CN117646034A - Construction and protein purification method of recombinant mIL13 lentivirus suspension serum-free stable cell strain - Google Patents

Abstract

The invention relates to the biomedical field, in particular to a construction method of a recombinant mIL13 lentivirus suspension serum-free stable cell strain, which comprises the steps of firstly inserting a mIL13 gene into a pLVX-AcGFP1-N1 lentivirus transfer vector plasmid to construct a mIL13 recombinant lentivirus transfer vector plasmid, then transfecting HEK293F cells under specific conditions by the recombinant plasmid, carrying out suspension culture in a serum-free culture medium to obtain mIL13 recombinant lentivirus liquid, finally infecting mammal cells by the mIL13 recombinant lentivirus liquid, carrying out suspension culture by using an OPM-293CD05 culture medium, and screening by using puromycin. The method not only solves the defects that adherent cells depend on FBS and are difficult to produce in an amplified way, but also avoids the defects that a lentivirus commercial expression system is high in cost and complex in operation, and meanwhile, can quickly and efficiently obtain stable cell strains, improve the productivity, reduce the production cost of recombinant proteins and obtain higher activity.

Description

Construction and protein purification method of recombinant mIL13 lentivirus suspension serum-free stable cell strain

Technical Field

The invention relates to the biomedical field, in particular to a construction and protein purification method of a recombinant mIL13 lentivirus suspension serum-free stable cell strain.

Background

Interleukin IL-13 is an immunomodulatory cytokine secreted primarily by activated Th2 cells and plays a key role in regulating inflammation, immune response and disease, and in addition, it inhibits the production of pro-inflammatory cytokines and chemokines, thereby down regulating macrophage activity. The IL13 protein is more important as a central medium of an immunoregulation process in various cell types, and the expression of the protein is still mainly expressed by a prokaryote at present, but the prokaryote expressed protein has the problem of low activity, so that the expression yield of mammalian cells is extremely low, and the transient yield is extremely unstable. In the research of gene functions, the stable expression cell strain can make up for the defect of short expression time of an exogenous gene in an instant infection (or transfection) experiment, is convenient for long-term observation of the interaction between proteins and the influence of genes on the cell functions, has better stability and smaller difference between batches, and is an important tool for developing gene function research, target spot verification and drug screening.

The construction of stable cell lines is generally divided into a lipid transfection method and a virus transfection method, the liposome transfection method has the problems of long transfection period and low efficiency, and the slow virus transfection screening of stable cell lines has higher efficiency and is suitable for most cells difficult to be transfected, so that the method is an ideal method for constructing stable cell lines. However, most existing lentivirus transfection processes adopt an adherent cell process (such as HEK 293T) which depends on bovine serum (FBS), and the following disadvantages often exist: (1) The serum from animal sources is used, so that the risk of foreign viral factor pollution is increased, the serum is complex in component and undefined in component, and the regulatory spirit of drug production registration is not met; (2) Peptide bovine serum (FBS) batches and places of production are susceptible to large differences, which can lead to batch-to-batch differences in the expressed lentiviruses; (3) The animal-derived trypsin is used for digesting the adherent cells, so that exogenous viral factors can be introduced, and the quality control difficulty is increased; (4) The wall-attached cell process is mostly a cell factory, so that the process is difficult to amplify, the yield is difficult to raise, the cost is difficult to reduce, and the labor force consumption is huge; (5) The cell line for producing lentivirus by using the adherent cells is usually HEK293T (high in virus titer), and because the cell line carries monkey vacuole virus 40 large T antigen (SV 40 large T antigen), potential cancerogenic risks exist, if the cell line cannot be well removed by downstream processes, a certain risk exists for clinical treatment, and the large T antigen has certain immunogenicity, the difficulty of clinical treatment can be increased, so that related residual detection of products is also needed.

At present, although the LV-MAX lentivirus expression system based on Thermo company can achieve high titer lentivirus, the culture medium and transfection reagent are expensive, and the large-scale use in production is difficult.

Disclosure of Invention

Based on the method, the invention provides a construction method of a recombinant mIL13 lentivirus suspension serum-free stable cell strain, which not only solves the defects that adherent cells depend on FBS and are difficult to produce in a magnifying way, but also avoids the defects that a lentivirus commercial expression system is high in cost and complex in operation, and meanwhile, can quickly and efficiently obtain the stable cell strain, improve the productivity, reduce the production cost of recombinant proteins and obtain higher activity.

