CN111286517A

CN111286517A - Packaging method of replication-defective retrovirus and application thereof

Info

Publication number: CN111286517A
Application number: CN201811500706.9A
Authority: CN
Inventors: 李靖; 韦苏珍; 曹春来; 陈康月; 贺华; 高佩璇; 史剑锐
Original assignee: Zhuhai United Laboratories Co Ltd
Current assignee: Zhuhai United Laboratories Co Ltd
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2020-06-16

Abstract

The invention provides a packaging method of replication-defective retroviruses and application thereof. The invention constructs a replication-defective retrovirus vector containing a target gene; the constructed viral vector and packaging vector were co-transfected into suspension culture CHO cells, and virus packaging was completed therein. The invention successfully packages the replication-defective retrovirus in the cells cultured in suspension, and utilizes the virus to infect the same suspension cell to obtain a stable cell strain capable of efficiently expressing recombinant proteins. The invention solves the problem of potential infectious factors caused by infecting suspension culture cells after the adherent culture cells are used for packaging viruses in the traditional method, and realizes that the virus packaging cells and protein expression cells in a serum-free culture system are the same cells. The packaging method provided by the invention is utilized to construct a stable cell strain, the yield of the secreted and expressed Fc fusion protein reaches more than 3g/L, and the protein yield of the cell strain after passage for 40 days is 95-100% before passage.

Description

Packaging method of replication-defective retrovirus and application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to a packaging method of replication-defective retroviruses and application thereof.

Background

Methods for transferring a foreign gene into a cell to express the gene include chemical transfection, electroporation, and viral infection. Among them, the virus infection method is the most efficient, and replication-defective retroviruses have become important tools for introducing foreign genes into mammalian cells.

Retroviruses contain two non-segmented, 7-12kb single-stranded RNAs encoding the gag, pol and env genes. The gag gene encodes the capsid protein of the virus, the pol gene encodes reverse transcriptase and integrase, and the env gene encodes the envelope protein of the virus. The artificially modified retrovirus vector retains the packaging signal of the virus genome and lacks genes required for packaging of virus particles, such as gag, pol and env. By expressing the three proteins through the packaging vector, virus particles can be packaged in packaging cells such as HEK-293 and NIH/3T3 cells, and the virus has no capability of generating progeny virus after infecting the cells, namely replication-defective retrovirus. After the replication-defective retrovirus infects cells, reverse transcription and integration processes of the viral RNA genome are completed through the retrovirus-carrying enzyme and integrase, and multiple copies of cDNA are integrated into the host cell genome. Multiple copies of a stably heritable specific nucleic acid sequence may be made available to the genome of the cell. This feature of retroviruses is used to allow, in principle, permanent expression of any foreign gene, such as a protein-encoding gene, in the cell.

The copy number of randomly integrated foreign genes in cell strains constructed by the traditional transfection method is low, the protein yield is low, and chemical drugs such as MTX or MSX are often required to be added to amplify the copy number of target genes. The screening process of obtaining the high-expression cell strain by utilizing the DHFR/MTX system to carry out gene amplification usually needs two rounds, and takes 2-4 months; the GS/MSX system requires one turn and takes 1-2 months. The gene amplification modes of the two systems enable the integrated exogenous gene to be amplified in a large quantity so as to improve the copy number. However, such amplification eventually leads to instability of the amplified gene and gene silencing.

Because the infection efficiency of retrovirus to mitotically active cells is close to 100%, the retrovirus system is used for constructing a stable cell strain, and the process of screening positive clones by antibiotics can be omitted. Retroviruses can mediate multi-copy gene integration, thereby omitting the pressurized screening amplification step. The monoclonal cells were sorted by limiting dilution on day 4 after cell infection. Compared with the traditional method, the method is shortened by 2-4 months. In addition, multiple copies of target genes introduced by the retrovirus vector are integrated in a transcription active region in a host genome, the constructed cell strain has high yield, and the long-term expression stability of the target protein is far superior to that of cells screened by MTX or MSX amplification.

In the conventional retrovirus packaging technology, it is necessary to co-transfect a replication-defective retrovirus vector and a packaging plasmid into an adherently growing packaging cell (such as HEK-293 and NIH/3T3) to complete the packaging of virus particles and obtain a supernatant containing the virus particles, and then infect a host cell to construct a cell strain expressing a target protein. In the biopharmaceutical field, recombinant protein expressing cells are typically cultured in suspension in serum-free medium such as CHO, NS0, per.c6, etc. In the above methods, the packaging cells are typically adherently grown cells in serum-containing media (e.g., HEK-293 and NIH/3T 3). One major drawback of conventional methods is the possibility of bringing infectious agents (non-packaged replication defective retroviruses) to the downstream infected cells, including bacteria, fungi, viruses, mycoplasma, chlamydia, prions, etc. These infectious agents originate from two aspects: (1) packaging cells, such as HEK-293 from human embryonic kidney, NIH/3T3 from mouse embryo, HEK-293 cells containing a defined adenovirus (see ATCC website introduction), in addition to the possibility of containing a variety of other undetected viruses; (2) serum in a packaging cell culture medium is a clear source of infectious factors such as viruses, mycoplasma, prion and the like; (3) in addition to the infectious agent, the serum has an inhibitory effect on the virus infection process, and the virus infection efficiency is reduced; suspension cells in the serum-free culture medium are infected by serum-containing virus liquid and then return to the serum-free culture medium again, so that growth arrest and apoptosis are easily caused, and the difficulty and the workload of cell strain construction are increased.

An important issue in the production of recombinant protein drugs expressed by mammalian cells is the control of viral contamination, and common viruses that can contaminate cell lines are: murine parvovirus (MMV), Reovirus type 3 (Reovirus 3, Reo 3), Murine Leukemia Virus (MLV), Adenovirus (Adenovirus, Ad), Pseudorabies virus (PRV), Human Rhinovirus (HRV), Bovine Polyoma Virus (BPV), Bovine Viral Diarrhea Virus (BVDV), and the like. Possible sources of such viral contamination are: animal derived components in the culture medium such as serum, animal peptone, growth factors, operators in the cell culture and production process, cell culture environment such as shaker, bioreactor, air, virus packaging cells, etc.

Therefore, there is a need to develop a new packaging method for replication defective retroviruses to prevent contamination of infectious agents from conventional replication defective retroviral packaging systems.

Disclosure of Invention

The primary object of the present invention is to overcome the disadvantages of the prior art, mainly the contamination of downstream suspension culture cells with infectious agents from heterologous packaging cells, and to provide a method for packaging replication-defective retroviruses.

Another object of the invention is to provide the use of said packaging method.

The purpose of the invention is realized by the following technical scheme:

a method of packaging a replication defective retrovirus comprising the steps of: constructing a replication-defective retrovirus vector containing a target gene; the retroviral vector containing the target gene and the packaging vector are cotransfected, suspended and cultured CHO cells, and the virus packaging is completed in the CHO cells.

