CN112646044A

CN112646044A - TFF2-Fc fusion protein and high-efficiency expression production method thereof

Info

Publication number: CN112646044A
Application number: CN202011560322.3A
Authority: CN
Inventors: 彭继先; 戴文宇; 柴辉; 钱文正; 闫韵秋; 李振
Original assignee: Shandong Jinghui Biotechnology Co ltd
Current assignee: Shandong Ruiying Pharmaceutical Group Co ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-04-13
Anticipated expiration: 2040-12-25
Also published as: CN112646044B

Abstract

The invention discloses a TFF2-Fc fusion protein and a high-efficiency expression production method thereof. The amino acid sequence of the TFF2-Fc fusion protein is shown in SEQ ID NO. 1. The high-efficiency expression production method comprises the following steps: artificially synthesizing a DNA sequence for encoding TFF2-Fc fusion protein, constructing a nucleic acid construct capable of being expressed in a eukaryotic cell, transfecting the eukaryotic cell, and further purifying and measuring. The TFF2-Fc fusion protein stock solution produced by the production method of the high-efficiency expression TFF2-Fc fusion protein has high quality, and the large-scale production of the TFF2 protein is realized.

Description

TFF2-Fc fusion protein and high-efficiency expression production method thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a TFF2-Fc fusion protein and a high-efficiency expression production method thereof.

Background

Trefoil factor (TFF) is a class of cysteine-rich small molecule polypeptides secreted mainly by gastrointestinal mucus cells, and is also called "Trefoil peptide" because of the presence of one or more Trefoil structures in the molecular conformation. TFF mainly includes the following three in mammals: breast cancer associated peptide (PS2, i.e., TFF1), spasmolytic polypeptide (SP, i.e., TFF2), and intestinal trefoil factor (ITF, i.e., TFF 3). TFF2 was isolated from porcine pancreas in 1982 by Jorgensen and colleagues and is highly conserved among different species. Wherein, the human trefoil factor 2(hTFF2) and the mouse trefoil factor 2(mTFF2) contain the same number of amino acids, and the homology of the amino acid sequence reaches 82%. The mature TFF2 protein consists of 106 amino acids, has a molecular weight of about 7-12kD, contains 4 exons and two symmetrical trefoil domains, is extremely stable in structure, is acid-resistant, heat-resistant and protease-resistant, and is mainly expressed in gastric mucosa neck goblet cells.

It has been shown that TFF2 acts primarily in the gastrointestinal tract, has gastrointestinal mucosal protection and epithelial repair functions, is involved in the regulation of gastric cancer, reduces bacterial damage to the gut, and protects mice from death due to DSS enteritis. In addition, TFF2 plays an important role in allergic reactions, and TFF2 can alleviate allergic reactions, such as asthma symptoms. However, the main mechanisms of action are not completely clear at present, and generally include interaction with mucin, promotion of cell migration, anti-apoptosis, inhibition of inflammatory response and the like; in recent research, TFF2 is a protein which is highly expressed after H9N2 infection and highly inhibited from H7N9 infection, and shows that the protein has repair functions of resisting inflammation and tissue injury in acute inflammatory diseases of respiratory tract. However, the current studies of hTFF2 are mainly focused on the early pathological stage, and no studies on the in vitro large-scale production of recombinant hTFF2 have been reported.

Disclosure of Invention

The invention aims to provide a TFF2-Fc fusion protein, and the coding gene thereof can be efficiently expressed in eukaryotic cells.

The invention also aims to provide a production method of the high-efficiency expression TFF2-Fc fusion protein, which comprises three stages of seed amplification, two-stage purification and separation and four stages of filtration, so that the stock solution of the produced TFF2-Fc fusion protein is high, the method is simple and feasible, and the method is suitable for mass production.

The invention also aims to provide a genetic engineering strain which can express the hTFF2-Fc fusion protein, has large and stable expression quantity, is easy for industrial production, and has low cost and good safety.

In order to achieve the purpose, the invention adopts the following technical scheme:

the TFF2-Fc fusion protein is characterized in that the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.

MKWVTFISLLFLFSSSSRAEKPSPCQCSRLSPHNRTNCGFPGITSDQCFDNGCCFDSSVTGVPWCFHPLPKQESDQCVMEVSDRRNCGYPGISPEECASRKCCFSNFIFEVPWCFFPKSVEDCHYGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:1)

Wherein the fusion is of the Fc fragment to the N-terminus of TFF2 or to the C-terminus of TFF 2.