The invention adopts the following technical scheme to realize the purposes: the construction method of the recombinant mIL13 lentivirus suspension serum-free stable cell strain comprises the following steps:

s1, inserting an mIL13 gene into a pLVX-AcGFP1-N1 lentiviral transfer vector plasmid to construct an mIL13 recombinant lentiviral transfer vector plasmid;

s2, diluting a transfection reagent PEIMax with an Opti-MEM culture medium, incubating for 4-5 min at room temperature, and carrying out 2.0-2.5 mug of total plasmid required per 1mL of cells according to mIL13 recombinant lentiviral transfer vector plasmid: viral packaging helper plasmid pCMV-VSV-G: pRSV-Rev: pspax2= (1.5 to 2.0): 1:1:1 was also diluted with Opti-MEM medium;

mixing the diluted plasmid with the transfection reagent after incubation uniformly, and continuing to incubate at room temperature for 10-20 min to obtain a DNA-PEIMax compound, wherein the mass ratio of the usage amount of PEImax to the total plasmid is (2-3): 1;

the DNA-PEIMax complex is added into living cells with good growth and the density of the living cells is 3.5 to 4.0x10 ⁶ cells/mL HEK293F cells and cultured in suspension with OPM-293CD05 cell culture medium;

1-10 mM sodium butyrate and 3-5% of OPM-293Profeed by volume ratio are added after 16-20h of transfection, slow viruses are harvested after 48-55h of transfection by centrifugation, cell fragments are removed by filtration, and then the mIL13 recombinant slow virus liquid is obtained;

s3, infecting mammal cells with mIL13 recombinant lentivirus liquid, performing suspension culture with an OPM-293CD05 culture medium, and screening with puromycin.

As a preferred embodiment, the method for constructing the mIL13 recombinant lentiviral transfer vector plasmid in step S1 is as follows: adding a Kozak sequence and a signal peptide sequence METDTLLLWVLLLWVPGSTG to the 5' -end of the amino acid sequence Ser26-Phe131 for encoding mIL13, synthesizing, and then inserting into a vector pLVX-AcGFP1-N1 to form a recombinant lentiviral transfer vector pLVX-mIL13;

the recombinant lentiviral transfer vector pLVX-mIL13 and the packaging plasmid pCMV-VSV-G, pRSV-Rev, pSPAX2 are respectively transformed into competent cells of escherichia coli to obtain single colonies; after single colony is selected for culture, recombinant lentiviral transfer vectors pLVX-mIL13, pCMV-VSV-G, pRSV-Rev and pSPAX2 without endotoxin are obtained by extraction with an endotoxin-free plasmid kit.

As a preferred embodiment, the E.coli competent cells include, but are not limited to, stbl3, TOP10 and DH 5. Alpha.

As a preferred embodiment, in step S2, HEK293F cells are prepared as follows: the day before transfection, P4-P20 generations are used for internal and living cell density is 3.5-5.5X10 ⁶ HEK293F cells with cells/mL and > 97% cell viability were diluted to 2.5X10 s with OPM-293CD05 medium ⁶ cells/mL, then subculturing for 24 hours; on the day of transfection, the viable cell density was diluted to 3.5-4.0X10 ⁶ cell/mL ready for use.

As a preferred embodiment, the pore size of the filter membrane used for removing cell debris by filtration is 0.45. Mu.m.

In a preferred embodiment, in step S3, the mammalian cells are Expi293F cells, and the preparation method of the Expi293F cells is as follows: the day before infection, P4-P20 times, the density of living cells is 3.0-5.0X10 ⁶ cell/mL and cell viability>97% of the cells were diluted to a viable cell density of 2.0X10 with OPM-293CD05 medium ⁶ cells/mL, then subcultured for another 24 hours; on the day of transfection, cells were collected by centrifugation and were pooled at 5.0X10 each ⁶ Cell suspensions were prepared at a ratio of 3.+ -. 0.5mL of medium to each living cell.

As a preferred embodiment, in step S3, a polybrene is added to the suspension of the Expi293F cells.

In a preferred embodiment, in step S3, the screening method is as follows: after 72h of infection, the cells were counted, centrifuged and fresh medium was added at 1.0X10 each ⁶ Puromycin is added in a ratio of 1 μg/mL puromycin to cells/mL; changing fresh culture medium containing puromycin every 2-3 days, screening for 4 times, passaging infected and screened cells, applying puromycin for maintenance screening culture, continuously screening and passaging for 3 times to obtain the puromycin-containing plant cell.

The invention also provides a purification method of the mIL13 protein, which comprises the following steps: constructing a mIL13 recombinant lentiviral transfer vector plasmid containing a histidine tag, constructing to obtain a cell strain according to the scheme, culturing the constructed cell strain, centrifuging and collecting the supernatant; and separating and purifying the target protein by adopting an affinity chromatography method.