The method for packaging a replication-defective retrovirus preferably specifically comprises the steps of:

(1) using the replication-defective retrovirus vector as a framework, subcloning a target gene onto the replication-defective retrovirus vector to obtain a replication-defective retrovirus recombinant vector containing the target gene;

(2) co-transfecting a replication-defective retrovirus recombinant vector containing a target gene and a packaging vector into suspension-cultured CHO cells;

(3) adding Sodium butyrate (Sodium butyrate) 18-20 hours after transfection to enhance the virus packaging efficiency; after sodium butyrate is treated for 10-12 hours, centrifuging, removing supernatant, and adding a fresh serum-free culture medium for culture;

(4) and harvesting 43-50 hours after transfection to obtain virus suspension.

The replication-defective retroviral vector in step (1) includes, but is not limited to, one or more of a pLPCX vector, another retroviral vector constructed with the pLPCX vector as a backbone, a pBabe vector, and a pLEGFP-C1 vector.

The other retroviral vector constructed by taking the pLPCX vector as a framework is preferably a pRDM vector, the pRDM vector is obtained by modifying the pLPCX vector, and a Puro-hCMV fragment on the pLPCX vector is replaced by Puro-mCMV-WRPE; wherein Puro is puromycin resistance gene, the sequence of mCMV is shown as SEQ ID NO.1, and the sequence of WRPE is shown as SEQ ID NO. 2; the preparation method specifically comprises the following steps:

(A) replacing the Puro-hCMV fragment on the pLPCX vector with Puro-mCMV to obtain pLPCX-Puro-mCMV;

(B) a Woodchuck Hepatitis Virus posttranscriptional Regulatory sequence (WPRE) was inserted downstream of the mCMV promoter on pLPCX-Puro-mCMV vector to obtain a pRDM vector.

The target protein in step (1) is a recombinant protein, including but not limited to monoclonal antibody, coagulation factor, growth factor, cytokine, Fc fusion protein, HSA fusion protein, etc.

The CHO cell used in the step (2) is preferably CHODG44, CHO-K1 or CHO-S.

The packaging plasmids described in step (2) are preferably pCMV-gag-pol and pMD2. G.

The method for co-transfecting the recombinant vector of the target gene and the packaging vector into the suspension culture CHO cells in the step (2) includes, but is not limited to, electroporation transfection, lipofection, PEI transfection, calcium phosphate transfection and the like.

In the co-transfection in the step (2), a DNA-transfection reagent compound is required to be prepared, wherein the DNA comprises a recombinant vector containing a target gene and 2 packaging vectors, and the mixing ratio of the DNA to the recombinant vector to the packaging vectors is 1-2: 1:1, preferably 5:3:3 or 2:1:1 or 1:1: 1; the ratio of the total amount of DNA to the number of cells to be transfected is 0.5-2.0. mu.g/(1X 10)⁶Individual cells).

The final concentration range of sodium butyrate described in step (3) is preferably 1-10 mM.

The medium described in step (3) is a CD medium, more preferably a CD FortiCHO medium, a CD OptiCHO medium, a CDM4CHO medium, or a CD11V medium.

The amount of the replaced fresh serum-free culture medium in the step (3) is preferably 1/4-1/2 of the volume of the culture medium during transfection.

The packaging method of the replication-defective retrovirus is applied to the construction of a monoclonal cell strain with stably expressed protein.

A method for constructing a stable monoclonal cell strain, comprising the following steps: the replication-defective retrovirus packaged by the packaging method is used for infecting the same kind of CHO cells used in the packaging method and screening a monoclonal cell strain; preferably comprising the steps of:

(5) infecting the same kind of CHO cells used in the packaging method with the replication-defective retrovirus suspension packaged by the packaging method, and supplementing a fresh culture medium 4-6 hours after infection to ensure sufficient and reasonable nutrition supply of the infected cells;

(6) replacing a fresh serum-free culture medium 40-50 hours after the retrovirus infection;

(7) the monoclonal cell lines were screened by limiting dilution at 84-120 hours after virus infection.

The construction method of the stable monoclonal cell strain further comprises the following steps:

(8) detecting the yield of the recombinant protein in the culture supernatant of the obtained monoclonal cell strain, taking the cell strain with higher yield, amplifying and culturing the cell strain step by step, and then carrying out shake flask fermentation culture to further evaluate the yield, thereby obtaining a high-yield monoclonal cell strain;

(9) and (3) carrying out subculture on the cell strain with high yield in the shake flask culture for 40 days (about 60 generations of proliferation), and comparing the yield of protein of the freshly recovered cell and the yield of protein of the cell after 40 days of subculture in the shake flask fermentation to obtain a stable and high-yield monoclonal cell strain.

In the infection process described in step (5), the amount of the replication defective retrovirus suspension used is 0.4X 10 per 1ml of the virus fluid⁶～0.6×10⁶And (4) cells.

In the infection process, an infection reinforcing agent polybrene is required to be added, and the dosage of the polybrene is preferably calculated according to the final concentration of 0.5-10 mu g/ml. The final concentration refers to the concentration in the infected system.

The amount of the fresh medium is preferably 4 to 5 times by volume based on the volume of the medium at the time of infection.

In the limiting dilution process described in step (7), the cell density of the plated plate is preferably 0.3-0.5 cells/well.

Compared with the traditional retrovirus packaging and stable cell strain construction technology, the invention has the following advantages:

(1) in the invention, the packaging cell and the infected cell are the same cell, so that the risk of infectious factors caused by heterologous packaging cells is completely eradicated;

(2) serum is not used in the whole process, so that infectious factors from the serum are completely eradicated;

(3) the invention solves the problem of the growth inhibition of suspension cells by serum withdrawal in the traditional method.

The invention provides a safe and efficient technical process for introducing exogenous genes into an expression cell genome, and in order to realize the invention, the following technical problems are solved: (A) the dosage proportion of the vector used for cotransfection in the virus packaging process ensures that the transcribed RNA genome, capsid protein, envelope protein, reverse transcriptase, integrase and the like are in proper proportion, and virus particles can be efficiently packaged; (B) optimizing the dosage of the sodium butyrate capable of improving the virus packaging efficiency; (C) the dosage of virus liquid, the dosage of polybrene and the dosage of CHO cells are optimized during infection, so that the cells can be effectively infected by the virus, the infection does not cause great negative effect on the growth of the cells, and the cell proliferation activity is good.

Drawings

FIG. 1 is a flow chart of the construction of pLPCX-Puro-mCMV-WPRE and pRDM-GLP-1-Fc recombinant retroviral vectors.

FIG. 2 is a graph showing the results of examining the yield of a monoclonal cell strain cultured in 15 ml-shake tube.

FIG. 3 is a graph showing the results of examining the yield of 50 ml-shake tube-cultured monoclonal cell lines.

FIG. 4 is a graph showing the results of measurements of the yield of a 250 ml-shake flask-cultured monoclonal cell strain.

FIG. 5 is a graph showing a comparison between the yields of freshly recovered monoclonal cell lines and the yields of monoclonal cell lines after 40 days of passaging.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

One, pLPCX-Puro-mCMV-WPRE (abbreviated as pRDM) recombinant vector construction, as shown in FIG. 1.

(1) The puromycin (puromycin) gene fragment was amplified by PCR using pLPCX plasmid (Clontech) as a template.

