A method for purifying BaEV envelope pseudotype lentivirus

Abstract

This invention belongs to the field of biotechnology, specifically relating to a purification method for BaEV enveloped pseudolentivirus. The purification method includes the following steps: (1) concentrating the clarified BaEV enveloped pseudolentivirus solution, centrifuging, discarding the supernatant, and obtaining a lentivirus precipitate; (2) resuspending the lentivirus precipitate in buffer solution, adding a totipotent nuclease for enzymatic digestion, and obtaining a crude pure sample; (3) purifying the crude pure sample by chromatography to obtain a refined pure sample; (4) concentrating the refined pure sample by ultrafiltration and sterilization to obtain purified BaEV enveloped pseudolentivirus. The BaEV enveloped pseudolentivirus purified using the method of this invention exhibits significantly improved infection titer and titer recovery rate, effectively reducing residual host cell protein, host cell DNA, and endotoxins. The purification method of this invention effectively removes impurities without affecting the activity of the purified lentivirus, and can be used to prepare high-titer and high-purity BaEV pseudolentivirus products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for purifying a BaEV envelope pseudotype lentivirus. Background Art

[0002] Lentiviral vector is a gene vector derived from human immunodeficiency virus, which is widely used for gene editing of eukaryotic cells such as hematopoietic stem cells and neural cells. With the long-term and in-depth research on gene delivery tools, lentivirus has been widely used as a gene vector in basic research and clinical practice. With the advent of the post-genomic era, gene therapy accounts for an increasingly large proportion in medical research. Some traditional diseases that are difficult to cure or alleviate can be alleviated or even cured through gene therapy. Lentivirus provides new tools and methods for the development of gene therapy in the future. Among them, vesicular stomatitis virus G protein (VSV-G) pseudotyped lentivirus has been widely used in gene therapy. However, the transduction efficiency of this pseudotyped lentivirus is relatively low when transducing hematopoietic stem cells and some lymphocytes.

[0003] Patent application No. 201280053749.1 discloses a method for producing a lentiviral vector pseudotyped with mutant baboon endogenous retrovirus (BaEV) glycoprotein. Although the BaEV glycoprotein pseudotyped lentiviral vector obtained by this method can effectively improve the transfection efficiency of hematopoietic stem cells and natural killer cells, and is used to prepare drugs for treating hematopoietic dysfunction or autoimmune diseases, in the preparation process of the pseudotyped lentiviral vector, the BaEV glycoprotein easily causes adherent cells to form syncytia and detach and die, which not only makes the titer of the prepared lentivirus low, but also leads to more host cell impurities remaining in the virus liquid. In order to solve the above problems, it is urgent to develop a method for obtaining high-titer and high-purity BaEV pseudotyped lentivirus. Summary of the invention

[0004] In view of the problems of the prior art, the present invention provides a method for purifying BaEV envelope pseudotype lentivirus.

[0005] The present invention provides a method for purifying a BaEV envelope pseudotype lentivirus, characterized in that the purification method comprises the following steps:

[0006] Step 1: Concentrate the BaEV envelope pseudotype lentivirus clarification solution, centrifuge, discard the supernatant, and obtain the lentivirus precipitate;

[0007] Step 2: Resuspend the lentiviral precipitate with buffer and add universal nuclease for digestion to obtain a crude pure sample;

[0008] Step 3: Purify the crude pure sample by chromatography to obtain a pure sample; the chromatography is Mustang Q XT membrane chromatography capsule filter chromatography or DEAE cellulose chromatography;

[0009] Step 4: The purified sample is filtered, concentrated, and sterilized to obtain the purified BaEV envelope pseudotype lentivirus.

[0010] Furthermore, in step 1, the BaEV envelope pseudotype lentivirus clarified fluid is obtained by centrifuging and filtering the BaEV envelope pseudotype lentivirus harvest fluid.

[0011] Furthermore, in step 1, the method for concentrating the BaEV envelope pseudotype lentivirus clarified liquid is: adding a polyethylene glycol solution to the BaEV envelope pseudotype lentivirus clarified liquid and mixing evenly; the volume ratio of the BaEV envelope pseudotype lentivirus clarified liquid and the polyethylene glycol solution is (2-8):1, the polyethylene glycol solution contains 20-50% g / L polyethylene glycol and 0.6-1.2 MNaCl, and the pH of the polyethylene glycol solution is 7.5-8.5.