As a preferred embodiment, the filler used for affinity chromatography is Ni-NTA, and the balance buffer is: 20mM Tris-HCL,250mM NaCl,10mM imidazole, 10% glycerol, pH 8.0;

the flushing buffer solution is as follows: 20mM Tris-HCL,250mM NaCl,40mM imidazole, 10% glycerol, pH 8.0;

elution buffer 1 was: 20mM Tris-HCL,250mM NaCl,80mM imidazole, 10% glycerol, pH 8.0;

elution buffer 2 was: 20mM Tris-HCL,250mM NaCl,250mM imidazole, 10% glycerol, pH 8.0;

elution buffer 3 was: 20mM Tris-HCL,250mM NaCl,500mM imidazole, 10% glycerol, pH 8.0.

The invention constructs mIL13 recombinant lentivirus transfer vector plasmid by inserting mIL13 into pLVX-AcGFP1-N1 vector, then transfects HEK293F cells or derived cells thereof under specific transfection conditions, reagents and culture medium to obtain recombinant lentivirus, and then infects the Expi293F cells with the recombinant lentivirus and carries out suspension culture screening to obtain a stable strain, and the method has at least the following advantages:

(1) During the slow virus production and infection process, HEK293F or cells derived from the HEK293F are used for suspension culture, and the cells do not contain SV40 large T antigen, so that the subsequent residual detection is reduced;

(2) The problem of low virus titer of HEK293F cell packaging viruses is solved by adopting specific transfection conditions, reagents and culture mediums to transfect HEK293F cells or derived cells thereof, and the titer of the HEK293F cell packaging viruses is higher than that of the packaging viruses obtained by adopting HEK293T cell wall-mounted culture in the tradition optimized by the applicant;

(3) In the slow virus production and infection process, animal-derived serum is not used, so that the risk of foreign virus factor pollution is reduced;

(4) In the slow virus production and infection process, cells are cultured in a suspension mode, the culture volume is easy to amplify, a large-scale production process can be met, and the production cost and labor force are reduced;

(5) All raw materials are chemically limited, so that the cell culture condition and the process stability in the production process can be ensured, and the cost is greatly saved by using a domestic culture medium and a conventional transfection reagent;

(6) The construction method uses lentivirus transfection to screen stable cell strains, has high efficiency and short time, can improve productivity, reduce batch-to-batch difference, saves cost and has high protein activity.

Drawings

FIG. 1 is a plasmid map of recombinant lentiviral transfer vector pLVX-mIL13 in example 1 of the present invention;

FIG. 2 shows the results of protein purification in example 2 of the present invention;

FIG. 3 shows the results of comparing virus titers of lentiviral fluid obtained by packing viruses with HEK293F suspension cells using the method of the present invention with those obtained by the conventional method using HEK293T cell adherence;

FIG. 4 is a fitted curve in the activity test of mIL13 protein cells in the assay of the invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.

In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

The reagents used in the examples of the present invention are all commercially available, without specific explanation. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as molecular cloning, as described in Sambrook et al: conditions described in the laboratory Manual (New York: coldSpring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.

The invention provides a construction method of a recombinant mIL13 lentivirus suspension serum-free stable cell strain, which comprises the following steps:

s1, inserting an mIL13 gene into a pLVX-AcGFP1-N1 lentiviral transfer vector plasmid to construct an mIL13 recombinant lentiviral transfer vector plasmid;

specifically, a Kozak sequence and a signal peptide sequence METDTLLLWVLLLWVPGSTG are added to the 5' -end of an amino acid sequence Ser26-Phe131 for encoding mIL13 and synthesized, and then the obtained product is inserted into a vector pLVX-AcGFP1-N1 to form a recombinant lentiviral transfer vector pLVX-mIL13; the recombinant lentiviral transfer vector pLVX-mIL13 and the packaging plasmid pCMV-VSV-G, pRSV-Rev, pSPAX2 are respectively transformed into competent cells of escherichia coli to obtain single colonies; after single colony is selected for culture, recombinant lentiviral transfer vectors pLVX-mIL13, pCMV-VSV-G, pRSV-Rev and pSPAX2 without endotoxin are obtained by extraction with an endotoxin-free plasmid kit.

In this method, E.coli competent cells that can be used are not particularly limited, and include, but are not limited to, stbl3, TOP10 and DH 5. Alpha. Among them, stbl3 competent cells are preferred because they carry recA13 mutation in their genome, which can help reduce the possibility of unnecessary recombination events between LTRs.