The primers for the PCR used were:

PuroF:5’–CTTACCGGTGCCGCCACCATCCCCTGACCCACGCCCCTGA-3’；

PuroR:5’-ACGCGTGAACTACAGAGTCTCGCTCAGGCACCGGGCTTGCGGGTCA-3’。

and (3) PCR reaction system: 50 μ l × 2

Template pLPCX4ng (2. mu.l), primer Purof 2. mu.l at a concentration of 5. mu.M, primer Puror 2. mu.l at a concentration of 5. mu.M, PrimerSTAR Max mix 25. mu.l, ddH₂Make up to 50. mu.l of O.

PCR procedure: 3min at 94 ℃; 34 cycles of 10s at 98 ℃, 10s at 53 ℃ and 1min at 72 ℃; 5min at 72 ℃; infinity at 4 ℃.

And purifying and recovering the PCR product by using the PCR product purification kit to obtain an AgeI-Puro-MluI fragment.

(2) PCR amplification was carried out using pUC57-mCMV plasmid (the sequence of mCMV is shown in Seq ID No.1, synthesized by Nanjing Kinshire and designated as pUC57-mCMV) as a template and mCMVF and mCMVR as primers, and the MluI-mCMV-BglII fragment was obtained by purification and recovery.

mCMVF:5’-GCGAGACTCTGTAGTTCACGCGTCTACTGAGTCATTAGGGACTTTCCA-3’；

mCMVR:5’-GGAAGATCTCCTGAGGCTGCGTTCTACG-3'

And (3) PCR reaction system: 50 μ l × 2

Template pUC57-mCMV 4ng (2. mu.l), primer mCMVF 2. mu.l at a concentration of 5. mu.M, primer mCMVR 2. mu.l at a concentration of 5. mu.M, PrimerSTAR Max mix 25. mu.l, ddH₂Make up to 50. mu.l of O.

And purifying and recovering the PCR product by using a PCR product purification kit to obtain the MluI-mCMV-BglII fragment.

(3) And carrying out fusion PCR reaction on AgeI-Puro-MluI and MluI-mCMV-BglII through primers Purof and mCMVR, and purifying to obtain an AgeI-HpaI-Puro-MluI-mCMV-BglII gene fragment.

And (3) PCR reaction system: 50. mu.l of 4AgeI-Puro-MluI fragment 4ng (4. mu.l), MluI-mCMV-BglII fragment 4ng (4. mu.l), primer Purof 2. mu.l at a concentration of 5. mu.M, primer MCMVR 2. mu.l at a concentration of 5. mu.M, PrimerSTAR max mix 25. mu.l, ddH₂Make up to 50. mu.l of O.

PCR procedure: 3min at 94 ℃; 34 cycles of 10s at 98 ℃, 10s at 57 ℃ and 2min at 72 ℃; 5min at 72 ℃; infinity at 4 ℃.

And purifying and recovering the PCR product by using the PCR product purification kit to obtain an AgeI-Puro-mCMV-BglII fragment.

(4) The AgeI-Puro-mCMV-BglII fragment and the pLPCX vector (Clontech) were double digested with AgeI and BglII restriction enzymes (Takara), and the fragment with open cohesive ends and the linearized vector were obtained by gel recovery purification experiments. Ligation was performed with T4DNA ligase (Takara). The ligation product is transformed into escherichia coli TOP10 competent cells, and the pLPCX-Puro-mCMV recombinant plasmid is finally obtained through a positive monoclonal screening experiment (bacterial liquid PCR, enzyme digestion identification and sequencing).

(5) NotI-WPRE-ClaI nucleic acid fragment (sequence shown in Seq ID No.2, and synthetic product pUC57-WPRE) was synthesized by Nanjing Kingsler. The NotI-WPRE-ClaI gene fragment with open cohesive ends and the pLPCX-Puro-mCMV linearized vector are obtained by carrying out gel recovery and purification on pUC57-WPRE and pLPCX-Puro-mCMV recombinant plasmids by using NotI and ClaI restriction endonucleases. Ligation was performed with T4DNA ligase. The ligation product is transformed into escherichia coli TOP10 competent cells, and through a positive monoclonal screening experiment (bacterial liquid PCR, enzyme digestion identification and sequencing), pLPCX-Puro-mCMV-WPRE recombinant plasmid (abbreviated as pRDM) is finally obtained, and the vector construction flow is shown in figure 1.

Construction of pRDM-GLP-1-Fc recombinant plasmid

(1) Plasmid pRDM was subjected to BglII and NotI double digestion, and the linear vector was recovered and purified by gel.

(2) BglII-GLP-1-Fc-NotI nucleic acid fragment (the GLP-1-Fc sequence is shown in Seq ID No.3, and the synthesized product is recombinant plasmid pUC57-GLP-1-Fc) was synthesized by Nanjing Kingsler. Carrying out BglII and NotI double enzyme digestion on the plasmid recombinant plasmid pUC57-GLP-1-Fc, and obtaining the GLP-1-Fc fragment with an open cohesive end by a glue recovery and purification method.

(3) The pRDM linearized vector is connected with a GLP-1-Fc fragment through T4DNA ligase, a connecting product is transformed into escherichia coli TOP10 competent cells, and pRDM-GLP-1-Fc recombinant plasmid can be finally obtained through a positive monoclonal screening experiment (bacterial liquid PCR, enzyme digestion identification and sequencing), wherein the vector construction flow is shown in figure 1.

EXAMPLE 2 packaging of retroviral vector pLEGFP-C1 in CHO-S cells and infection of CHO-S cells (optimized for vector dose ratio)

(1) Plasmid extraction: the retrovirus vector pLEGFP-C1(Clontech) and the packaging vector pMD2.G (Addgene), pCMV-gag-pol (cell Biolabs) were subjected to plasmid extraction to obtain high-concentration high-purity endotoxin-free plasmids.

(2) Cell culture: the CHO-S (cGMPBanked,

) The cells were suspended and prepared for virus packaging experiments after 3 passages in CD FortiCHO (containing 8mM L-glutamine) medium using conventional subculture methods.

(3) Packaging and infection of retroviral recombinant vector pLEGFP-C1:

(A) 22-24 hours before cotransfection, CHO-S cells were cultured at 2X 10⁶Cell/ml, 30ml

Expression Medium (C)

Expression Medium, containing 4mM L-glutamine, 0.3% Poloxamer188), passaged; on the day of transfection, CHO-S cells were diluted to 2X 10⁶One cell/ml, and 5 ml/tube into 9 50ml shake tubes.

(B) The total amount of co-transfected plasmid DNA was 5. mu.g, where the mass ratios of pLEGFP-C1, pCMV-gag-pol and pMD2.G are shown in Table 1:

TABLE 1 pLEGFP-C1, pCMV-gag-pol and pMD2.G proportioning protocol in co-transfection

pLEGFP-C1, pCMV-gag-pol and pMD2.G in mass ratio	pLEGFP-C1, pCMV-gag-pol, pMD2.G actual dose (. mu.g)
		1:1:1	1.67、1.67、1.67
2:1:1	2.5、1.25、1.25
		5:3:3	2.3、1.35、1.35
3:2:1	2.5、1.7、0.8
		5:2:1	3.1、1.25、0.625
1:1:2	1.25、1.25、2.5
		3:3:5	1.35、1.35、2.3
1:2:3	0.8、1.7、2.5
		1:2:5	0.625、1.25、3.1

Plasmid was added to 500. mu.l according to the above formulation

Complex-forming solution (A)

Complex format Solution), mixing gently; then 5. mu.l of transfection reagent was added

Reagent, mixing gently; the mixture was allowed to stand at room temperature for 5 minutes to allow the formation of a DNA-transfection reagent complex.