[0012] Furthermore, in step 1, the polyethylene glycol is PEG8000, the volume ratio of the BaEV envelope pseudotype lentivirus clarification liquid and the polyethylene glycol solution is 4:1, and the preparation method of the polyethylene glycol solution comprises the following steps: mixing polyethylene glycol and NaCl in a buffer solution, adjusting the pH, and sterilizing; the polyethylene glycol solution contains 25%~45% g / L polyethylene glycol and 0.5~0.7 M NaCl, and the pH of the polyethylene glycol solution is 7.5~8.0.

[0013] Furthermore, the polyethylene glycol solution contains 40% g / L polyethylene glycol and 0.6 M NaCl, the pH of the polyethylene glycol solution is 8.0, and the sterilization method is sterilization by filtering through a 0.45 µm filter.

[0014] Furthermore, in step 2, the buffer is a buffer containing 40-60 mM Tris-HCl and 1-5 mM MgCl2, the pH value of the buffer is 7.5-8.5; and the activity of the universal nuclease is 5-50 U / ml.

[0015] Furthermore, in step 2, the buffer is a buffer containing 50 mM Tris-HCl and 2 mM MgCl2, and the pH value of the buffer is 8.0; the activity of the universal nuclease is 40 U / ml.

[0016] Furthermore, in step 3, the chromatography is Mustang Q XT membrane chromatography capsule filter chromatography, and the Mustang Q XT membrane chromatography capsule filter chromatography comprises the following steps:

[0017] (1) Equilibration: Equilibrate the Mustang Q XT membrane using equilibration buffer.

[0018] (2) Sampling: Load the crude pure sample. After loading, continue to balance with the equilibration buffer until UV 280 The value is stable;

[0019] (3) Elution: Elution is performed using an eluent, and the eluate is collected; the eluent is composed of an elution buffer A and an elution buffer B; the elution buffer A includes 20-50 mM Tris-HCl and 0-0.3 M NaCl, with a pH value of 7.5-8.0; the elution buffer B includes 20-50 mM Tris-HCl and 1-2 M NaCl, with a pH value of 7.5-8.0; the elution procedure is: first, linear elution is performed, the volume ratio of the elution buffer B in the eluent is increased from 0% to 60%, and then isocratic elution is performed, and the eluent UV 280 Start collecting when >50 mAU, until UV 280 The collection was terminated when the concentration was less than 50 mAU.

[0020] Further, in step (1), the equilibration buffer comprises 50 mM Tris-HCl and 0.3 M NaCl, with a pH value of 7.5-8.0;

[0021] The elution buffer A includes 50 mM Tris-HCl and 0.15-0.3 M NaCl, with a pH value of 7.5-8.0; the elution buffer B includes 50 mM Tris-HCl and 1-1.5 M NaCl, with a pH value of 7.5-8.0;

[0022] The elution procedure is as follows: first, linear elution is performed at a flow rate of 5 ml / min, the volume ratio of elution buffer B in the eluent is increased from 0% to 60%, and then isocratic elution is performed, and the eluent UV 280 Start collecting when >50 mAU, until UV 280 The collection was terminated when the concentration was less than 50 mAU.

[0023] Furthermore, in step 4, the diafiltration and concentration comprises the following steps: concentrating with a centrifugal concentrator tube and diafiltration with PBS; the molecular retention capacity of the centrifugal concentrator tube is 100-500 KDa; and the sterilization method is sterilization by filtration through a 0.22 µm filter.

[0024] Furthermore, the molecular cutoff of the centrifugal concentrator is 300 KDa.

[0025] In the present invention, the BaEV pseudotyped lentivirus refers to a lentiviral vector pseudotyped with a mutant BaEV glycoprotein, the viral envelope of which has the unique structure and surface markers of the BaEV envelope.