S2, diluting a transfection reagent PEIMax with an Opti-MEM culture medium, incubating for 4-5 min at room temperature, and carrying out recombination on lentiviral transfer vector plasmid according to mIL13 by 2.0-2.5 mug of total plasmid required by each 1mL of cells: viral packaging helper plasmid pCMV-VSV-G: pRSV-Rev: pspax2= (1.5 to 2.0): 1:1:1 was also diluted with Opti-MEM medium;

mixing the diluted plasmid with the transfection reagent after incubation uniformly, and continuing to incubate at room temperature for 10-20 min to obtain a DNA-PEIMax compound, wherein the mass ratio of the usage amount of PEImax to the total plasmid is (2-3): 1;

the DNA-PEIMax complex is added into living cells with good growth and the density of the living cells is 3.5 to 4.0x10 ⁶ cells/mL HEK293F cells and cultured in suspension with OPM-293CD05 cell culture medium;

1-10 mM sodium butyrate and 3-5% of OPM-293Profeed by volume ratio are added after 16-20h of transfection, slow viruses are harvested after 48-55h of transfection by centrifugation, cell fragments are removed by filtration, and then the mIL13 recombinant slow virus liquid is obtained;

in this step, HEK293F cells were prepared as follows: the day before transfection, P4-P20 generations are used for internal and living cell density is 3.5-5.5X10 ⁶ cells/mL and is fineHEK293F cells with > 97% cell viability were diluted to 2.5X10 with OPM-293CD05 medium ⁶ cells/mL, then subculturing for 24 hours; on the day of transfection, the viable cell density was diluted to 3.5-4.0X10 ⁶ cell/mL ready for use.

S3, infecting mammal cells with mIL13 recombinant lentivirus liquid, performing suspension culture with an OPM-293CD05 culture medium, and screening with puromycin.

In the step, the screening method comprises the following steps: after 72h of infection, the cells were counted, centrifuged and fresh medium was added at 1.0X10 each ⁶ Puromycin is added in a ratio of 1 μg/mL puromycin to cells/mL; changing fresh culture medium containing puromycin every 2-3 days, screening for 4 times, passaging infected and screened cells, applying puromycin for maintenance screening culture, continuously screening and passaging for 3 times to obtain the puromycin-containing plant cell.

In this step, the mammalian cells are not particularly limited, and one or more of CHO (Chinese hamster ovary, chinese hamster ovary cells), NSO (Mo μse myela, mouse myeloma cells), HEK293 (h μman embryonic kidney293, human embryonic kidney293 cells) may be selected. Among them, preferred mammalian cells are selected from the group consisting of Expi293 cells, and have high infection efficiency.

The preparation method of the Expi293F cells comprises the following steps: the day before infection, P4-P20 times, the density of living cells is 3.0-5.0X10 ⁶ cell/mL and cell viability>97% of the cells were diluted to a viable cell density of 2.0X10 with OPM-293CD05 medium ⁶ cells/mL, then subcultured for another 24 hours; on the day of transfection, cells were collected by centrifugation and were pooled at 5.0X10 each ⁶ Cell suspensions were prepared at a ratio of 3.+ -. 0.5mL of medium to each living cell.

Furthermore, the final concentration of the polybrene is 8 mug/mL, namely, 4 mug of 10mg/mL polybrene solution, 3-4 mL of cell suspension and 1-2mL of virus solution are added to 5mL of total volume (cell suspension and virus solution).

A method for purifying a mll 13 protein, comprising the steps of: constructing a mIL13 recombinant lentiviral transfer vector plasmid containing a histidine tag, constructing to obtain a cell strain according to the scheme, culturing the constructed cell strain, centrifuging and collecting the supernatant; and separating and purifying the target protein by adopting an affinity chromatography method. Further carrying out ultrafiltration concentration and split charging on the affinity purification product to obtain the recombinant mIL protein.

Wherein, the filler used for affinity chromatography is Ni-NTA, and the balance buffer solution is: 20mM Tris-HCL,250mM NaCl,10mM imidazole, 10% glycerol, pH 8.0; the flushing buffer solution is as follows: 20mM Tris-HCL,250mM NaCl,40mM imidazole, 10% glycerol, pH 8.0; elution buffer 1 was: 20mM Tris-HCL,250mM NaCl,80mM imidazole, 10% glycerol, pH 8.0; elution buffer 2 was: 20mM Tris-HCL,250mM NaCl,250mM imidazole, 10% glycerol, pH 8.0; elution buffer 3 was: 20mM Tris-HCL,250mM NaCl,500mM imidazole, 10% glycerol, pH 8.0.