Wherein the Fc fragment is an Fc fragment derived from an immunoglobulin IgG. Preferably, the IgG is selected from any one of IgG1, IgG2, IgG3 and IgG 4. More preferably, the IgG is IgG 4.

Wherein the Fc fragment comprises a hinge region, a CH2 region and a CH3 region, and the fusion between the TFF2 and the Fc fragment is direct fusion or fusion through a linker sequence.

Wherein GGGGS is a sequence of a linker moiety, the sequence before GGGGS is an amino acid sequence of TFF2, and the sequence after GGGGS is an amino acid sequence of an Fc fragment of IgG 4.

A gene encoding TFF2-Fc protein for efficient expression in eukaryotic cells, wherein the gene encodes TFF2-Fc fusion protein.

In a specific embodiment, the fusion is of an Fc fragment to the N-terminus of TFF 2; in another specific embodiment, the fusion is of an Fc fragment to the C-terminus of TFF 2. In a preferred embodiment, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1. In a specific embodiment, the encoding gene of the TFF2-Fc fusion protein has the nucleotide sequence shown in SEQ ID NO. 2; or a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID NO. 2 and capable of encoding the amino acid sequence shown in SEQ ID NO. 1. In another specific example, the gene encoding TFF2-Fc fusion protein has a nucleotide sequence in which one or more nucleotide residues are inserted, deleted or added into the sequence of SEQ ID NO. 2 and which is capable of encoding the amino acid sequence shown in SEQ ID NO. 1.

GAGAAGCCTTCTCCTTGTCAGTGTTCTAGGCTGTCCCCTCACAACAGAACCAATTGCGGATTCCCAGGCATCACATCCGATCAGTGTTTCGATAACGGCTGTTGCTTCGATTCTAGCGTGACAGGAGTCCCTTGGTGTTTTCACCCTCTGCCTAAGCAGGAATCAGATCAGTGCGTGATGGAGGTGTCCGATAGAAGAAATTGCGGCTACCCAGGAATTTCTCCAGAGGAGTGCGCTTCCAGAAAGTGTTGCTTCTCCAACTTCATCTTCGAGGTCCCTTGGTGTTTCTTTCCTAAGAGCGTGGAAGATTGTCATTACGGAGGAGGAGGCTCCGAGTCTAAGTACGGACCTCCTTGTCCTCCTTGTCCAGCTCCAGAATTTCTGGGAGGACCTTCCGTGTTCCTGTTTCCTCCTAAGCCTAAGGACACCCTGATGATCTCTAGAACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCTCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGAGTGGAAGTGCATAACGCTAAAACCAAGCCTAGAGAGGAGCAGTTTAACTCCACCTATAGAGTGGTGTCAGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGAAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGACTGCCTTCTTCCATCGAGAAGACCATCTCCAAGGCTAAGGGACAGCCTAGAGAACCTCAGGTGTATACACTGCCTCCTTCTCAGGAGGAGATGACAAAGAACCAGGTCTCTCTGACTTGCCTGGTGAAGGGATTTTACCCTTCCGATATCGCCGTGGAATGGGAATCTAACGGACAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCTAGGCTGACAGTGGATAAGTCTCGTTGGCAGGAAGGCAACGTGTTCTCTTGTTCCGTGATGCACGAGGCTCTGCATAATCACTACACACAGAAGTCCCTGTCTCTGTCTCTGGGAAAA(SEQ ID NO:2)

A nucleic acid construct comprising the nucleotide sequence of the invention as described previously herein. In a specific embodiment, the nucleic acid construct is a vector capable of mediating gene expression in a eukaryotic host cell, the vector being selected from one or more of an adenoviral vector, an adeno-associated viral vector, and a plasmid vector; in another specific embodiment, the plasmid vector is selected from one or more of pcDNA3.1, pCMV, pTRE, pSL and pBudCE4.1, and most preferably the plasmid vector is pcDNA3.1.

A genetically engineered host cell comprising a nucleic acid sequence as described previously herein or comprising a nucleic acid construct as described previously herein, said host cell being capable of efficient expression to produce a TFF2-Fc fusion protein. In a specific embodiment, the host cell is a eukaryotic cell selected from any one of a yeast cell, a plant cell, an insect cell, and a mammalian cell; preferably, the host cell is a mammalian cell, more preferably a cell selected from one or more of BHK cells, VERO cells and CHO cells, more preferably a CHO cell, most preferably a CHO-K1 cell.