[0026] Compared with VSVG pseudotyped lentivirus, transfection of mutant BaEV envelope glycoprotein particles easily causes adherent cells to form syncytia, leading to cell detachment and death 48 hours after transfection. This not only reduces the lentivirus packaging titer, but also introduces a large amount of host DNA residues and host protein residues, seriously affecting the quality of the lentivirus.

[0027] The purification method of the BaEV envelope pseudotype lentivirus of the present invention adopts a specific chromatography column, optimizes the PEG solution formula used when concentrating the lentivirus and the buffer formula used during chromatography, and the infection titer and titer recovery rate of the BaEV envelope pseudotype lentivirus purified by the method of the present invention are significantly improved, and HCP, HCD and endotoxin residues can be effectively reduced. The purification method of the present invention effectively removes impurities without affecting the activity of the purified lentivirus, and can be used to prepare high-titer and high-purity BaEV pseudotype lentivirus products.

[0028] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0029] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 These are fluorescent images of 293T cells infected with BaEV envelope pseudotyped lentivirus before purification (denoted as clarified solution) and after purification (denoted as purified solution). DETAILED DESCRIPTION

[0031] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0032] The universal nuclease used in the example is Pannarase universal nuclease, which is from Shanghai Zhudian Biotechnology Co., Ltd., with the product number CYG002F0010.

[0033] UV 280 The values ​​refer to the UV absorbance at a wavelength of 280 nanometers.

[0034] Example 1: Purification method of BaEV envelope pseudotyped lentivirus

[0035] 1. Preparation of lentiviral clarified solution

[0036] Preparation of BaEV envelope pseudotyped lentivirus using 293T suspension cells:

[0037] (1) The density of 293T suspension cells is 3.0×10 6 / ml for plasmid transfection, and the plasmid DNA transfection ratio was 0.8μg / 10 6 cells,

[0038] (2) Take the shuttle plasmid (pCDH-GFP), packaging plasmid (pSpax2), and envelope plasmid (pCMV-BaEV-Rless) at a ratio of 1.2:1.2:1, mix the above three plasmids, add Opti-MEM medium, mix carefully, and let stand for 5 minutes. This is recorded as solution A.

[0039] (3) Dilute PEIpro with Opti-MEM medium (the ratio of PEIpro to the total mass of the three plasmids is 1:1), mix carefully, and let stand for 5 minutes. This is called solution B.

[0040] (4) Slowly add solution B into solution A, mix slowly, and let stand for 10 minutes to obtain a mixed solution;

[0041] (5) Slowly add the mixture into the cells, mix gently, and start transfection;

[0042] (6) 24 hours after transfection, add one volume of fresh culture medium;

[0043] (7) 48 hours after transfection, the cell culture medium was harvested to obtain the BaEV envelope pseudotype lentivirus harvest medium, which was centrifuged at low speed and filtered through a 0.45 µm filter to obtain the lentivirus clarified medium.

[0044] 2. Preparation of optimized PEG8000 solution

[0045] Use PBS to prepare PEG8000 solution with a concentration of 40% (w / v, unit: g / L), then add NaCl with a concentration of 0.6 M, dissolve in a 50°C water bath, adjust the pH to 8.0, and filter through a 0.45 µm filter to obtain a 40% optimized PEG8000 solution.

[0046] 3. Optimized PEG8000 solution to concentrate lentivirus

[0047] To a 50 ml centrifuge tube, add 36 ml of lentivirus clarification solution and 9 ml of 40% optimized PEG8000 solution and mix evenly. Mix once every 20 min for a total of 4 times and let stand at 4°C for 6 h. Then centrifuge at 3000×g, 4°C for 30 min, discard the supernatant and obtain the precipitate.

[0048] 4. Add 0.5 ml buffer (50 mM Tris-HCl, 2 mM MgCl2, pH 8.0) to resuspend the precipitate, mix thoroughly, add 40 U / ml universal nuclease, treat at 37°C for 1 h to obtain a crude pure sample.