(C) Adding the DNA-transfection reagent complex mixture into the suspension of CHO-S cells to be transfected, and continuing culturing.

(D) The next day after co-transfection (18-20 hours later), sodium butyrate was added at a final concentration of 10 mM.

(E) After further culturing for 10-12 hours, the cells were centrifuged at 800rpm for 5 minutes, the supernatant was removed, and 2ml of fresh water was added

The culture was continued after resuspension of the expression medium (containing 4mM L-glutamine, 0.3% Poloxamer 188).

(G) And after 15-18 hours, centrifuging at 800rpm for 5 minutes, and filtering the obtained supernatant through a 0.45-micrometer filter membrane to obtain the retrovirus liquid.

(4) Retroviral infection with the GFP gene:

(A) before virus infection, the density of CHO-S cells to be infected is 2.1 × 10⁶The cell per ml, the cell survival rate is 99 percent, the diameter is 11.4 mu m, the observation state under the microscope is good, and the cell can be used for virus infection.

(B) The retrovirus fluid was taken to 50 ml-shake tube, 1 ml/tube, and labeled at the same time. Polybrene was added to a final concentration of 2. mu.g/ml. Finally, 0.4X 10 of the mixture is added respectively⁶The CHO-S cells to be infected were gently mixed, and then mixed at 110rpm with 8% CO₂The culture was carried out at 37 ℃.

(C) 4 hours after viral infection, 4ml of fresh feed was added

Expression Medium, 130rpm, 8% CO₂The culture was continued at 37 ℃.

(D) 2 days after viral infection (about 48 hours), centrifugation was carried out at 800rpm for 5 minutes, and after removing the supernatant, the mixture was added5ml of fresh

The expression medium was continued to be cultured.

(5) The experimental results are as follows: 30-90% of the cells fluoresced green under a fluorescent microscope 4 days after infection (about 96 hours), and the proportion of cells expressing green fluorescence was from high to low in 5:3:3 group (80-90%) > 2:1:1 group (80-90%) >1:1:1 group (70-80%) >3:2:1 group (60-70%) >5:2:1 group (60-70%) >3:3:5 group (40-50%) >1:1:2 group (40-50%) >1:2:3 group (30-40%) >1:2: 5 group (30-40%).

Example 3 packaging and infection of the retroviral vector pLEGFP-C1 CHO DG44 cells in CHO DG44 cells (optimized for sodium butyrate)

(2) Cell culture: resuscitation chopg 44(cGMPBanked,

) The cells were suspended and prepared for virus packaging experiments after 3 passages in CDDG44(8mM L-glutamine) medium by conventional subculture methods.

(3) Packaging and infection of retroviral recombinant vector pLEGFP-C1:

(A) 22-24 hours before cotransfection, CHODG44 cells were cultured at 2X 10⁶Cell/ml, 30ml

Expression Medium (C)

Expression Medium containing 4mM L-glutamine, 0.3% Poloxamer188 (Poloxamer188)), 1X HT additive)); on the day of transfection, cells were diluted to 2X 10⁶One cell/ml, and 5 ml/tube into 8 50ml shake tubes for use.

(B) Mu.g of pLEGFP-C1, 1.25. mu.gg of pCMV-gag-pol and 1.25. mu.g of pMD2.G (mass ratio 2:1:1) were added to 500. mu.l

Complex-forming solution (A)

In complete format Solution), gently mixing; then 5. mu.l of transfection reagent was added

(C) The DNA-transfection reagent complex mixture was added to the suspension of CHODG44 cells to be transfected, and the culture was continued.

(D) The next day after co-transfection (18-20 hours later), sodium butyrate was added to 6 tubes of virus packaging cells at final concentrations of 0, 1, 2, 5, 10, 15, 20mM, respectively.

(E) After further culturing for 10-12 hours, centrifuging at 800rpm for 5 minutes, discarding the supernatant, and adding 2ml of fresh

(F) And after 15-18 hours, centrifuging at 800rpm for 5 minutes, and filtering the obtained supernatant through a 0.45-micrometer filter membrane to obtain the retrovirus liquid.

(4) Retroviral infection:

(A) before virus infection, the cell density of CHODG44 to be infected is 2.2X 10⁶The cell per ml, the cell survival rate is 99 percent, the diameter is 11.8 mu m, the observation state under the mirror is good, and the cell can be used for virus infection.

(B) The virus solution was taken to 50 ml-shake tube, 1 ml/tube. Polybrene was added to a final concentration of 0.5. mu.g/ml. Finally, 0.4X 10 of the mixture is added respectively⁶The CHODG44 cells to be infected were gently mixed, and then mixed at 110rpm with 8% CO₂The culture was carried out at 37 ℃.

(C) 4 hours after viral infection, supplement4ml of fresh

Expression Medium, 130rpm, 8% CO₂The culture was continued at 37 ℃.

(D) 2 days after viral infection (about 48 hours), centrifugation was carried out at 800rpm for 5 minutes, and after removing the supernatant, 5ml of fresh DNA was added

The expression medium was continued to be cultured.

(5) The experimental results are as follows: 4 days after infection (about 96 hours), under a fluorescence microscope, 40-90% of cells have green fluorescence, and the proportion of cells expressing fluorescence is from high to low in a 5mM sodium butyrate group (80-90%) >10 mM sodium butyrate group (80-90%) >2mM sodium butyrate group (70-80%) >1mM sodium butyrate group (60-70%) >15mM sodium butyrate group (50-60%) >0mM sodium butyrate group (40-50%).

EXAMPLE 4 packaging and infection of CHO-S cells with the retroviral vector pLEGFP-C1 (optimized for polybrene)

(1) Cell culture: the CHO-S (cGMPBanked,

(2) Packaging and infection of retroviral recombinant vector pLEGFP-C1:

Expression Medium (C)

Expression Medium, containing 4mM L-glutamine, 0.3% Poloxamer188), passaged; on the day of transfection, CHO-S cells were diluted to 2X 10⁶Each cell/ml, and subpackaged in 5 ml/tube to7 tubes of 50ml were placed back in the shaker for further use.

(B) Mu.g of pLEGFP-C1, 1.25. mu.g of pCMV-gag-pol and 1.25. mu.g of pMD2.G (mass ratio 2:1:1) were added to 500. mu.l

Complex-forming solution (A)

(C) The DNA-transfection reagent complex mixture was added to the suspension of CHO-S cells to be transfected and the culture continued.

(D) The next day after co-transfection (18-20 hours later), sodium butyrate was added at a final concentration of 5 mM.

(3) Retroviral infection with the GFP gene:

(A) before virus infection, the density of CHO-S cells to be infected is 2.6 multiplied by 10⁶The cell per ml, the cell survival rate is 99 percent, the diameter is 11.2 mu m, the observation state under the microscope is good, and the cell can be used for virus infection.