A production method for efficiently expressing TFF2-Fc fusion protein, which produces TFF2-Fc fusion protein by culturing the genetically modified host cell. In one particular implementation, the method comprises the steps of: a) an upstream cell culture process, which comprises seed recovery, shake flask amplification, Wave system amplification, reactor seed amplification, reactor fed-batch culture and clarification filtration; b) downstream protein purification procedures including affinity chromatography, low pH virus inactivation, anion exchange chromatography, virus removal filtration, ultrafiltration or wash filtration processes and stock preparation.

Wherein the volume of the Wave system is 10-25L. Preferably, the Wave system is the "ReadyToProcess Wave 25 Wave bioreaction System". The Wave system works in a swinging mode, liquid swings up and down at a certain angle in a culture bag of the reaction system to achieve the effects of mixing and mass transfer, and gas is introduced from the surface and is commonly used for cell amplification.

Preferably, the reactor in the process is an XDR200 or XDR500 reactor, which differs from Wave systems mainly in the aeration and agitation means. XDR200 and XDR500 reactors, the stirring paddle is arranged below the reactors and is driven by a magnetic stirrer to achieve the liquid mixing effect, and gas is generally introduced at the bottom of the liquid.

Specifically, the production method of the high-efficiency expression TFF2-Fc fusion protein comprises the following steps:

a. seed recovery: thawing the frozen tube of the host cell containing the expression vector of the TFF2-Fc fusion protein in water bath, and recovering;

b. cell expansion: sequentially carrying out shake flask amplification, Wave system amplification and reactor amplification on the recovered host cells step by step;

c. feeding culture in a reactor: culturing the seed cells after the amplification in a fed-batch manner for 12-18 days to obtain a culture solution;

d. clarifying and filtering: filtering the culture solution;

e. affinity chromatography: eluting with 45-55mmol/L acetic acid-sodium acetate buffer solution with pH of 3-4, and collecting eluate;

f. low pH viral inactivation: diluting the collected eluate to less than or equal to 15g/L, adjusting pH to 2.50-4.50, inactivating, deep-filtering, and collecting filtrate;

g. anion exchange chromatography: eluting the filtrate with an eluent of pH7.5 containing 20mmol/L Tris-HCl, 50mmol/L arginine hydrochloride and 1mol/L sodium chloride, and collecting the eluent with ultraviolet absorption value of 280nm of 150mAu/mm-150 mAu/mm;

h. virus removal filtration and concentration: filtering the eluate again to remove virus, and concentrating;

i. preparing stock solution: diluting the concentrated protein solution to protein concentration of 45-55mg/ml, filtering, packaging, and freezing for storage.

Wherein, the expression "collecting the eluent with ultraviolet 280nm absorption value of 150mAu/mm-150 mAu/mm" means that the eluent is collected from the time when the ultraviolet absorption value rises to 150mAu/mm at 280nm, and the eluent is stopped collecting when the ultraviolet absorption value is reduced to 150mAu/mm again.

In a specific embodiment, the seed resuscitation comprises: and (3) thawing the frozen tube of the host cell containing the expression vector of the TFF2-Fc fusion protein in water bath at 36.5-38.5 ℃ and recovering.

In a specific embodiment, the shake flask amplification comprises: at 0.3X 10⁶-0.6×10⁶Inoculating the recovered cells at an inoculation density of one cell/ml at 36.5 + -0.5 deg.C and 6.0 + -1.0% CO₂And culturing for 2-4 days at 130 +/-10 rpm for subculture until the cell amount required by Wave system seed amplification is reached.

In a specific embodiment, the Wave system amplification comprises: at 0.5X 10⁶-0.8×10⁶Inoculating the cells after the cell expansion in the shake flask at the inoculation density of one cell/ml, and shaking at the temperature of 35.5-37.5 ℃ at the shaking speed of 16-20r/min at the angle of 6-10 degrees and CO₂The flow rate is 30-50ml/min, and the air flow rate is 340-.

In a specific embodiment, the reactor seed amplification comprises: initial inoculum density at 0.6X 10⁶-1.0×10⁶Inoculating the cells amplified by the Wave system at the inoculation density of each cell/ml, and amplifying the cells under the conditions of pH 6.5-7.2, DO 40%, temperature 35.5-37.5 ℃ and stirring speed of 60-150 r/min, wherein DO control is related to the bottom, and pH control is related to carbon dioxide.