[0049] 5. Mustang Q XT5 membrane chromatography capsule filter chromatography purification

[0050] (1) Equilibration: 5 CV (column volume) of equilibration buffer was used to equilibrate Mustang Q XT5 (cytiva, XT5MSTGQPM6) at a flow rate of 10 ml / min until the conductivity and pH value were stable; the equilibration buffer was 50 mM Tris-HCl, 0.3 M NaCl, pH 8.0;

[0051] (2) Injection: Inject 10CV of crude pure sample through the infusion valve at a flow rate of 5 ml / min. After the injection is completed, continue to pump in 10CV of equilibrium buffer at a flow rate of 5 ml / min until UV 280 The value is stable;

[0052] (3) Elution: The infusion valve was switched to an eluent consisting of elution bufffer-A and bufffer-B, and linear elution was performed at a flow rate of 5 ml / min. The volume ratio of bufffer-B in the eluent increased from 0% to 60%, and the elution volume was 6 CV. The elution salt concentration was increased from 0.3 M to 1.32 M, and then maintained stable for another 5 CV. UV 280 >50 mAU, start collecting eluate, UV 280 When the concentration is less than 50 mAU, the collection is stopped. After the eluate is collected, PBS is immediately added to the eluate to dilute the NaCl concentration to less than 0.3 M. Among them, the elution buffer A solution is 50 mM Tris-HCl, 0.3 M NaCl, pH 8.0; the elution buffer B solution is 50 mMTris-HCl, 1.5 M NaCl, pH 8.0;

[0053] (4) CIP: The infusion valve was switched to CIP-bufffer, and 6 CV of solution was pumped in at a flow rate of 10 ml / min for in-situ cleaning and regeneration; CIP-bufffer was 1 M NaOH aqueous solution.

[0054] 6. Diafiltration and concentration

[0055] The diluted eluate was immediately transferred to a 300 KDa centrifugal concentrator tube (sartorius, VS2052), centrifuged and concentrated at 3000×g, 4°C, and then washed and filtered twice with PBS (i.e., removing the supernatant, adding PBS to resuspend, centrifuging and concentrating, removing the supernatant, adding PBS to resuspend, and centrifuging and concentrating), and finally adding X-VIVO 15 medium to resuspend to 20 ml. The concentrated lentivirus was sterilized by filtering through a 0.22 µm filter to obtain the purified BaEV envelope pseudotype lentivirus.

[0056] Example 2: Purification method of BaEV envelope pseudotype lentivirus

[0057] 1. Preparation of lentiviral clarified solution

[0058] (1) 293T cells were seeded in a 15 cm culture dish and cultured in 10% fetal bovine serum (Gibco) medium so that the confluence was 70-80% at the time of transfection.

[0059] (2) 1 hour before transfection, replace the cell culture medium with Opti-MEM medium;

[0060] (3) Take the shuttle plasmid (pCDH-GFP), packaging plasmid (pSpax2), and envelope plasmid (pCMV-BaEV-Rless) at a ratio of 1.2:1.2:1, mix a total of 25 μg of the above three plasmids, add Opti-MEM medium to 500 μl, mix carefully, and let stand for 5 minutes. This is called solution A.

[0061] (4) Dilute PEIpro to 500 μl with Opti-MEM medium (the ratio of PEIpro to the total mass of the three plasmids is 1:1), mix carefully, and let stand for 5 minutes. This is called solution B.

[0062] (5) Slowly add liquid B into liquid A, mix slowly, and let stand for 10 minutes to obtain a mixed solution;

[0063] (6) Slowly add the mixture into the cell culture medium, mix gently, and start transfection;

[0064] (7) Six hours after transfection, the culture medium was replaced with 10% fetal bovine serum (Gibco). At 48 hours after transfection, the cell culture supernatant was harvested and centrifuged at low speed and filtered using a 0.45 µm filter to obtain the lentiviral clarified fluid.

[0065] 2. Preparation of optimized PEG8000 solution

[0066] Use PBS to prepare PEG8000 solution with a concentration of 40% (w / v, unit: g / L), then add NaCl with a concentration of 0.6 M, dissolve in a 50°C water bath, adjust the pH to 8.0, and filter through a 0.45 µm filter to obtain a 40% optimized PEG8000 solution.

[0067] 3. Optimized PEG8000 solution to concentrate lentivirus

[0068] To a 50 ml centrifuge tube, add 36 ml of lentivirus clarification solution and 9 ml of 40% optimized PEG8000 solution and mix evenly. Mix once every 20 min for a total of 5 times and place in a 4°C refrigerator for 6 h. Then centrifuge at 4000×g, 4°C for 50 min, discard the supernatant, and obtain the precipitate.

