequence, wherein the amino acid sequence of the recombinant human immunoglobulin epsilon and gamma Fc fusion protein is shown in SEQ ID No. 2:

wherein 1-21 is a secretion signal peptide sequence of an Ig kappa chain; 22-25, 346 and 347 are amino acid residues encoded by a vector sequence; 26-345 are amino acid sequences of human IgE Fc (CH2-CH3-CH 4); 346-579 are amino acid sequences of human IgG Fc (Hinge-CH2-CH 3).

In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence of claim 1. The specific operation steps are as follows:

(1) designing overlap PCR primers, obtaining IgE Fc and IgG Fc gene fragments from pSecTaq-AAFP plasmids through a first round of overlap PCR, and connecting the two fragments through a second round of overlap PCR to obtain a sequence shown as SEQ ID NO. 1. The overlap PCR requires 5 primers, forward1 is shown as SEQ ID NO.3, forward2 is shown as SEQ ID NO.4, forward3 is shown as SEQ ID NO.5, reverse1 is shown as SEQ ID NO.6, reverse2 is shown as SEQ ID NO. 7:

(2) performing double enzyme digestion on the PCR product and the pGS plasmid by using XhoI enzyme and MluI enzyme, and then integrating the AAFP gene fragment and the vector fragment of pGS into a closed recombinant plasmid under the action of T4 ligase;

(3) the method for verifying whether the plasmid loads the target gene comprises the steps of transferring the plasmid into an escherichia coli competent cell DH5 alpha, carrying out colony PCR, culturing a colony by using a culture medium containing ampicillin, selecting a positive colony to carry out colony PCR, and verifying whether the positive colony contains the target vector;

(4) and (3) identifying whether the vector is successfully constructed by carrying out double enzyme digestion on the positive clone extracted plasmid by using MluI enzyme and XhoI enzyme, wherein the constructed vector is named as pGS-AAFP.

In a fourth aspect, the present invention provides a CHO-K1 cell line capable of producing recombinant human immunoglobulin epsilon and gamma Fc fusion protein, which is constructed by the following steps:

(1) the pGS-AAFP vector is digested by BcgI to be linearized;

(2) the linearized pGS-AAFP vector was transfected into CHO-K1 host cells using the Lipofectamine (Invitrogen) transfection method.

(3) The transfected CHO-K1 cells were subjected to MSX pressure screening, and the expression level of positive clones was screened by ELISA to obtain CHO-K1 cells according to claim 4.

In a fifth aspect, the present invention provides conditions for carrying out scale-up culture of a cell line of the AAFP gene fragment.

Specifically, cell strains containing AAFP gene fragments are recovered, subjected to three-stage seed culture, and inoculated into a production culture reactor for culture.

The CHO cell referred to in the present invention includes commercial CHO cell lines such as CHO-K1, CHO-DG44 and the like.

The expression vector used in the present invention may be selected from various known vectors such as commercial vectors: pCDNA3.1, pSecTag, and the like. The recombinant expression vector is transferred into host cells by an electrotransfection or chemical transfection method, and stable recombinant cells are screened by proper conditions to be used as engineering cell strains for preparing the Fc fusion protein of the recombinant human immunoglobulin epsilon and gamma.

Drawings

FIG. 1 is a flow chart of the pressure screening method for CHO-K1 cell line according to the present invention.

FIG. 2 is a flow chart of the cell culture process containing AAFP gene fragment according to the present invention.

FIG. 3 shows the PCR electrophoresis results of recombinant plasmid transformed E.coli DH5 alpha colony, which shows that the selected DH5 alpha positive clones all contain target genes and the construction of pGS-AAFP plasmid is completed.

Detailed Description

Examples

1. Construction and screening of CHO-K1 cell strain containing AAFP gene fragment

1.1 amplification of the AAFP Gene

In order to remove non-human sequences doped in the AAFP gene sequence in the pSecTaq-AAFP vector, we designed 3 pairs of primers:

the overlapPCR method was used to amplify the genes of our desired AAFP. In the first round of PCR, two PCR reactions were included, and the first reaction was performed using pSecTag-AAFP as a template to amplify the sequence of IgE Fc fragment. Where F1 was fully paired with the 5' end of IgE Fc, the 3 ' end of R1 was paired with the 5' end of the IgE Fc antisense chain, and the 5' end of R1 was paired with the 5' end of IgG Fc. The second reaction also amplified IgG Fc fragment sequences using pSecTag-AAFP as template. Wherein the 3 ' end of F2 is paired with the 5' end of IgG Fc, the 5' end is paired with the 5' end of R1, the 3 ' end of R2 is paired with the 5' end of IgG Fc antisense chain, and the 5' end is MluI cleavage sequence and protective base. The reaction system of the two PCRs involved in the first round is as follows:

PCR conditions were 94 ℃ pre-denaturation for 2min, 1 thermal cycle; thermal denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 80s, and 30 thermal cycles; renaturation at 72 deg.C for 10 min; storing at 4 ℃.