(B) The retrovirus fluid was taken to 50 ml-shake tube, 1 ml/tube, and labeled at the same time. Polybrene was added to final concentrations of 0, 0.5, 1, 5, 10, 15, 20. mu.g/ml, respectively. Finally, 0.6X 10 of the mixture is added respectively⁶An individual CHO-S cell to be infected, gently mixedAfter homogenization, 110rpm, 8% CO₂The culture was carried out at 37 ℃.

(C) 4 hours after viral infection, 4ml of fresh feed was added

Expression Medium, 130rpm, 8% CO₂The culture was continued at 37 ℃.

(D) 2 days after virus infection (about 48 hours), centrifugation was carried out at 800rpm for 5 minutes, and after removing the supernatant, culture was continued by adding 5ml of fresh CHOgro expression medium.

(5) The experimental results are as follows: 20-90% of the cells fluoresced green under a fluorescence microscope at 4 days (about 96 hours) after infection, and the proportion of cells expressing green fluorescence was from high to low 5. mu.g/ml of the polyamine group (80-90%) > 1. mu.g/ml of the polyamine group (70-80%) > 0.5. mu.g/ml of the polyamine group (70-80%) > 10. mu.g/ml of the polyamine group (60-70%) > 0. mu.g/ml of the polyamine group (50-60%) > 15. mu.g/ml of the polyamine group (20-40%) > 20. mu.g/ml of the polyamine group (20-40%).

Example 5 packaging of retroviral vector pLEGFP-C1 in HEK-293T cells and infection of CHO DG44 and HEK-293T cells

(1) Cell culture: HEK-293T (ATCC) cells were recovered and prepared for virus packaging experiments after 2 passages in DMEM + 10% (v/v) FBS medium using conventional subculture methods.

(2) Packaging and infection of retroviral recombinant vector pLEGFP-C1:

(A) 18-24 hours before cotransfection, 1.5X 10⁶A number of HEK-293T cells were seeded in T25 cell culture flasks and the confluency of cells at transfection should be 85-95%.

(B) Mu.g of pLEGFP-C1, 1.25. mu.g of pCMV-gag-pol and 1.25. mu.g of pMD2.G (mass ratio 2:1:1) were added to 750. mu.l of OptiMEM serum-free medium to obtain a DNA/medium mixture. Mu.l Turbofect (Thermo-Fisher) transfection reagent was added to the DNA/media mixture and gently mixed on the tube wall. Incubation is carried out for 15-20 minutes at room temperature to allow the formation of DNA-transfection reagent complexes.

(C) Adding the DNA-transfection reagent compound into a cell culture medium to be transfected, gently mixing uniformly, and placing in a cell culture box.

(D) The next day after co-transfection (18-20 hours later), sodium butyrate was added to a final concentration of 10 mM.

(F) After the culture is continued for 10-12 hours, the culture is continued by replacing 3ml of fresh DMEM + 10% (v/v) FBS medium.

(G) And after 15-18 hours, taking the supernatant, and filtering the supernatant through a 0.45-micrometer filter membrane to obtain the retrovirus liquid. At the moment, the HEK-293T cells are subjected to large-scale rounding, falling and death and partial cells have wire drawing phenomena in microscopic examination, which indicates that the retrovirus is greatly packaged and released in the cells. The cells showed strong green fluorescence under fluorescence microscopy.

(3) Retrovirus infection of CHODG44 and HEK-293T cells:

(A) before viral infection, the cell density of CHO-DG44 to be infected is 1.8X 10⁶The cell per ml, the cell survival rate is 98 percent, the diameter is 11.2 mu m, the observation state under the mirror is good, and the cell can be used for virus infection. HEK-293T cells grow in a T25 culture flask, the confluence degree is 30-40%, the cell morphology is normal, and the growth state is good.

(B) The retrovirus fluid was taken to 50 ml-shake tube, 1 ml/tube. Polybrene was added to a final concentration of 5. mu.g/ml. Adding 0.6X 10⁶The CHO DG44 cells to be infected were gently mixed, and then mixed at 110rpm with 8% CO₂The culture was carried out at 37 ℃. The supernatant from the HEK-293T flask was aspirated, 2ml of virus solution was added, and polybrene was added to a final concentration of 5. mu.g/ml. 5% CO₂And then, the mixture was subjected to static culture at 37 ℃.

(C) 4 hours after viral infection, 4ml of fresh CD DG44 (containing 8 mML-glutamine) medium was added to the CHODG44 cell shake tube at 130rpm with 8% CO₂Continuing culturing at 37 ℃; 6ml fresh DMEM + 10% (v/v) FBS medium was added to HEK-293T flasks with 5% CO₂The culture was continued at 37 ℃.

(D) At 2 days (about 48 hours) after viral infection, CHO DG44 cells were centrifuged at 800rpm for 5 minutes, DMEM + 10% (v/v) FBS and CD DG44 mixed media were removed, and 5ml of fresh CD DG44 medium was added, at which time the cells were transferred to complete serum-free media for culture. The HEK-293T cell culture medium was discarded, and the culture was continued by adding 8ml of fresh DMEM + 10% FBS medium.

(5) The experimental results are as follows: cell morphology, survival rate and fluorescence intensity were observed daily 4-8 days after retroviral infection. The HEK-293T cell has normal morphology and normal growth, and no obvious cell death and shedding are observed. Under a fluorescence microscope, green fluorescence was observed. CHO DG44 cells were poorly shaped and survived between 10-20%. Weak green fluorescence under ultraviolet lamp and fluorescence microscope. The reason why the CHO DG44 cell survival rate is low without neomycin screening after the infection is that: (1) cytopathic effects of viral infection; (2) cell growth inhibition and apoptosis when cells were switched from serum-containing infection medium to completely serum-free CD DG44 medium.

Example 6 packaging of retroviral vector pRDM-GLP-1-Fc in CHO-S cells and infection of CHO-S cells

(1) Cell culture: CHO-S suspension cells (cGMP Banked,

) The cells were prepared for virus packaging experiments after 3 passages in CD FortiCHO complete medium (containing 8mM L-glutamine) using conventional subculture methods.

(2) Retroviral recombinant vector pRDM-GLP-1-Fc packaging and infection

(A) One day before cotransfection (about 24 hours), CHO-S cells were cultured at 0.5X 10⁶～0.6×10⁶Passaging individual cells/ml, 30ml of CD FortiCHO complete medium (containing 8mM L-glutamine); on the day of transfection, CHO-S cells were diluted to 1X 10⁶Each cell/ml, and 5 ml/tube into 50 ml-shake tube, and put back to the shaker for use.

(B) Add 8.3. mu.l of FreeStyle MAX Reagent to 241.7. mu.l of serum-free medium OptiPROSFM (250. mu.l final volume), mix gently; at the same time, 3.7. mu.g pRDM-GLP-1-Fc, 2.3. mu.g pCMV-gag-pol, 2.3. mu.g pMD2.G (mass ratio 5:3:3) were added to another OptiPRO^TMSFM and make the final volume 250. mu.l, mix gently.

(C) Meanwhile, a positive control group and a negative control group were separately provided, the positive control group co-transfected with pLEGFP-C1 plasmid and packaging vector (transfection at a mass ratio of 5:3:3, and 8.3. mu.g in total) and the negative control group (transfection of only packaging vectors pCMV-gag-pol and pMD2.G, and co-transfection at a mass ratio of 1:1, and 8.3. mu.g in total) in the same manner.