[0069] 4. Add 1 ml of PBS to resuspend the precipitate, mix the resuspended solution thoroughly, add 20 U / ml universal nuclease, treat at 37°C for 1 h to obtain a crude pure sample.

[0070] 4. DEAE chromatography purification

[0071] (1) Equilibration: Equilibrate the column with 5 CV of equilibration buffer at a flow rate of 3 ml / min until the conductivity and pH value are stable. The equilibration buffer is 50 mM Tris-HCl and 0.15 M NaCl, pH 8.0.

[0072] (2) Injection: Inject 10CV of crude pure sample through the infusion valve at a flow rate of 3 ml / min. After the injection is completed, continue to pump in 10CV of equilibrium buffer at a flow rate of 3 ml / min until UV 280 The value is stable;

[0073] (3) Elution: The infusion valve was switched to an eluent consisting of elution bufffer-A and bufffer-B, and linear elution was performed at a flow rate of 3 ml / min. The volume ratio of bufffer-B in the eluent increased from 0% to 60%, and the elution volume was 6 CV. The elution salt concentration was increased from 0.15 M to 0.66 M, and then maintained stable for 5 CV. UV 280 >50 mAU, start collecting eluate, UV 280 When the concentration is less than 50 mAU, the collection is terminated; after the eluate is collected, PBS is immediately added to the eluate to dilute the NaCl concentration in the eluate to less than 0.3 M; the elution bufffer-A solution is 50 mM Tris-HCl and 0.15 M NaCl, pH 8.0; the elution bufffer-B solution is 50 mM Tris-HCl and 1 M NaCl, pH 8.0;

[0074] (4) CIP: The infusion valve was switched to CIP-bufffer, and 6 CV of solution was pumped in at a flow rate of 3 ml / min for in-situ cleaning and regeneration; CIP buffer was 1 M NaOH aqueous solution;

[0075] (5) Cleaning: Pump 10 CV of equilibrium buffer at a flow rate of 3 ml / min to clean the column until the conductivity and pH value are stable;

[0076] (6) Column storage: Switch the infusion valve to pump in 8 CV of 20% ethanol at a flow rate of 3 ml / min for equilibration, and then remove the column and store it at 4°C.

[0077] 5. Diafiltration and concentration

[0078] Immediately after elution, the diluted eluate was transferred to a 300 KDa centrifugal concentrator tube, concentrated by centrifugation at 3000×g and 4°C, then washed with PBS for 3 times and finally concentrated to 3 ml. The concentrated lentivirus was sterilized by filtration through a 0.22 µm filter to obtain the purified BaEV envelope pseudotype lentivirus.

[0079] Example 3: Purification method of BaEV envelope pseudotype lentivirus

[0080] 1. Preparation of lentiviral clarified solution

[0081] Same as step 1 in Example 2.

[0082] 2. Preparation of optimized PEG8000 solution

[0083] Use PBS to prepare PEG8000 solution with a concentration of 25% (w / v, unit: g / L), then add 0.6 M NaCl, dissolve in a 50°C water bath, adjust the pH to 8.0, and filter through a 0.45 µm filter to obtain a 25% optimized PEG8000 solution.

[0084] 3. Optimized PEG8000 solution to concentrate lentivirus

[0085] To a 50 ml centrifuge tube, add 36 ml of lentivirus clarification solution and 9 ml of 25% optimized PEG8000 solution and mix evenly. Mix once every 20 min for a total of 4 times and place in a 4°C refrigerator overnight. On the next day, centrifuge at 3000×g at 4°C for 30 min, discard the supernatant and obtain the precipitate.

[0086] 4. Add 1 ml PBS to resuspend the precipitate, mix thoroughly, add 20 U / ml universal nuclease, and treat at 37°C for 1 h to obtain a crude pure sample.

[0087] 5. DEAE chromatography purification

[0088] (1) Equilibration: Equilibrate the column with 5 CV of equilibration buffer at a flow rate of 3 ml / min until the conductivity and pH are stable. The equilibration buffer consists of 50 mM Tris-HCl and 0.15 M NaCl, pH 8.0.