After the first round of reaction, PCR products are separated and purified by using a PCR product extraction kit, and the PCR products are used as templates for carrying out second round PCR. The forward F3 primer of the second round of PCR consists of protective basic group, XhoI enzyme cutting sequence, Kozak sequence, mouse Kappa chain secretion signal peptide sequence and 5' end sequence of IgE Fc in sequence, the reverse primer is R2, and the template is the product IgE Fc and IgG Fc obtained by the extraction of the first round of PCR.

The second round of reaction system is as follows:

IgE Fc PCR product (after 100-fold dilution)	0.5μL
		IgG Fc PCR product (after 100-fold dilution)	0.5μL
Primer F3 working solution	1μL
		Primer R2 working solution	1μL
5×PS buffer	5μL
		dNTP	2μL
PrimeStar enzyme	0.25μL
		Water (I)	14.75μL

PCR conditions were 94 ℃ pre-denaturation for 2min, 1 thermal cycle; thermal denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 2min, and 35 thermal cycles; renaturation at 72 deg.C for 10 min; storing at 4 ℃.

1.2 double digestion of PCR products

Performing double enzyme digestion on the PCR product in the step 1.1 by using XhoI and MluI, and recovering a target fragment, wherein the double enzyme digestion reaction system is as follows:

enzyme digestion is carried out for 2hr at 37 ℃, and after enzyme digestion, 1.0% gel electrophoresis separation and gel recovery are carried out to obtain the AAFP gene.

1.3pGS plasmid double digestion

XhoI and MluI are used for carrying out double enzyme digestion on pGS plasmids, and target fragments are recovered, wherein the double enzyme digestion reaction system is as follows:

Buffer3(NEB)	5μL
		MluI	2μL
XhoI	2μL
		pGS plasmid	2μL
Water (W)	39μL

And (3) carrying out enzyme digestion at 37 ℃ for 2hr, and carrying out 1.0% gel electrophoresis separation and gel recovery after enzyme digestion to obtain a pGS vector fragment.

1.4 ligation of pGS-AAFP vectors

The AAFP gene fragments obtained in 1.2 and 1.3 and the vector fragment of pGS are integrated into a closed recombinant plasmid under the action of T4 ligase. The DNA ligation reaction system is as follows:

T4 ligase	1μL
		Buffer	1μL
AAFP Gene fragment	7μL
		Vector fragment of pGS	1μL

The reaction was carried out at 4 ℃ overnight.

1.5 colony PCR

Competent E.coli DH 5. alpha. was transformed with the ligation products and positive clones were selected on LB plates containing Amp. Selecting positive clones, inoculating the positive clones in 0.5ml LB culture medium, fermenting for 4 hours, taking the bacterial liquid as a template, and carrying out clone PCR reaction by taking F3 and R2 as primers, wherein the reaction system is as follows:

cloning of fermentation broth	1μL
		Primer F3 working solution	1μL
Primer R2 working solution	1μL
		2×Taq PCR MasterMix	10μL
Sterilizing deionized water	7μL

PCR conditions were 95 ℃ pre-denaturation for 5min, 1 thermal cycle; thermal denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 25 thermal cycles; renaturation at 72 deg.C for 7 min; storing at 4 ℃.

And identifying the PCR product by agarose gel electrophoresis, wherein a fragment with the size of 2KB is a positive clone, further carrying out MluI + XhoI double-enzyme digestion identification and gene sequencing on the positive clone, confirming that the positive clone is consistent with a target sequence, and naming the obtained plasmid as pGS-AAFP.

1.6 linearization of pGS-AAFP plasmid

Through analyzing the gene sequence of pGS-AAFP plasmid, BcgI can be used for single enzyme digestion of pGS-AAFP, and cannot influence the functional units on the pGS-AAFP plasmid. Therefore, before transfection of CHO-K1S cells, pGS-AAFP was first digested with BcgI and the linearized plasmid was recovered using a PCR extraction kit.

1.7 Liposome transfection and cell line selection

The linearized pGS-AAFP plasmid was transfected into CHO-K1 host cells using the Lipofectamine (Invitrogen) transfection method. After transfection, cells were plated in 6-well plates and screened by adding 25MMSX, and cells that were not successfully transfected with the plasmid were killed. After cell adaptation, a 96-well cell culture plate was seeded with 1 cell per well by limiting dilution for monoclonal screening, and wells marked to contain only a single cell were selected under the microscope. After single cells grow out of clones, comparing the AAFP expression level of each single clone by using an ELISA method, selecting the first 96 clones, transplanting the clones into 196 pore plate, continuing to culture, when the cells grow full, comparing the expression level of the 96 clones by using the ELISA method again, taking 24 clones with the highest expression level, amplifying the clones into a 24 pore plate for continuous culture, after the cells grow full, performing ELISA again to compare the expression level among the pores, selecting 5 pores with the highest expression level, inoculating the cells into a 6 pore plate, amplifying and culturing, then inoculating the cells into a 125mL shake flask to culture and compare the growth of the cells and the expression level of the AAFP, and selecting a cell strain with the highest expression level as a production cell strain.