In a specific embodiment, the reactor fed-batch culture comprises: at 0.8X 10⁶-1.2×10⁶Inoculating the cells after the seed amplification of the reactor at the inoculation density of each cell/ml, and controlling the culture temperature of 35.5-37.5 ℃ and the culture temperature of D0-D5 and the temperature of 32-34.0 ℃ after D5 at the pH of 6.5-7.2 and DO40 percent; culturing under the condition of stirring rotating speed of 60-150 r/min, and associating D at the bottomControlling O and pH with carbon dioxide, and continuously culturing for 12-18 days to obtain culture solution.

In a specific embodiment, the clarifying filtration comprises: clarifying and filtering the harvest solution with two-stage clarifying and filtering membrane package and capsule filter (preferably 0.22 μm capsule filter), wherein the filtering pressure is less than or equal to 1.5bar, and the loading of the one-stage membrane package is less than or equal to 71.3L/m²The secondary film packing amount is less than or equal to 142.6L/m²The flux is adjusted to be less than or equal to 100L/m²The culture broth was filtered.

In a specific embodiment, the affinity chromatography comprises: the height of the chromatographic column is 10-27 cm, the retention time is 3-8min, the loading capacity is less than or equal to 25.1g/L, and the eluent with 50mAu/mm-50mAu/mm at the ultraviolet 280nm position is collected during elution.

In a specific embodiment, the low pH viral inactivation comprises: diluting the eluate to less than or equal to 15g/L, adjusting pH to 2.50-4.50, standing at 18-26 deg.C for 60-90min, and adjusting pH to 5-7; using a depth filtration membrane (preferably, the maximum loading is 85-110L/m)²) And carrying out deep filtration on the virus inactivation collected liquid, and temporarily storing the obtained filtrate at 2-8 ℃.

In a specific embodiment, the anion exchange chromatography comprises: adjusting the pH value of a sample to 7.50 +/-0.10, adjusting the conductivity to be less than or equal to 6mS/cm, keeping the time for 3-8min, loading the sample to be less than or equal to 30g/L, performing gradient elution to collect the eluent with the ultraviolet 280nm absorption value of 150mAu/mm-150mAu/mm, adjusting the pH value to 7.00 +/-0.10, and filtering by using a bag filter.

In a specific embodiment, the virus removal filtering comprises: adopting a prefiltering filter and a virus removing filter, wherein the loading capacity is less than or equal to 2.4kg/m2, rinsing the filter by 10L/m2 after filtering, the pressure in the whole process is not more than 29psi, merging the two filtrates, and filtering by a capsule filter to obtain a filtrate.

In a specific embodiment, the concentrating in step h comprises: and (3) concentrating the filtrate to 20.0-30.0 g/L, starting washing and filtering, continuously changing the filtrate and concentrating to more than 50g/L, rinsing the ultrafiltration system and the pipeline, collecting the membrane washing solution, and combining the concentrated solution and the membrane washing solution to obtain a mixed solution.

In a specific embodiment, the stock solution preparation comprises: diluting the mixed solution to protein concentration of 45-55mg/ml, mixing, filtering with capsule filter, and subpackaging in sterile liquid storage bag at-30 deg.C.

Preferably, in the reactor fed-batch culture, the temperature is adjusted to 32-34 ℃ when the culture reaches 5 days (D5); in the fermentation culture process, when the glucose concentration is less than 5g/L, the sugar is supplemented to 5-10 g/L; feed medium A of 3.0 to 8.0% by volume of the initial medium and feed medium B of 0.15 to 0.8% by volume of the initial medium were fed on day 3 (D3), day 5 (D5), day 7 (D7), day 9 (D9) and day 11 (D11), respectively.

Preferably, in the reactor fed-batch culture, the reactor is a 500L disposable bioreactor; the fed-batch culture medium is ActiPro, the feed culture medium A is Cell Boost7a, and the feed culture medium B is Cell Boost 7B (all three culture media are produced by Sitoufang bioengineering technology, Guangzhou). Optionally, the feeding medium may be dynamis (gibco).

Preferably, in affinity chromatography, the affinity elution buffer is 40-60mmol/L acetic acid-sodium acetate pH 3.5.