[0089] (2) Injection: Inject 10CV of crude pure sample through the infusion valve at a flow rate of 3ml / min. After the injection is completed, continue to pump in 10CV of equilibrium buffer at a flow rate of 3ml / min until UV 280 The value is stable;

[0090] (3) Elution: The infusion valve was switched to an eluent consisting of elution bufffer-A and bufffer-B, and linear elution was performed at a flow rate of 3 ml / min. The volume ratio of bufffer-B in the eluent increased from 0% to 60%, and the elution volume was 6 CV. The elution salt concentration increased from 0.1 M to 0.66 M, and then remained stable for 5 CV. UV 280 >50 mAU, start collecting eluate, UV 280 When the concentration is less than 50 mAU, the collection is stopped. After the eluate is collected, PBS is immediately added to the eluate to dilute the NaCl concentration in the eluate to less than 0.3 M. Among them, the elution buffer-A solution is 50 mM Tris-HCl and 0.15 M NaCl, pH 8.0; the elution buffer-B solution is 50 mM Tris-HCl and 1 M NaCl, pH 8.0;

[0091] (4) CIP: The infusion valve was switched to CIP-bufffer, and 6 CV of solution was pumped in at a flow rate of 3 ml / min for in-situ cleaning and regeneration; the CIP buffer was a 1 M NaOH aqueous solution;

[0092] (5) Cleaning: Pump 10 CV of equilibrium buffer at a flow rate of 3 ml / min to clean the column until the conductivity and pH value are stable;

[0093] (6) Column storage: Switch the infusion valve to pump in 8 CV of 20% ethanol at a flow rate of 3 ml / min for equilibration, and then remove the column and store it at 4°C.

[0094] 5. Wash and filtration concentration: Immediately after elution, transfer the diluted eluate into a 100 KDa ultrafiltration centrifuge tube, centrifuge and concentrate at 3000×g, 4°C, then wash and filter with PBS three times, and finally concentrate to 3 ml. The concentrated lentivirus is sterilized by filtration through a 0.22 µm filter to obtain the purified BaEV envelope pseudotype lentivirus.

[0095] The technical effects of the present invention are demonstrated by experimental examples below.

[0096] Experimental Example 1: Recovery rate of infection titer before and after optimization of PEG8000 concentration conditions

[0097] 1. Experimental samples

[0098] Sample before concentration: clarified lentivirus solution of Example 1;

[0099] Optimized PEG8000 concentrated sample: concentrated lentiviral precipitate obtained according to step 3 of Example 1;

[0100] Unoptimized PEG8000 concentrated sample: Referring to the method in step 3 of Example 1, the 40% optimized PEG8000 solution was replaced with the unoptimized PEG8000 solution to obtain a concentrated lentiviral precipitate; wherein the unoptimized PEG8000 solution was prepared by using ultrapure water to prepare the PEG8000 solution at a concentration of 25% (w / v, unit g / L), and then adding NaCl at a concentration of 0.8 M, and sterilizing at 121° C. for 30 min to obtain an unoptimized PEG8000 solution.

[0101] 2. Experimental Methods

[0102] 1. Count 293T cells after digestion and adjust the density to 2.0×10 5 / ml, inoculated into 12-well plates, 1ml in each well.

[0103] 2. Make 10-fold gradient dilutions of the virus solution in a 1.5ml centrifuge tube. The dilution method is: prepare 5 1.5ml tubes for each virus, add 135μl of culture medium to each tube, add 15μl of virus stock solution to the first tube, mix well, then pipette 15μl and add to the second tube to mix well. And so on, make 5 dilutions. After the cells adhere to the wall, discard the original culture medium in the 12-well plate and replace it with 0.9ml of new complete culture medium. Add 100μl of diluted virus solution to each well and mark it. Repeat 3 wells for each dilution, continue to culture for 48h, and then replace with fresh culture medium.

[0104] 3. The lentivirus itself expresses green fluorescent protein. 72 hours after infection, the cells were digested and the percentage of cells expressing green fluorescence was detected by flow cytometry to calculate the lentivirus titer.