EXAMPLE 1 construction of recombinant lentiviral transfer plasmids

(1) Gene synthesis and cloning construction

The 5' -front end of the amino acid sequence encoding mIL13 (NP-032381.1) Ser26-Phe131 was added with a Kozak sequence (GCCACC) and a signal peptide sequence METDTLLLWVLLLWVPGSTG, while the C-end of the objective gene sequence was added with a 6His tag sequence (HHHH) for easy purification, and the nucleotide sequence was synthesized by general organism (Anhui) Inc. (hereinafter abbreviated as "Anhui general") and inserted into the vector pLVX-AcGFP1-N1, and the synthetic sequence was substituted for the AcGFP1 sequence. The recombinant lentivirus transfer vector pLVX-mIL13 is formed, the above processes are all completed by the general use of Anhui, and the recombinant plasmid and the detection report are returned, and the plasmid map is shown in figure 1.

(2) Extraction of recombinant endotoxin-free plasmid

The transfer plasmid pLVX-mIL13 obtained in the step (1) and the packaging plasmids pCMV-VSV-G, pRSV-Rev and pSPAX2 are respectively transformed into competent cells of Stbl3 to obtain single colonies. Single colonies were picked and cultured overnight in TB medium, and extracted with endotoxin-free plasmid miniextraction medium kit (cat. No. DP118-02, beijing Tiangen Biochemical) to give 2.2. Mu.g/. Mu.L pLVX-mIL13, 1.8. Mu.g/. Mu.L pCMV-VSV-G, 1.6. Mu.g/. Mu.L pRSV-Rev, 1.8. Mu.g/. Mu.L pSPAX2 endotoxin-free plasmid.

(3) Packaging of suspension serum-free mIL13 lentiviruses

The obtained pLVX-mIL13, pCMV-VSV-G, pRSV-Rev and pSPAX2 endotoxin-free plasmids are transfected into HEK293F cells, and the specific steps are as follows:

the day before transfection, HEK293F cells are substituted for P4-P20 generations, and the density of living cells is 3.5-5.5X10% ⁶ cell/mL and cell viability>97% high density cells were diluted to a viable cell density of 2.5X10 ⁶ cells/mL, then subcultured for another 24 hours;

on the day of transfection, viable cell densities were diluted to 4.0X10 in 50mL cell culture tubes ⁶ Transfection mixtures were prepared as follows for cells/ml4.25 mL: the total plasmid amount required per 1mL of cells was 2.5. Mu.g/mL, and plasmid pLVX-mIL13 was transferred: pSPAX2: pCMV-VSV-G: pRSV-Rev=2:1:1:1, transfection reagent PEIMax: total plasmid=2.4:1 (m/m). 135. Mu.g of endotoxin transfer plasmid pLVX-mIL, 2.5. Mu.g of packaging plasmids pCMV-VSV-G, pRSV-Rev and pSPAX2 are added into a 1.5mL microcentrifuge tube containing 0.25mL of Opti-MEM medium, 30. Mu.L of PEIMax (1 mg/mL, pH 7.0) is added into another 1.5mL microcentrifuge tube containing 0.25mL of Opti-MEM, the mixture is incubated at room temperature for 5min, diluted PEImax is added into diluted recombinant plasmid DNA, the mixture is gently mixed and incubated at room temperature for about 15min, and the DNA/PEImax complex is slowly added into diluted cells;

after 16-20h of transfection 3mM sodium butyrate and 4% (v/v) OPM-293Profeed were added, 48-55h after transfection, medium was collected, cells were centrifuged at 1300 Xg in a horizontal rotor centrifuge for 15min, the supernatant was transferred to a fresh tube, lentiviruses were harvested, filtered with 0.45 μm PES filter membrane, cell debris was further removed, and the filtered lentiviruses were stored at-80 ℃.

Wherein, HEK293F cell culture conditions: 200rpm,8% CO ₂ 37 ℃ and humidity>85%; the medium was OPM-293CD05 (Shanghai ao Pu Mai).

(4) Infection of Expi293F cells with mIL13 lentiviral fluid

The obtained mIL13 lentiviral liquid is infected with an Expi293F cell, and the specific steps are as follows:

the day before infection, the Expi293F cells were substituted for P4Within the P20 generation, the viable cell density is 3.0-5.0X10 ⁶ cell/mL and cell viability>97% high density cells were diluted to a viable cell density of 2.0X10 ⁶ cells/mL, then subcultured for another 24 hours;

on the day of infection, cell viability was established in 50mL cell culture tubes>97% of 5.0X10 ⁶ Adding 3.0mL of culture medium plus 4 mu L of 10mg/mL of Polybrene (Polybrene) mixed solution plus 2.0mL of the obtained mIL13 slow virus liquid after centrifugation of the living cells, wherein the final concentration of the Polybrene is 8 mu g/mL;

after infection for 16-18 h, 5mL of fresh medium was centrifuged.

Among them, the Expi293F cell culture conditions: 200rpm,8% CO ₂ 37 ℃ and humidity>85%; the medium was OPM-293CD05 (Shanghai ao Pu Mai).