Preferably, in anion exchange chromatography, the anion elution buffer is a mixture of 10-30mmol/L Tris-HCl, 40-60mmol/L arginine hydrochloride and 0.5-2mol/L sodium chloride, pH 7.5.

Preferably, in the concentration step, the ultrafiltration equilibration buffer is a mixture of 15-35mmol/L PB and 50-200mmol/L NaCl, pH 7.0.

Preferably, in the stock solution preparation step, the stock solution dilution buffer is a mixture of 15 to 35mmol/L PB and 50 to 200mmol/L sodium chloride, pH 7.0.

The invention has the beneficial effects that: the invention firstly synthesizes a fusion protein of TFF2 and Fc, which realizes high-efficiency expression in eukaryotic cells. In the production method of the high-efficiency expression TFF2-Fc fusion protein, a host cell strain transfected with a TFF2-Fc fusion gene is in a stable growth stage in a self-adaptive state through a gradually increasing seed amplification process, when the strain is amplified to a stable target density, the strain is subjected to fed-batch culture, and a culture medium is correspondingly supplemented along with the continuous growth of the strain, so that the enrichment or waste of all required nutrient elements during one-time addition is avoided; in addition, the effects of purification and concentration step by step are realized by the initial elution of affinity chromatography, the inactivation of low-pH virus, the further elution of anion exchange chromatography and the filtration of virus. Therefore, the TFF2-Fc fusion protein stock solution produced by the production method can be applied to large-scale production in a 500L reactor, and the produced TFF2-Fc fusion protein stock solution has high quality and good consistency of repeated batches.

Drawings

FIG. 1 is a schematic flow chart of the production method of the stock solution of highly expressed TFF2-Fc fusion protein of the present invention.

Detailed Description

Example 1: preparation of cell line expressing TFF2-Fc fusion protein

1. Obtaining of the encoding Gene

Selecting Fc polypeptide fused to C-terminal of TFF2, connecting with connecting peptide to obtain TFF2-Fc fusion polypeptide, and constructing its coding gene sequence according to the structure:

1) the coding gene of TFF2 protein cloned according to the method disclosed in patent application CN201610104936.8 is the starting sequence;

2) modifying an Fc polypeptide sequence of IgG4 disclosed by UniProt in a protein database by using a coding sequence of the Fc polypeptide sequence, and obtaining a coding sequence by using a CHO (Chinese hamster ovary) preference codon;

3) selecting GGGGS as a joint to obtain a coding sequence of the joint;

4) the nucleic acid sequences of 1) -3) were ligated according to the structure of TFF 2-linker-Fc and optimized according to the codon preference of CHO-K1 to obtain the nucleotide sequence shown in SEQ ID NO. 2.

2. Vector construction

1) The nucleotide sequence obtained above is delivered to Nanjing Kingsrei Biotech Co.Ltd to synthesize a fragment;

2) adding restriction enzyme cutting sites at both ends of the sequence by PCR;

3) performing double digestion on the pcDNA3.1 and the sequence obtained in the step 2) by using the same endonuclease, and performing agarose gel electrophoresis, and then cutting the gel to recover a coding fragment and a linear plasmid fragment;

4) passing the two fragments obtained in step 3) through T4 ligase at 16 ℃ overnight;

5) transfecting a ligation product with DH5, selecting a positive clone, extracting a plasmid after PCR verification, and sequencing;

6) carrying out amplification culture on the positive clone strains, and extracting plasmids by using a medium-sized extraction kit;

7) the plasmid was transfected into cells CHO-K1 by electrotransfection, screened with MSX, and monoclonals were obtained by single cell plating and deposited in liquid nitrogen.

Example 2: production method for efficiently expressing TFF2-Fc fusion protein

1. Seed recovery: the frozen tube of the transfected cell CHO-K1 prepared in example 1 was thawed and recovered in a water bath at 37.5 ℃;

2. and (3) flask amplification: at 0.5X 10⁶Inoculating the recovered cells at cell/ml inoculation density, wherein the shake culture conditions are 36.5 + -0.5 deg.C and 6.0 + -1.0% CO₂Culturing at 130 +/-10 rpm for 3 days for passage, and transferring to a Wave system after the cell amount required by Wave seed amplification is reached;

wave system amplification: at 0.7X 10⁶Inoculating the cells after the cell amplification in the shake flask at the inoculation density of cells/ml, and shaking at the temperature of 36.5 ℃, the shaking speed of 18r/min, the angle of 8 degrees and CO₂Amplifying under the conditions of 40ml/min of flow and 360ml/min of air flow;