[0105] Lentivirus titer calculation formula:

[0106] Virus infection titer (TU / ml) = number of cells × fluorescence percentage × 10 3 / Volume of virus stock solution (μl);

[0107] Total infectious titer = viral infectious titer × virus volume;

[0108] Infectious titer recovery rate = total infectious titer after concentration / total infectious titer before concentration × 100%.

[0109] 3. Experimental Results

[0110] The results are shown in Table 1. The infection titer recovery rate of the optimized PEG8000 group was significantly improved compared with the unoptimized PEG8000 group. The experimental results show that the use of optimized PEG8000 solution to concentrate BaEV envelope pseudotype lentivirus can significantly increase the total virus infection titer and titer recovery rate.

[0111] Table 1 Recovery rate of infection titer before and after optimization of PEG8000 concentration conditions

[0112]

[0113] Experimental Example 2: Lentivirus infection titer recovery rate and quality evaluation

[0114] 1. Experimental samples

[0115] The clear solution and purified solution prepared in Examples 1-3, wherein the clear solution refers to the clear solution of the lentivirus before purification, and the purified solution refers to the purified BaEV envelope pseudotype lentivirus.

[0116] 2. Experimental Methods

[0117] The virus infection titer test method refers to Experimental Example 1;

[0118] Infectious titer recovery rate = (purified fluid volume × purified fluid infectious titer) / (clarified fluid volume × clarified fluid infectious titer) × 100%;

[0119] The kinetic colorimetric method known in the art was used to detect the endotoxin residue in the purified solution, the ELISA method known in the art was used to detect the host cell protein (HCP) content in the purified solution, and the qPCR method known in the art was used to detect the host cell DNA (HCD) content in the purified solution.

[0120] 3. Experimental Results

[0121] The results are shown in Table 2. It can be seen that compared with the lentivirus clarification liquid before purification, the infection titer of the BaEV envelope pseudotype lentivirus obtained by the purification method of the BaEV envelope pseudotype lentivirus of the present invention is significantly improved, and the purification method of the present invention can effectively reduce HCP, HCD and endotoxin residues.

[0122] Further comparison revealed that, compared with Examples 2 and 3, the BaEV envelope pseudotype lentivirus obtained in Example 1 had the highest infection titer, the highest infection titer recovery rate, and the lowest HCP, HCD and endotoxin residues.

[0123] Table 2. Lentivirus infection titer recovery rate and quality evaluation

[0124]

[0125] Experimental Example 3: Activity of BaEV-encapsulated pseudotyped lentivirus

[0126] 1. Experimental samples

[0127] Example 1 shows lentivirus before and after purification, wherein the lentivirus before purification refers to the lentivirus clarified liquid, and the purified lentivirus refers to the purified BaEV envelope pseudotype lentivirus purified liquid.

[0128] 2. Experimental Methods

[0129] 1. Count 293T cells after digestion and adjust the density to 2.0×10 5 / ml, inoculated into 12-well plates, with 1 ml of cell suspension in each well.

[0130] 2. Take 100 μl of lentiviral clarified solution and 0.5 μl of lentiviral purified solution and add them to the cells in the 12-well plate, mark them, continue to culture for 48 hours, and then replace with fresh culture medium.

[0131] 3. The lentivirus itself expresses green fluorescent protein. 72 hours after infection, the green fluorescence expression of cells was observed under a fluorescence microscope and photographed.

[0132] 3. Experimental Results

[0133] The results are as follows Figure 1 As shown, compared with the clarified solution group, the green fluorescence of the purified solution group increased significantly, indicating that the cell infection titer of the BaEV envelope pseudotype lentivirus purified by the method of the present invention was significantly improved.