(D) The plasmid mixture diluted in step (B) was added to the transfection reagent dilution in step (B), gently mixed (final volume 500. mu.l), and allowed to stand at room temperature for 10 minutes to form a DNA-transfection reagent complex. Adding the DNA-transfection reagent complex mixture into the CHO-S cells to be transfected, and continuing to culture.

(E) The next day after co-transfection (18-20 hours later), sodium butyrate was added to a final concentration of 1 mM.

(F) And continuing to culture for 10-12 hours, centrifuging at 800rpm for 5 minutes, removing supernatant, adding 2ml of fresh CD FortiCHO culture medium to perform basic suspension, and continuing to culture.

(4) CHO-S cell infected by retrovirus containing GLP-1-Fc recombinant gene

(A) Before virus infection, the density of CHO-S cells to be infected is 1.8 multiplied by 10⁶The cell per ml, the cell survival rate is 98 percent, the diameter is 11.9 mu m, the observation state under the mirror is good, and the cell can be used for virus infection.

(B) The retrovirus fluid was taken to 50 ml-shake tube, 1 ml/tube. Polybrene was added to a final concentration of 1. mu.g/ml. Finally, 0.4X 10 of the mixture is added respectively⁶The CHO-S cells to be infected are mixed gently and evenly and then are mixed at 110rpm and 8% CO₂The culture was continued at 37 ℃.

(C) 4 hours after viral infection, 4ml of fresh CD FortiCHO complete medium (containing 8mM L-glutamine) was supplemented with 8% CO at 130rpm₂The culture was continued at 37 ℃.

(D) 2 days after virus infection (about 48 hours), centrifugation was carried out at 800rpm for 5 minutes, and after removing the supernatant, culture was continued by adding 5ml of fresh CDFortCHO medium. In this step, cells of the positive control group collected by centrifugation and visible under an ultraviolet lamp have stronger green fluorescence, and the negative control group does not have fluorescence.

(E) 4 days after virus infection, samples were taken to determine viable cell density, cell viability and cell status under a microscope. In this example, the detection results are shown in table 2:

TABLE 2 detection results of groups of cells 4 days after viral infection

	Viable cell density (× 10)⁶Cells/ml)	Activity (%)	Diameter (μm)
				pRDM-GLP-1-Fc	4.5	98	12.3
pLEGFP-C1	3.8	95	12.8
				NC	5.2	97	11.8

The state of the cells was observed well under the mirror. Without puromycin selection, the cell culture system was expanded from 5ml to 30ml using CD FortiCHO complete medium (containing 8mM L-glutamine) at 130rpm with 8% CO₂The culture was continued at 37 ℃ while collecting the culture supernatant of 5ml culture system to examine the production of GLP-1-Fc protein by ELISA. The sandwich ELISA method is briefly described as follows: 96-well plate JingyangAnti-human IgG Fc antibody (Cat. 109-. An HRP-labeled mouse anti-GLP-1 monoclonal antibody (cat # AP84512-HRP, Shanghai' an organism) was then added to bind the primary anti-captured GLP-1-Fc. The yield of GLP-1-Fc in the cell supernatant was detected as: 15.445 μ g/ml. A part of the cells were taken out from the 30ml culture system and subjected to cell cryopreservation while positive single clones were screened by limiting dilution. Except for direct amplification culture, one part of the CHO-S cells infected by the virus containing the GLP-1-Fc gene are selected and screened for 7 days by puromycin with the concentration of 5 mu g/ml, the average survival rate of the cells in the screening process is over 90 percent, which shows that the virus infection efficiency is high, and only a small part of the cells are not infected and have no puromycin resistance. In view of the high efficiency of viral infection, puromycin screening is usually omitted and infected cells can be directly subjected to limiting dilution screening of monoclonal cells.

Example 7 packaging of retroviral vector pRDM-GLP-1-Fc in HEK-293T cells and infection of CHO-S and HEK-293T cells

(1) Cell culture: HEK-293T (ATCC) cells were thawed and prepared for co-transfection virus packaging experiments after 2 passages in DMEM + 10% (v/v) FBS medium using conventional subculture methods.

(2) Retroviral vector pRDM-GLP-1-Fc packaging and infection:

(A) 18-24 hours before cotransfection, 1.5X 10⁶HEK-293T cells were seeded in T25 cell culture flasks and the confluency of cells at transfection should be 85-95%.

(B) 2.5. mu.g of pRDM-GLP-1-Fc, 1.25. mu.g of pCMV-gag-pol and 1.25. mu.g of pMD2.G (mass ratio 2:1:1) were added to 750. mu.l of OptiMEM serum-free medium to obtain a DNA/medium mixture. Mu.l Turbofect (Thermo-Fisher) transfection reagent was added to the DNA/media mixture and gently mixed on the tube wall. And (5) incubating for 15-20 minutes at room temperature.

(C) The transfection reagent/DNA/media mixture was added to the cell culture media, gently mixed and placed in a cell incubator.

(G) And after 15-18 hours, taking the supernatant, and filtering the supernatant through a 0.45-micrometer filter membrane to obtain the retrovirus liquid. At the moment, the HEK-293T cells are subjected to large-scale rounding, falling and death and partial cells have wire drawing phenomena in microscopic examination, which indicates that the retrovirus is greatly packaged and released in the cells.

(3) Retroviral infection of CHO-S and HEK-293T cells:

(A) before virus infection, the density of CHO-S cells to be infected is 2.6 multiplied by 10⁶The cell per ml, the cell survival rate is 98 percent, the diameter is 11.2 mu m, the observation state under the mirror is good, and the cell can be used for virus infection. HEK-293T cells grow in a T25 culture flask, the confluence degree is 30-40%, the cell morphology is normal, and the growth state is good.

(B) Collecting retrovirus liquid, placing into 50ml shaking tube, adding into the solution at a ratio of 1 ml/tube, and adding into the solution at a ratio of 0.5 × 10⁶Adding polybrene with final concentration of 10 μ g/ml into CHO-S cells to be infected, mixing, and stirring at 110rpm with 8% CO₂And cultured at 37 ℃. The supernatant from the HEK-293T flask was aspirated, 2ml of virus solution was added, and polybrene at a final concentration of 10. mu.g/ml, 5% CO was added₂And then, the mixture was subjected to static culture at 37 ℃.

(C) 4 hours after viral infection, 4ml of fresh CDFortiCHO (8mM L-glutamine) medium was added to the CHO-S cell shake tube at 130rpm with 8% CO₂Continuing culturing at 37 ℃; 6ml fresh DMEM + 10% (v/v) FBS medium was added to HEK-293T flasks with 5% CO₂The culture was continued at 37 ℃.

(D) 2 days after virus infection (about 48 hours), centrifugation was carried out at 800rpm for 5 minutes, the CHO-S mixed medium (DMEM + 10% (v/v) FBS and CDFortiCHO) was removed, and culture was continued by adding 5ml of fresh CD FortiCHO medium, at which time the cells were transferred to complete serum-free medium. The HEK-293T cell culture medium was discarded, and the culture was continued by adding 8ml of fresh DMEM + 10% FBS medium.