4. and (3) amplification of reactor seeds: at 0.8X 10⁶Inoculating the cells amplified by the Wave system at the inoculation density of cells/ml, amplifying at the conditions of pH7, DO 40%, temperature 36.5 ℃, stirring speed of 100r/min, and associating DO control at the bottom and pH control with carbon dioxide;

5.500L Disposable bioreactor Fed-batch culture: at 1.0 × 10⁶Inoculating the cells after the seed amplification of the reactor at the inoculation density of cells/ml, culturing the cells under the conditions of pH7, DO40 percent, temperature 36.5 ℃, stirring speed of 100r/min, and associating DO control at the bottom and pH control with carbon dioxide; when the culture is carried out to D5, adjusting the temperature to 33 ℃; during the fermentation culture, when the glucose concentration is higher<When the sugar content is 5g/L, the sugar content is 7 g/L; feeding 5.0% of feed medium A and 0.3% of feed medium to D3, D5, D7, D9 and D11 respectivelyA medium B, wherein the fed-batch culture medium is ActiPro, the feed culture medium A is Cell Boost7a, and the feed culture medium B is Cell Boost 7B;

6. a clarification and filtration stage: clarifying and filtering the harvest liquid by adopting a two-stage clarifying and filtering membrane package and a 0.22 mu m bag filter, wherein the filtering pressure is less than or equal to 1.5bar, and the loading capacity of the first-stage membrane package is less than or equal to 71.3L/m²The secondary film packing amount is less than or equal to 142.6L/m²The flux is adjusted to be less than or equal to 100L/m²H, filtering the harvest liquid;

7. affinity chromatography: the height of the chromatographic column is 20cm, the retention time is 5min, the loading capacity is less than or equal to 25.1g/L, the chromatographic column is eluted by 50mmol/L acetic acid-sodium acetate buffer solution with the pH value of 3.5, 50mAu/mm-50mAu/mm of eluent at the position of 280nm of ultraviolet is collected during elution, and the eluent is collected;

8. low pH viral inactivation: diluting the sample to less than or equal to 15g/L, adjusting pH to 3.50, standing at 22 deg.C for 70min, adjusting pH to 6.00, and performing adsorption and deep filtration with deep filtration membrane to obtain virus inactivation collection solution with maximum loading of 97.3L/m²Filtering the collected liquid by a 0.22 mu m bag filter, and temporarily storing at 4 ℃;

9. anion exchange chromatography: adjusting the pH value of a sample to 7.50 +/-0.10, adjusting the conductivity to be less than or equal to 6mS/cm, keeping for 5min, eluting with a buffer solution containing 20mmol/L Tris-HCl, 50mmol/L arginine hydrochloride and 1mol/L sodium chloride, having a pH value of 7.5, collecting an eluate with an ultraviolet absorption value of 150mAu/mm-150mAu/mm by gradient elution, adjusting the pH value to 7.00 +/-0.10, and filtering the eluate by a 0.22um bag filter;

10. virus removal and filtration: adopting a prefiltering filter and a virus removing filter, wherein the loading capacity is less than or equal to 2.4kg/m²And after filtration, the filter is rinsed by 10L/m²Combining the filtrates, filtering with 0.22um capsule filter, and collecting the filtrate;

11. ultrafiltration concentration liquid change: when the filtrate obtained in the step 10 is concentrated to 25g/L, washing and filtering the filtrate by using a buffer solution which is pH7.0 and contains 25mmol/L PB and 100mmol/L sodium chloride, continuously changing the solution and concentrating the solution to more than 50g/L, rinsing an ultrafiltration system and a pipeline by using the same buffer solution, collecting a membrane washing solution, and combining the concentrated solution and the membrane washing solution to obtain a mixed solution;

12. preparing stock solution: diluting the mixture to 50mg/ml protein concentration with pH7.0 dilution buffer containing 25mmol/L PB and 100mmol/L sodium chloride, mixing, filtering with 0.22 μm capsule filter, and storing at-30 deg.C or below in sterile storage bag, wherein the whole preparation process is shown in FIG. 1.