Thus, the target AAFP cell line was obtained.

The specific construction steps are shown in the attached figure 1 in the specification in detail.

2. Cell culture containing AAFP gene fragment

2.1 determination of the culture Medium

The CD CHO culture medium of Invitrogen company can support the rapid growth of CHO-K1 cells, but the expression amount of recombinant protein is low, while the growth speed of CHO-K1 cells in the PFCHO culture medium of Hyclone company is relatively slow, but the expression amount of protein is high. Therefore, a CD CHO culture medium of Invitrogen company and a PFCHO culture medium of Hyclone company are selected and mixed according to the ratio of 1:1, and GSsupplement, F-68 and NaHCO are added₃And the like as a basic culture medium for cell culture, and is applied to a seed culture stage. On the other hand, in the construction of genetically engineered cells, we used the GS expression system, and therefore, cell lines could be grown in glutamine-free medium. In serum-free medium at 1:1CD/PF, 1 × GS Supplement was supplemented without additional glutamine. In production fed-batch cultures, to support cells in reactor cultures to higher cell densities, maintain longer production times, and achieve higher product concentrations, the nutrient-rich feed medium is fed-batch beginning in the middle of the culture. The basic components of the feed medium are added into DMEM/F12(BDF medium) without salt, glutamine and sugarHigh concentration of glucose, amino acids, vitamins and nucleotides. The basic culture medium formula is as follows:

CD CHO AGT	121.3g
		PF-CHO part1	30g
PF-CHO part2	52g
		F-68	5g
50*GS supplement	200ml
		NaHCO3	10g
ultrapure water H2O	10L

2.2 class I and II seed culture (Shake flask culture)

According to a conventional cell recovery method, a frozen cell is taken from a working cell bank and is quickly put into warm water at 37 ℃ for thawing. After thawing completely, the cells in the frozen tube were aseptically transferred to a 10ml centrifuge tube, 5ml of pre-warmed medium 37 ℃ was added, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 5ml of pre-warmed medium 37 ℃ was added to resuspend the cells. Suspending the cells in a liquidTransferring into 125ml shake flask, supplementing with 25ml culture medium, and placing in 5% CO₂And culturing in a constant temperature incubator at 37 ℃. The shaker speed was set at 120 rpm. The cell density and activity were monitored daily and after 4 days of culture, the cell density was about 3.0X 10⁶cells/ml were passaged into 1000ml shake flasks and supplemented with pre-warmed fresh medium at 37 ℃ to 270 ml. After further 3 days of culture, the cell density was about 3X 10⁶cells/mL, inoculated into a 5L reactor to start grade III seed culture.

2.3 class III seed culture

Transferring about 300ml of cell fluid from the shake flask to a NBS7.5L or BBraun 5L bioreactor containing 2.7L1:1CD/PF +1 × GS Supplement medium at a culture volume of about 3L, a temperature of 36.8 deg.C, dissolved oxygen of 50%, pH7.1, a stirring speed of 80rpm, and a cell density of about 3.0 × 10 for 3 days⁶cells/ml, and inoculating into a production tank for production.

2.4 production culture

Pilot production was carried out in a C30 tank from B Braun, with a total reactor volume of 42L and a working volume of 30L. For inoculation, approximately 3L of cell fluid from a 7.5L or 5L reactor was transferred to a C30 bioreactor containing 20L of basal medium (1:1CD/PF +1 GSsupplement), initially set at 36.8 deg.C, dissolved oxygen 50%, pH 7.1. + -. 0.05, and agitation speed 60 rpm. When the concentration of residual sugar in the culture medium is reduced to about 1.5g/L, feeding culture is started, and the optimized and determined feed culture medium 10 XBDF +5 XGS +45g/LGlc is continuously fed. In order to extend the production cycle as much as possible, the cell density reaches 1X 10⁷At cell/ml, the incubation temperature was lowered to 31 ℃. According to the sugar measurement result of daily sampling, the glucose content in the culture solution is controlled to be 1-2 g/L, and the culture is finished when the cell viability is reduced to be below 80%.

The specific steps are shown in the attached figure 2 in the specification in detail.