(E) And (3) adding puromycin to carry out a screening experiment, wherein according to the result of a prophase killing curve, the puromycin concentration suitable for HEK-293T cells is 2 mug/ml, the puromycin concentration suitable for CHO-S cells is 5 mug/ml, and the liquid is changed every two days. Meanwhile, HEK-293T and CHO-S cells without virus infection are set as negative controls of puromycin killing screening experiments.

(5) The experimental results are as follows: cell morphology and survival were observed daily 4-8 days after retroviral infection. The HEK-293T cell has normal shape and growth, no obvious cell death and shedding is seen, and about 80 percent of cells survive in a puromycin screening experiment. CHO-S cells have poor morphology, and only about 5-10% of the cells survive after puromycin screening. After CHO-S is infected by the packaging replication-defective retrovirus in the HEK-293T cell, the cell infection rate is low, most cells die after puromycin screening, and partial cell death reasons can be cell injury caused by infection and cell apoptosis caused by changing into a serum-free culture medium after infection.

EXAMPLE 8 Limited dilution screening of Positive monoclonals

(1) Cloning medium containing 6mM L-glutamine (per 100 ml): 97ml of CD FortiCHO basal medium, 3ml of 200mM L-glutamine, and preheating the prepared basal medium at 37 ℃ for later use.

(2) Samples were taken to examine the density, cell viability and growth status of CHO-S live cells (prepared in example 6, and GLP-1-Fc stably expressing cell line obtained by retroviral infection). The cells were gradually diluted to 0.3 cells/40. mu.l with the cloning medium, and the diluted cells were seeded into a 96-well plate with a discharging gun at 40. mu.l/well, i.e., 0.3 cells/well.

(3) Inoculated 96-well plate was placed in CO₂Incubator at 37 ℃ and 5% CO₂And absolute static culture under saturated humidity conditions for 4 hours, microscope under hole by hole observation, confirmed and labeled only containing a cell hole and the culture medium to 200 u l. Place the cells back in CO₂The incubator continues static culture.

(4) Absolutely statically culturing for 13 days, observing the growth condition of the monoclonal cells under a microscope, finding out a hole with obvious cell proliferation, marking, and supplementing 50 mu l/hole of CD FortiCHO culture medium complete culture medium (containing 8 mML-glutamine).

(5) And (4) after continuously standing and culturing for 3-4 days, sucking culture supernatant of the single cell colony, and performing SDS-PAGE detection. According to the SDS-PAGE detection result, a monoclonal antibody with a more obvious target band is selected and transferred to a 15 ml-shaking tube, and 3ml of CDFortiCHO complete medium (containing 8 mML-glutamine) is used for amplification culture in the 15 ml-shaking tube.

(6)15 ml-shake tube culture for 3 days, sampling to detect cell viability and viable cell density, observing cell growth state under a mirror, selecting monoclonal cell culture supernatant with good growth state, and performing ELISA detection, wherein ELISA detection results are shown in figure 2.

(7) According to the ELISA detection result, the monoclonal cells with higher yield are selected and transferred to a 50 ml-shaking tube, and 8ml of CDFortiCHO complete culture medium (containing 8 mML-glutamine) is used for amplification culture.

(8) Culturing in a 50 ml-shaking tube system for 4 days, sampling to detect the cell viability, the viable cell density and observing the cell growth state under a mirror. After centrifugation at 800rpm for 5 minutes, culture supernatants were subjected to ELISA, and the results of ELISA are shown in FIG. 3.

(9) According to the ELISA detection result, selecting the monoclonal cells with higher yield, transferring the monoclonal cells to a 125 ml-shake flask, and carrying out amplification culture on 30ml of CD11V complete culture medium (containing 8mM L-glutamine); and simultaneously performing cell strain cryopreservation.

Example 9 evaluation of shake flask fermentation culture yield

(1) After 3 days of cell culture from 50 ml-shake transfer to 125 ml-shake flask expansion, cells were incubated at 0.5X 10⁶One cell, 50ml CD11V complete medium (containing 8mM L-glutamine) was inoculated into a 250 ml-shake flask at 130rpm with 8% CO₂Cultures were performed at 37 ℃ and the day of inoculation was recorded as day 0.

(2) Starting on day 3 of culture, 2ml were sampled daily for the following parameter tests: viable Cell Density (VCD), cell viability, cell diameter, pH, osmotic pressure, lactate concentration, glutamine concentration, ammonia concentration, and glucose concentration, while observing the cell state under a microscope.

(3) When the cell density is higher than 2.5X 10⁷At individual cells/ml, the culture temperature was lowered to 33 ℃.

(4) When the glucose content in the culture medium is lower than 2g/L, the glucose solution is supplemented to 4 g/L.

(5) The feeding medium PFF 063%, 4%, 5% (V/V) was added on

days

3, 5, 7, 9, 11, 13 of the shake flask culture, respectively.

(6) Culture supernatants were collected at 9, 11, and 14 days of culture, respectively, and the concentration and purity of the target protein were determined by high performance size exclusion chromatography.

(7) The experiment was terminated on day 14 of culture or when the cell viability was below 80%.

(8) The shake flask culture yields of the monoclonal cell lines in this example are shown in FIG. 4. The experiment is a shake flask fermentation experiment which selects 11 cell strains with higher yield on the basis of the ELISA detection result of a 50 ml-shake tube culture system. The shake flask fermentation experiment obtains higher protein yield: the highest protein yield reaches more than 3.1g/L, and the yield of all cell lines is higher than 2.5 g/L.

EXAMPLE 10 Stable passage experiment of monoclonal cell lines

(1) 11 monoclonal cell lines were picked and resuscitated in CD FortiCHO complete medium (8mm L-glutamine).

(2) When the cell density is more than 2X 10⁶At a cell/ml, at 0.2X 10⁶Individual cells/ml, 5ml were inoculated for passage.

(3) The cells were in good condition and stable doubling time by day 6 of culture (after 2 passages), and the inoculum was evaluated for shake flask culture yield as yield data for the cell lines at the beginning of resuscitation.

(4) The shake flask culture yield evaluation experiment was performed again after 40 days of subculture in CD FortiCHO complete medium (containing 8mm L-glutamine).

(5) The comparison of the yield of shake flask fermentation experiments performed on stable cell lines immediately after recovery and after 40 days of passage (about 60 generations of multiplication) is shown in FIG. 5. Through comparison of the results of2 times of shake flask fermentation yields, after 40 days of subculture (about 60 times of multiplication), the protein yield of the cell line is 95-100% of the protein yield of the cell line at the beginning of recovery, which indicates that the protein expression of the cell line is stable.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Zhuhai Federal pharmaceutical Co Ltd

<120> packaging method of replication defective retrovirus and use thereof

<130>1

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<211>526

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCMV promoter

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tactgagtca ttagggactt tccaatgggt tttgcccagt acataaggtc aataggggtg 60

aatcaacagg aaagtcccat tggagccaag tacactgagt caatagggac tttccattgg 120

gttttgccca gtacaaaagg tcaatagggg gtgagtcaat gggtttttcc cattattggc 180

acgtacataa ggtcaatagg ggtgagtcat tgggtttttc cagccaattt aattaaaacg 240

ccatgtactt tcccaccatt gacgtcaatg ggctattgaa actaatgcaa cgtgaccttt 300

aaacggtact ttcccatagc tgattaatgg gaaagtaccg ttctcgagcc aatacacgtc 360

aatgggaagt gaaagggcag ccaaaacgta acaccgcccc ggttttcccc tggaaattcc 420

atattggcac gcattctatt ggctgagctg cgttctacgt gggtataaga ggcgcgacca 480

gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga acgcag 526

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<211>610

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<223> WPRE sequences

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gcggccgctc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 60

taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 120

tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 180

ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 240

cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 300

tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 360

aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt 420

tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 480

cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 540

tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 600

gcctgatcga 610

<210>3

<211>1085

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<213> Artificial Sequence (Artificial Sequence)