The quality detection result of the TFF2-Fc fusion protein stock solution prepared by the steps is shown in the following table 1:

TABLE 1

Example 3

In this example, the procedure and the operating conditions were the same as in example 2 except that "33 ℃ adjusted temperature in D5" in step 5 of example 2 was replaced with "34 ℃ adjusted temperature in D5".

The quality detection result of the prepared TFF2-Fc fusion protein stock solution is shown in the following table 2:

TABLE 2

Example 4

In this example, the procedure and the operating conditions were the same as in example 2 except that "the temperature was adjusted to 33 ℃ in the case of culturing to D5" in step 5 of example 2 was replaced with "the temperature was adjusted to 32 ℃ in the case of culturing to D5".

The quality detection result of the prepared TFF2-Fc fusion protein stock solution is shown in the following table 3:

TABLE 3

Example 5

In this example, the fermentation culture step was adjusted as follows according to step 5 of example 2: feeding culture in a 500L disposable bioreactor: at 1.0 × 10⁶Inoculating the cells after the seed amplification of the reactor at the inoculation density of cells/ml, culturing the cells under the conditions of pH7, DO40 percent, temperature 36.5 ℃, stirring speed of 100r/min, and associating DO control at the bottom and pH control with carbon dioxide; when the culture is carried out to D5, adjusting the temperature to 33 ℃; during the fermentation culture, when the glucose concentration is higher<When the sugar content is 5g/L, the sugar content is supplemented to 5 g/L; 8.0% of feed medium A and 0.8% of feed medium B were fed to D3, D5, D7, D9 and D11, respectively, wherein feed medium A was Cell Boost7a and feed medium B was Cell Boost 7B.

The remaining steps and operating conditions were the same as in example 2.

The quality detection result of the prepared TFF2-Fc fusion protein stock solution is shown in the following table 4:

TABLE 4

Example 6

In this example, the fermentation culture step was adjusted as follows according to step 5 of example 2: feeding culture in a 500L disposable bioreactor: at 1.0 × 10⁶Inoculating the cells after the seed amplification of the reactor at the inoculation density of cells/ml, culturing the cells under the conditions of pH7, DO40 percent, temperature 36.5 ℃, stirring speed of 100r/min, and associating DO control at the bottom and pH control with carbon dioxide; when the culture is carried out to D5, adjusting the temperature to 33 ℃; during the fermentation culture, when the glucose concentration is higher<When the sugar content is 5g/L, the sugar content is supplemented to 10 g/L; 3.0% of feed medium A and 0.15% of feed medium B were fed to D3, D5, D7, D9 and D11, respectively, wherein feed medium A was Cell Boost7a and feed medium B was Cell Boost 7B.

The remaining steps and operating conditions were the same as in example 2.

The quality detection result of the prepared TFF2-Fc fusion protein stock solution is shown in the following table 5:

TABLE 5

The above examples demonstrate that the stepwise incremental seed amplification mode and fed-batch culture method of the present invention are advantageous for stable growth of host cell strains, so that the purity of the protein in the finally prepared TFF2-Fc fusion protein stock solution measured by three purity measurement methods is more than 93%, and the residual toxin in the protein stock solution is excellently reduced and the contents of exogenous DNA residue and protein a residue are also extremely low due to the adoption of double chromatographic separation and four filtration steps. Thus, the method of the invention realizes 500L or more large-scale production by combining specific amplification, culture, filtration and purification steps, and has high consistency of repeated batches.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the present invention, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the present invention.

Sequence listing

<110> Shandong Jinghui Biotechnology Ltd

<120> TFF2-Fc fusion protein and high-efficiency expression production method thereof

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<170> SIPOSequenceListing 1.0

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Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu Ser Pro His Asn Arg

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Thr Asn Cys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Asp Asn

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Gly Cys Cys Phe Asp Ser Ser Val Thr Gly Val Pro Trp Cys Phe His

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Pro Leu Pro Lys Gln Glu Ser Asp Gln Cys Val Met Glu Val Ser Asp

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Arg Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu Cys Ala Ser

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Arg Lys Cys Cys Phe Ser Asn Phe Ile Phe Glu Val Pro Trp Cys Phe

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Phe Pro Lys Ser Val Glu Asp Cys His Tyr Gly Gly Gly Gly Ser Glu