(5) Screening of mIL 13-stable cells

After 72h of infection of the cells, the cells were counted, centrifuged, fresh medium was added and puromycin (1.0X10) ⁶ 1. Mu.g/mL puromycin was added to cells/mL). Fresh culture medium containing puromycin is changed every 2-3 days, after 4 times of screening, the infected and screened cells are passaged, and puromycin is continuously applied for maintenance screening culture, after 3 times of continuous screening and passaging, the stable cell strain is frozen and preserved.

EXAMPLE 2 expression and purification of mIL 13-stabilized cells

(1) mIL13 stable cell expression

Cells after 4 times of continuous screening and 3 generations of continuous screening are expressed, and cells in logarithmic growth phase are regulated to a density of 1.0X10 by using a preheated OPM-293CD05 culture medium ⁶ cells/mL of living cells, when the cell density in the culture system reaches 3.0-4.0X10 ⁶ At cells/mL, 3% (v/v) OPM-293Profeed feed (Shanghai ao Pu Mai) and 4g/L glucose were added for the first time. 3% (v/v) OPM-293Profeed feed was added every other day to harvest cells, with cell viability around 80%.

(2) mIL13 stable transfer cell purification

Supernatant was collected and purified by centrifugation at 12000rpm for 10 min. His tag affinity chromatography is adopted to separate and purify target protein, and the used filler is Ni-NTA (GE). The obtained supernatant was added with 0.1mM AEBSF,10mM imidazole, bound to a 2mL Ni-NTA column equilibrated beforehand with 20mM Tris-HCl,250mM NaCl,10mM imidazole, 10% glycerol pH 8.0, and then the protein was washed off with 10 volumes of Tris containing 10mM imidazole, and gradient elution was performed with a solution containing 40, 80, 250, 500mM imidazole. The collected flow-through and eluent samples were analyzed by SDS-PAGE and the purification results are shown in FIG. 2.

The buffers used in the affinity chromatography procedure were as follows:

equilibration buffer: 20mM Tris-HCL,250mM NaCl,10mM imidazole, 10% glycerol, pH 8.0.

Flushing buffer: 20mM Tris-HCL,250mM NaCl,40mM imidazole, 10% glycerol, pH 8.0.

Eluent 1:20mM Tris-HCL,250mM NaCl,80mM imidazole, 10% glycerol, pH 8.0.

Eluent 2:20mM Tris-HCL,250mM NaCl,250mM imidazole, 10% glycerol, pH 8.0.

Eluent 3:20mM Tris-HCL,250mM NaCl,500mM imidazole, 10% glycerol, pH 8.0.

Method for packaging lentivirus by using traditional HEK293T cells after optimization of comparative example

The method for culturing and packaging lentivirus by using optimized traditional HEK293T cells in a adherence manner comprises the following specific processes:

16-18 h before transfection, digesting the logarithmic growth phase by trypsin, and the cell viability>95% of 293T cells were subjected to cell density adjustment to 0.4 to 0.5X10 with fresh medium (DMEM+10% FBS) ⁶ cells/mL 2mL/Well was inoculated into 6-Well cell culture plates (to achieve a confluency of cells of about 70-80% at the time of transfection). Mixing, standing at 37deg.C, 5% CO ₂ The incubator is internally provided with a plurality of culture chambers;

2h before transfection, the cell culture medium was replaced with serum-free medium (DMEM);

mu.g of endotoxin-free plasmid (transfer plasmid pLVX-mIL 13.6. Mu.g, packaging plasmid pCMV-VSV-G, pRSV-Rev, pSPAX2 each 0.8. Mu.g) was added to a sterile 1.5mL microcentrifuge tube containing 100. Mu.L of Opti-MEM medium;

will 12 mu LPEImax (1 mg/mL, pH 7.0) was added to another sterile 1.5mL microcentrifuge tube containing 100. Mu.L of Opti-MEM medium, gently mixed and incubated at room temperature for 5min, diluted PEImax was added to diluted recombinant plasmid DNA, gently mixed and incubated at room temperature for about 15min, DNA/PEImax complex was slowly added to diluted cells, gently mixed with shaking in8 words, and left to stand at 37℃with 5% CO ₂ The incubator is internally provided with a plurality of culture chambers;

6-8 hours after transfection, removing the culture medium containing DNA/PEImax; 2ml of fresh medium (DMEM+10% FBS+3mM sodium butyrate) was added to each well and allowed to stand at 37℃with 5% CO ₂ Culturing in the incubator.