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<223> GLP-1-Fc sequences

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agatctgctg actagcgttt aaacttaagc ttagcgcaga ggcttggggc agccgagcgg 60

cagccaggcc ccggcccggg cctcggttcc agaagggaga ggagcccgcc aaggcgcgca 120

agagagcggg ctgcctcgca gtccgagccg gagagggagc gcgagccgcg ccggccccgg 180

acggcctccg aaaccatggg cgtgaaggtc ctgttcgccc tgatttgcat cgccgtcgca 240

gaggcacacg gcgagggcac cttcacctcc gacgtgtcct cctatctcga agagcaggcc 300

gccaaggaat tcatcgcctg gctggtgaag ggcggcggcg gtggtggtgg ctccggaggc 360

ggcggctctg gtggcggtgg cagcgctgag tccaaatatg gtcccccatg cccaccctgc 420

ccagcacctg aggccgccgg gggaccatca gtcttcctgt tccccccaaa acccaaggac 480

actctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt gagccaggaa 540

gaccccgagg tccagttcaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagaca 600

aagccgcggg aggagcagtt caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 660

caccaggact ggctgaacgg caaggagtac aagtgcaagg tctccaacaa aggcctcccg 720

tcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagagcc acaggtgtac 780

accctgcccc catcccagga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 840

aaaggcttct accccagcga catcgccgtg gagtgggaaa gcaatgggca gccggagaac 900

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcagg 960

ctaaccgtgg acaagagcag gtggcaggag gggaatgtct tctcatgctc cgtgatgcat 1020

gaggctctgc acaaccacta cacacagaag agcctctccc tgtctctggg ttgataagcg 1080

gccgc 1085

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<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> primer Purof

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cttaccggtg ccgccaccat cccctgaccc acgcccctga 40

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<211>46

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acgcgtgaac tacagagtct cgctcaggca ccgggcttgc gggtca 46

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<211>48

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<223> primer mCMVF

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gcgagactct gtagttcacg cgtctactga gtcattaggg actttcca 48

<210>7

<211>28

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<213> Artificial Sequence (Artificial Sequence)

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<223> primer mCMVR

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ggaagatctc ctgaggctgc gttctacg 28

Claims

1. A method of packaging a replication defective retrovirus, comprising the steps of: constructing a replication-defective retrovirus vector containing a target gene; the replication-defective retrovirus vector containing the gene of interest is cotransfected with a packaging vector to suspension culture CHO cells, and virus packaging is completed therein.

2. The method of packaging a replication deficient retrovirus according to claim 1, comprising the steps of:

(3) adding sodium butyrate 18-20 hours after transfection to enhance the virus packaging efficiency; after sodium butyrate is treated for 10-12 hours, centrifuging, removing supernatant, and adding a fresh serum-free culture medium for culture;

(4) and harvesting 43-50 hours after transfection to obtain virus suspension.

3. The method of packaging a replication deficient retrovirus according to claim 2, wherein:

the replication-defective retroviral vector in step (1) includes but is not limited to one or more of a pLPCX vector, other retroviral vectors constructed by taking the pLPCX vector as a framework, a pBabe vector and a pLEGFP-C1 vector;

the target protein in the step (1) is a recombinant protein;

the CHO cell in the step (2) is CHODG44, CHO-K1 or CHO-S;

the packaging plasmids in the step (2) are pCMV-gag-pol and pMD2. G;

the culture medium in the step (3) is a CD culture medium.

4. The method of packaging a replication deficient retrovirus according to claim 3, wherein:

the other retrovirus vector constructed by taking the pLPCX vector as a framework is a pRDM vector, the pRDM vector is obtained by modifying the pLPCX vector, and a Puro-hCMV fragment on the pLPCX vector is replaced by Puro-mCMV-WRPE; wherein Puro is puromycin resistance gene, the sequence of mCMV is shown as SEQ ID NO.1, and the sequence of WRPE is shown as SEQ ID NO. 2;

the recombinant protein includes but is not limited to monoclonal antibody, coagulation factor, growth factor, cell factor, Fc fusion protein, HSA fusion protein;

the CD culture medium is a CD FortiCHO culture medium, a CD OptiCHO culture medium, a CDM4CHO culture medium or a CD11V culture medium.

5. The method of packaging a replication deficient retrovirus according to claim 2, wherein:

the method for co-transfecting the target gene recombinant vector and the packaging vector into the suspension culture CHO cells in the step (2) comprises but is not limited to a shock transfection method, a liposome transfection method, a PEI transfection method and a calcium phosphate transfection method;

preparing a DNA-transfection reagent compound in the co-transfection in the step (2), wherein the DNA comprises a recombinant vector containing a target gene and 2 packaging vectors, and the mixing ratio of the DNA to the recombinant vector to the packaging vectors is 1-2: 1: 1; the ratio of the total amount of DNA to the number of cells to be transfected is 0.5-2.0. mu.g/(1X 10)⁶Individual cells);

the final concentration range of the sodium butyrate in the step (3) is 1-10 mM;

the amount of the replaced fresh serum-free culture medium in the step (3) is 1/4-1/2 of the volume of the culture medium during transfection.

6. Use of the method of packaging a replication defective retrovirus according to any one of claims 1 to 5 to construct a monoclonal cell line in which the protein is stably expressed.

7. A method for constructing a stable monoclonal cell strain, which is characterized by comprising the following steps: the packaging method according to any one of claims 1 to 5, wherein the replication-defective retrovirus packaged by the packaging method is used to infect CHO cells of the same kind used in the packaging method, and a monoclonal cell strain is selected.

8. The method of claim 7, comprising the steps of:

(5) infecting the same CHO cells used in the packaging method with the replication defective retrovirus suspension packaged according to any one of claims 1 to 5, and supplementing fresh medium 4 to 6 hours after infection to ensure adequate and rational nutrient supply to the infected cells;

9. The method of claim 8, further comprising the steps of:

(9) and (3) carrying out subculture on the high-yield cell strain in the shake flask culture for 40 days, and comparing the yield of protein obtained by shake flask fermentation of the freshly recovered cell and the cell after subculture for 40 days to obtain a stable high-yield monoclonal cell strain.

10. The method of constructing a stable monoclonal cell strain according to claim 8 or 9, wherein:

in the infection process described in step (5), the amount of the replication defective retrovirus suspension used is 0.4X 10 per 1ml of the virus fluid⁶～0.6×10⁶(ii) individual cells;

an infection reinforcing agent polybrene is required to be added in the infection process, and the dosage of the polybrene is calculated according to the final concentration of 0.5-10 mug/ml;

the dosage of the fresh culture medium is calculated according to the volume of 4-5 times of the volume of the culture medium during infection.