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

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

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

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

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

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ser Leu Gly Lys

340

<210> 2

<211> 1020

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagaagcctt ctccttgtca gtgttctagg ctgtcccctc acaacagaac caattgcgga 60

ttcccaggca tcacatccga tcagtgtttc gataacggct gttgcttcga ttctagcgtg 120

acaggagtcc cttggtgttt tcaccctctg cctaagcagg aatcagatca gtgcgtgatg 180

gaggtgtccg atagaagaaa ttgcggctac ccaggaattt ctccagagga gtgcgcttcc 240

agaaagtgtt gcttctccaa cttcatcttc gaggtccctt ggtgtttctt tcctaagagc 300

gtggaagatt gtcattacgg aggaggaggc tccgagtcta agtacggacc tccttgtcct 360

ccttgtccag ctccagaatt tctgggagga ccttccgtgt tcctgtttcc tcctaagcct 420

aaggacaccc tgatgatctc tagaacccca gaagtgactt gcgtggtggt ggacgtgtct 480

caggaagatc ccgaggtgca gttcaattgg tacgtggacg gagtggaagt gcataacgct 540

aaaaccaagc ctagagagga gcagtttaac tccacctata gagtggtgtc agtgctgaca 600

gtgctgcatc aggattggct gaacggaaag gagtacaagt gcaaggtgtc caacaaggga 660

ctgccttctt ccatcgagaa gaccatctcc aaggctaagg gacagcctag agaacctcag 720

gtgtatacac tgcctccttc tcaggaggag atgacaaaga accaggtctc tctgacttgc 780

ctggtgaagg gattttaccc ttccgatatc gccgtggaat gggaatctaa cggacagcca 840

gagaacaact acaagaccac acctccagtg ctggactccg acggctcctt cttcctgtac 900

tctaggctga cagtggataa gtctcgttgg caggaaggca acgtgttctc ttgttccgtg 960

atgcacgagg ctctgcataa tcactacaca cagaagtccc tgtctctgtc tctgggaaaa 1020

Claims

1. The TFF2-Fc fusion protein is characterized in that the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.

2. The fusion protein of claim 1, wherein the fusion is of the Fc fragment to the N-terminus of TFF2 or to the C-terminus of TFF 2.

3. A gene encoding TFF2-Fc protein for efficient expression in eukaryotic cells, wherein the encoding gene encodes the fusion protein of claim 1 or 2.

4. The encoding gene of claim 3, wherein the encoding gene has:

1) a nucleotide sequence shown as SEQ ID NO. 2;

2) a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the nucleotide sequence of SEQ ID NO. 2 and capable of encoding the amino acid sequence set forth in SEQ ID NO. 1; or

3) One or more nucleotide residues are inserted, deleted or added in the nucleotide sequence shown in SEQ ID NO. 2, and the nucleotide sequence can encode the amino acid sequence shown in SEQ ID NO. 1.

5. A nucleic acid construct comprising the nucleotide sequence of claim 3 or 4.

6. The nucleic acid construct of claim 5, wherein the nucleic acid construct is a vector capable of mediating gene expression in a eukaryotic host cell, the vector being selected from one or more of an adenoviral vector, an adeno-associated viral vector, and a plasmid vector; preferably, the plasmid vector is selected from one or more of pcDNA3.1, pCMV, pTRE, pSL and pBudCE4.1; more preferably, the plasmid vector is pcDNA3.1.

7. A genetically engineered host cell comprising a nucleotide sequence according to claim 3 or 4 or comprising a nucleic acid construct according to claim 5 or 6, and wherein said host cell expresses a TFF2-Fc fusion protein.

8. The host cell of claim 5, wherein the host cell is a eukaryotic cell selected from any one of a yeast cell, a plant cell, an insect cell, and a mammalian cell; preferably, the host cell is a mammalian cell, more preferably a cell selected from one or more of BHK cells, VERO cells and CHO cells, more preferably a CHO cell, most preferably a CHO-K1 cell.

9. A method for producing a highly expressed TFF2-Fc fusion protein, wherein the TFF2-Fc fusion protein is produced by culturing the genetically modified host cell according to claim 6 or 7.

10. The production method according to claim 9, characterized in that it comprises the steps of: a) an upstream cell culture process, which comprises seed recovery, shake flask amplification, Wave system amplification, reactor seed amplification, reactor fed-batch culture and clarification filtration; b) downstream protein purification procedures including affinity chromatography, low pH virus inactivation, anion exchange chromatography, virus removal filtration, ultrafiltration or wash filtration processes and stock preparation.