Claims

1. A method for purifying a BaEV envelope pseudotype lentivirus, characterized in that: The purification method comprises the following steps: Step 1: Concentrate the BaEV envelope pseudotype lentivirus clarification liquid, centrifuge, discard the supernatant, and obtain a lentivirus precipitate; the method for concentrating the BaEV envelope pseudotype lentivirus clarification liquid is: add a polyethylene glycol solution to the BaEV envelope pseudotype lentivirus clarification liquid and mix evenly; the polyethylene glycol is PEG8000; the volume ratio of the BaEV envelope pseudotype lentivirus clarification liquid and the polyethylene glycol solution is 4:1; the preparation method of the polyethylene glycol solution comprises the following steps: mix polyethylene glycol and NaCl in a buffer solution, adjust the pH, and sterilize; the polyethylene glycol solution contains 25-45% g / L polyethylene glycol and 0.5-0.7M NaCl; the pH of the polyethylene glycol solution is 7.5-8.0; Step 2: Resuspend the lentiviral precipitate with buffer and add universal nuclease for digestion to obtain a crude pure sample; Step 3: Purify the crude pure sample by chromatography to obtain a pure sample; the chromatography is Mustang QXT membrane chromatography capsule filter chromatography or DEAE cellulose chromatography; Step 4: The purified sample is filtered, concentrated, and sterilized to obtain the purified BaEV envelope pseudotype lentivirus.

2. The purification method according to claim 1, characterized in that In step 1, the BaEV envelope pseudotype lentivirus clarified fluid is obtained by centrifuging and filtering the BaEV envelope pseudotype lentivirus harvest fluid.

3. The purification method according to claim 1, characterized in that In step 1, the polyethylene glycol solution contains 40% g / L polyethylene glycol and 0.6 M NaCl, the pH of the polyethylene glycol solution is 8.0, and the sterilization method is sterilization by filtering through a 0.45 μm filter.

4. The purification method according to claim 1, characterized in that In step 2, the buffer is a buffer containing 40-60 mM Tris-HCl and 1-5 mM MgCl2, the pH value of the buffer is 7.5-8.5; the activity of the universal nuclease is 5-50 U / ml.

5. The purification method according to claim 4, characterized in that In step 2, the buffer is a buffer containing 50 mM Tris-HCl and 2 mM MgCl2, and the pH value of the buffer is 8.0; the activity of the universal nuclease is 40 U / ml.

6. The purification method according to claim 1, characterized in that In step 3, the chromatography is Mustang Q XT membrane chromatography capsule filter chromatography, and the Mustang Q XT membrane chromatography capsule filter chromatography comprises the following steps: (1) Equilibration: Equilibrate the Mustang Q XT membrane using equilibration buffer; (2) Sampling: Load the crude pure sample. After loading, continue to balance with the equilibration buffer until UV 280 The value is stable; (3) Elution: Elution is performed using an eluent, and the eluate is collected; the eluent is composed of an elution buffer A and an elution buffer B; the elution buffer A comprises 20 to 50 mM Tris-HCl and 0 to 0.3 M NaCl, with a pH value of 7.5 to 8.0; the elution buffer B comprises 20 to 50 mM Tris-HCl and 1 to 2 M NaCl, with a pH value of 7.5 to 8.0; the elution procedure is: firstly, linear elution is performed, the volume proportion of the elution buffer B in the eluent is increased from 0% to 60%, and then isocratic elution is performed, and the eluent UV 280 Start collecting when >50mAU, until UV 280 The collection was terminated when the concentration was less than 50 mAU.

7. The purification method according to claim 6, characterized in that In step (1), the equilibration buffer comprises 50 mM Tris-HCl and 0.3 M NaCl, and the pH value is 7.5-8.0; The elution buffer A includes 50 mM Tris-HCl and 0.15-0.3 M NaCl, with a pH value of 7.5-8.0; the elution buffer B includes 50 mM Tris-HCl and 1-1.5 M NaCl, with a pH value of 7.5-8.0; The elution procedure is as follows: firstly, linear elution is performed at a flow rate of 5 ml / min, the volume ratio of elution buffer B in the eluent is increased from 0% to 60%, and then isocratic elution is performed, and the eluent UV 280 Start collecting when >50mAU, until UV 280 The collection was terminated when the concentration was less than 50 mAU.

8. The purification method according to any one of claims 1 to 7, characterized in that In step 4, the diafiltration and concentration comprises the following steps: concentrating with a centrifugal concentrator tube and diafiltration with PBS; the molecular retention capacity of the centrifugal concentrator tube is 100-500KDa; and the sterilization method is sterilization by filtration through a 0.22μm filter.

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