After further culturing for 48h, the cell supernatant was collected and centrifuged at 1300 Xg in a horizontal rotor centrifuge for 15min, the supernatant was transferred to a fresh tube, lentiviruses were harvested, filtered with a 0.45 μm PES filter membrane, further cell debris was removed, and the filtered lentiviruses were stored at-80 ℃.

Detection and analysis:

(1) As shown in FIG. 3, it is apparent from FIG. 3 that the virus titer of the mIL13 lentiviral solution obtained in the examples and comparative examples was higher by packing the virus with HEK293F suspension cells according to the method of the present invention than by the conventional method of attaching HEK293T cells according to the following GFP expression method.

The method for measuring the virus titer by using the GFP expression method comprises the following steps:

a. four hours before infection (about 10:00), at 7X 10 in 100. Mu.L of medium (MEM modified +10% FBS) ³ cells/Well density HT1080 cells were seeded in 96 Well plates (confluency of cells at about 30% at the time of infection).

b. 15mL of fresh medium (MEM+10% FBS) was mixed with 12. Mu.L of 10mg/mL Polybrene (final concentration 8. Mu.g/mL), diluted medium was prepared, and then vortexed.

c. About half an hour before infection, in a 1.5mLEP tube, 15. Mu.L of the virus stock was sequentially diluted into 135. Mu.L of the dilution medium to prepare 9 logarithmic serial dilutions (10 ^-1 To 10 ^-9 ) Each log was repeated 2 times.

d、Infected cells (15:00 or so), the inoculation medium was removed from HT1080 cells, then 100. Mu.L of virus dilutions were transferred to corresponding wells, 96 well infected cell plates were centrifuged at 900 Xg for 30min at room temperature, then 37℃at 5% CO ₂ Incubate overnight.

e. The next morning (around 9:00), the medium was removed from HT1080 infected cells and replaced with 100. Mu.L of fresh medium without Polybrene.

f. After 72h incubation of the infected cells, fluorescent cells were observed with a fluorescence microscope.

(2) mIL13 protein cell Activity assay

IL13 has secondary proliferation effect on TF-1 cells (human erythroleukemia cells), biological activity of IL13 can be quantitatively measured by detecting proliferation of TF-1 cells, recombinant mIL13 protein obtained in example 2 is subjected to ultrafiltration tube liquid exchange concentration, and split charging to obtain protein for activity test, and the specific steps are as follows:

TF-1 cells were cultured in complete medium (1640+10% FBS+2ng/mL GM-CSF), experiments were started when the cell viability was above 90%, after a certain amount of cells were collected, washed 2 times with RPMI 1640, resuspended in appropriate amount of detection medium (RPMI 1640+10% FBS) and transferred to T25 flasks, factor starved culture overnight;

the above treated cells were collected, washed 2 times with RPMI 1640, resuspended and counted in 1-2ml of detection medium (RPMI 1640+10% FBS) to prepare a cell density of 4.0X10 ⁵ cell suspension of cells/mL was added to a 96-well plate, 2X 10 ⁴ cells/well；

Protein was diluted 5-fold with detection medium (RPMI 1640+10% fbs), 10 concentration gradients (containing 0 ng/ml), 96 wells, 50 μl/well to final protein concentration: 1000,200,40,8,1.6,0.32,0.064,0.0128,0.00256,0ng/ml. Shaking, mixing, and culturing at 37deg.C with 5% CO2 for 48 hr.

10ul of CCK8 was added to each well, placed in an incubator, OD450 (reference wavelength 630 nm) was detected after 2 and 4 hours, and data were processed using Origin8 to obtain a fitted curve and ED50, as shown in FIG. 4, and the activity of the recombinant protein was calculated to be 23.9-95.6ng/mL.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The construction method of the recombinant mIL13 lentivirus suspension serum-free stable cell strain is characterized by comprising the following steps:

s1, inserting an mIL13 gene into a pLVX-AcGFP1-N1 lentiviral transfer vector plasmid to construct an mIL13 recombinant lentiviral transfer vector plasmid;

s2, diluting a transfection reagent PEIMax with an Opti-MEM culture medium, incubating for 4-5 min at room temperature, and carrying out 2.0-2.5 mug of total plasmid required per 1mL of cells according to mIL13 recombinant lentiviral transfer vector plasmid: viral packaging helper plasmid pCMV-VSV-G: pRSV-Rev: pspax2= (1.5 to 2.0): 1:1:1 was also diluted with Opti-MEM medium;

mixing the diluted plasmid with the transfection reagent after incubation uniformly, and continuing to incubate at room temperature for 10-20 min to obtain a DNA-PEIMax compound, wherein the mass ratio of the usage amount of PEImax to the total plasmid is (2-3): 1;

the DNA-PEIMax complex is added into living cells with good growth and the density of the living cells is 3.5 to 4.0x10 ⁶ cells/mL HEK293F cells and cultured in suspension with OPM-293CD05 cell culture medium;

1-10 mM sodium butyrate and 3-5% of OPM-293Profeed by volume ratio are added after 16-20h of transfection, slow viruses are harvested after 48-55h of transfection by centrifugation, cell fragments are removed by filtration, and then the mIL13 recombinant slow virus liquid is obtained;

s3, infecting mammal cells with mIL13 recombinant lentivirus liquid, performing suspension culture with an OPM-293CD05 culture medium, and screening with puromycin.