Sequence listing

<110> Shanghai Jingfeng pharmacy Co., Ltd

<120> expression method of Fc fusion protein of recombinant human immunoglobulin epsilon and gamma

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cccccgacca tccagctcct gtgcctcgtc tctgggtaca ccccagggac tatcaacatc 180

acctggctgg aggacgggca ggtcatggac gtggacttgt ccaccgcctc taccacgcag 240

gagggtgagc tggcctccac acaaagcgag ctcaccctca gccagaagca ctggctgtca 300

gaccgcacct acacctgcca ggtcacctat caaggtcaca cctttgagga cagcaccaag 360

aagtgtgcag gtacgttccc acctgccctg gtggccgcca cggaggccag agaagagggg 420

cgggtgggcc tcacacagcc ctccggtgta ccacagattc caacccgaga ggggtgagcg 480

cctacctaag ccggcccagc ccgttcgacc tgttcatccg caagtcgccc acgatcacct 540

gtctggtggt ggacctggca cccagcaagg ggaccgtgaa cctgacctgg tcccgggcca 600

gtgggaagcc tgtgaaccac tccaccagaa aggaggagaa gcagcgcaat ggcacgttaa 660

ccgtcacgtc caccctgccg gtgggcaccc gagactggat cgagggggag acctaccagt 720

gcagggtgac ccacccccac ctgcccaggg ccctcatgcg gtccacgacc aagaccagcg 780

gtgagccatg ggcaggccgg ggtcgtgggg gaagggaggg agcgagtgag cggggcccgg 840

gctgacccca cgtctggcca caggcccgcg tgctgccccg gaagtctatg cgtttgcgac 900

gccggagtgg ccggggagcc gggacaagcg caccctcgcc tgcctgatcc agaacttcat 960

gcctgaggac atctcggtgc agtggctgca caacgaggtg cagctcccgg acgcccggca 1020

cagcacgacg cagccccgca agaccaaggg ctccggcttc ttcgtcttca gccgcctgga 1080

ggtgaccagg gccgaatggg agcagaaaga tgagttcatc tgccgtgcag tccatgaggc 1140

agctagcccc tcacagaccg tccagcgagc ggtgtctgta aatcccggta aagagcccaa 1200

atcttgtgac aaaactcaca catgcccacc gtgcccaggt aagccagccc aggcctcgcc 1260

ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac aggccccagc 1320

cgggtgctga cacgtccacc tccatctctt cctcagcacc tgaactcctg gggggaccgt 1380

cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg 1440

tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg 1500

tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca 1560

cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt 1620

acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc atctccaaag 1680

ccaaaggtgg gacccgtggg gtgcgagggc cacatggaca gaggccggct cggcccaccc 1740

tctgccctga gagtgaccgc tgtaccaacc tctgtcccta cagggcagcc ccgagaacca 1800

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1860

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1920

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1980

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 2040

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 2100

aaatga 2106

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

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Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile

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Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile

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

65 70 75 80

Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr

85 90 95

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

100 105 110

Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp

115 120 125

Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe

130 135 140

Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp

145 150 155 160

Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser

165 170 175

Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn

180 185 190

Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp

195 200 205

Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro

210 215 220

Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala

225 230 235 240

Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp

245 250 255

Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile

260 265 270

Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His

275 280 285

Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe

290 295 300

Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe

305 310 315 320

Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln

325 330 335

Arg Ala Val Ser Val Asn Pro Gly Lys Gly Ser Glu Pro Lys Ser Cys

340 345 350

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Pro Gly Lys

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttcaccccgc ccaccgtgaa g 21

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tctgtaaatc ccggtaaaga gcccaaatct tgtgac 36

<210> 5

<211> 99

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaatctcgag gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 60

tccaggttcc actggtgact tcaccccgcc caccgtgaa 99

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtcacaagat ttgggctctt taccgggatt tacaga 36

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtcacgcgtt catttacccg gagacag 27

Claims (6)

1. A nucleotide sequence of Fc fusion protein for coding recombinant human immunoglobulin epsilon and gamma is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. An Fc fusion protein of recombinant human immunoglobulin epsilon and gamma, which is characterized in that the amino acid sequence of the Fc fusion protein of recombinant human immunoglobulin epsilon and gamma is shown as SEQ ID NO.2 and is obtained by coding and expressing the nucleotide sequence of claim 1.

3. A method for expressing the fusion protein of claim 2, comprising the steps of: constructing recombinant plasmid, introducing into CHO cell, screening to obtain positive clone, and expressing fusion protein.

4. The use of the fusion protein according to claim 2 in antiallergic drugs.

5. A CHO host cell strain characterized by containing the expression vector described in claim 3.

6. An expression vector, wherein the expression vector comprises the nucleotide sequence of claim 1 and is constructed by genetic engineering means; the construction method is overlap PCR; the PCR primers used in the construction are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

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