2. The method according to claim 1, wherein the method for constructing the mIL13 recombinant lentiviral transfer vector plasmid in step S1 is as follows: adding a Kozak sequence and a signal peptide sequence METDTLLLWVLLLWVPGSTG to the 5' -end of the amino acid sequence Ser26-Phe131 for encoding mIL13, synthesizing, and then inserting into a vector pLVX-AcGFP1-N1 to form a recombinant lentiviral transfer vector pLVX-mIL13;

the recombinant lentiviral transfer vector pLVX-mIL13 and the packaging plasmid pCMV-VSV-G, pRSV-Rev, pSPAX2 are respectively transformed into competent cells of escherichia coli to obtain single colonies; after single colony is selected for culture, recombinant lentiviral transfer vectors pLVX-mIL13, pCMV-VSV-G, pRSV-Rev and pSPAX2 without endotoxin are obtained by extraction with an endotoxin-free plasmid kit.

3. The method of construction according to claim 2, wherein the e.coli competent cells include, but are not limited to, stbl3, TOP10 and DH5 a.

4. A method of constructing HEK293F cells according to any of claims 1 to 3, wherein in step S2 the method of preparing HEK293F cells is as follows: the day before transfection, P4-P20 generations are used for internal and living cell density is 3.5-5.5X10 ⁶ HEK293F cells with cells/mL and > 97% cell viability were diluted to 2.5X10 s with OPM-293CD05 medium ⁶ cells/mL, then subculturing for 24 hours; on the day of transfection, the viable cell density was diluted to 3.5-4.0X10 ⁶ cell/mL ready for use.

5. The method according to any one of claims 1 to 3, wherein the pore size of the filter membrane used for removing cell debris by filtration is 0.45. Mu.m.

6. A method according to any one of claims 1 to 3, wherein in step S3 the mammalian cells are Expi293F cells and the preparation of Expi293F cells is as follows: the day before infection, P4-P20 times, the density of living cells is 3.0-5.0X10 ⁶ cell/mL and cell viability>97% of the cells were diluted to a viable cell density of 2.0X10 with OPM-293CD05 medium ⁶ cells/mL, then subcultured for another 24 hours; day of transfectionCells were collected by centrifugation and washed every 5.0X10 ⁶ Cell suspensions were prepared at a ratio of 3.+ -. 0.5mL of medium to each living cell.

7. The method according to claim 6, wherein in step S3, the suspension of the Expi293F cells is supplemented with polybrene.

8. A method according to any one of claims 1 to 3, wherein in step S3, the screening method comprises: after 72h of infection, the cells were counted, centrifuged and fresh medium was added at 1.0X10 each ⁶ Puromycin is added in a ratio of 1 μg/mL puromycin to cells/mL; changing fresh culture medium containing puromycin every 2-3 days, screening for 4 times, passaging infected and screened cells, applying puromycin for maintenance screening culture, continuously screening and passaging for 3 times to obtain the puromycin-containing plant cell.

A method for purifying a mbil 13 protein, comprising the steps of: constructing a mIL13 recombinant lentiviral transfer vector plasmid containing a histidine tag, constructing to obtain a cell strain according to the scheme of any one of claims 1-8, culturing the constructed cell strain, centrifuging and collecting the supernatant; and separating and purifying the target protein by adopting an affinity chromatography method.

10. The method according to claim 9, wherein the filler used for affinity chromatography is Ni-NTA, and the equilibration buffer is: 20mM Tris-HCL,250mM NaCl,10mM imidazole, 10% glycerol, pH 8.0;

the flushing buffer solution is as follows: 20mM Tris-HCL,250mM NaCl,40mM imidazole, 10% glycerol, pH 8.0;

elution buffer 1 was: 20mM Tris-HCL,250mM NaCl,80mM imidazole, 10% glycerol, pH 8.0;

elution buffer 2 was: 20mM Tris-HCL,250mM NaCl,250mM imidazole, 10% glycerol, pH 8.0;

elution buffer 3 was: 20mM Tris-HCL,250mM NaCl,500mM imidazole, 10% glycerol, pH 8.0.