Preparation method of recombinant adenovirus

Abstract

The invention discloses a preparation method of recombinant adenovirus, which comprises the following steps: adding chloroform into collected HEK 293T cells packaged with adenovirus and a culture medium for cracking at an amplification cracking stage of the adenovirus, extracting by using chloroform at a later purification stage, precipitating by using PEG8000, finally adding nuclease, and extracting by using chloroform. By adopting the preparation method disclosed by the invention, the yield of the adenovirus is high, 1mL of virus liquid with the titer greater than 5 * 10 < 10 > vg can be obtained in a 10cm vessel, and the requirements of cell experiments and animal experiments can be completely met; the method does not need complex equipment, is simple and rapid to operate and low in cost, can be used for large-scale production and purification of the recombinant adenovirus, and has important fundamental research and clinical application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of adenovirus, and more specifically, the present invention relates to a method for preparing a recombinant adenovirus. Background Art

[0002] Adenovirus, adeno-associated virus, retrovirus, and lentivirus are currently the most commonly used viral gene therapy vectors. Retroviruses can only infect continuously dividing cells, limiting their scope of application; lentiviruses will randomly integrate into the host genome, with potential carcinogenic risks; adeno-associated viruses have a wide host range, but their gene loading (up to 4.7 kb at most) is relatively low. In contrast, adenovirus (AdVenovirus, AdV) vectors can infect both dividing and non-dividing cells, with an infection efficiency as high as 100%, do not integrate into the host genome, have a gene loading as high as 8 kb - 36 kb, and have a high virus production titer, being easy to mass-produce, and have been widely used in gene therapy and vaccine development. Currently, a series of progress has been made in recombinant adenovirus vectors in basic research and clinical trials. Tumor therapy and vaccine vector trials based on adenovirus account for 50% of global virus vector-related clinical trials, and it is expected that this proportion will further increase over time.

[0003] Currently, the method for preparing recombinant adenovirus is to harvest infected cells - lyse the cells to release the virus - and then separate by density gradient centrifugation or chromatography column, which has the following defects:

[0004] 1. Usually, methods such as repeated freezing and thawing, homogenization, sonication, and detergents are used to release the virus. However, these methods are all relatively cumbersome, and it is difficult to achieve rapid and uniform processing of a large number of samples by repeated freezing and thawing. At the same time, appropriate freezing and thawing temperatures, pH values, and salt ion concentrations need to be explored; the homogenization pressure needs to be appropriate. Too high pressure will cause a large loss of virus and there are also problems with aseptic treatment; sonication will generate a large amount of free radicals that react with the virus capsid protein, affecting its infection efficiency. This method is difficult to use in large-scale production and also has problems with aseptic treatment; non-ionic detergents (such as Tween, Triton, and Brij) can be used to dissolve cell membranes and release adenovirus from cells, but additional steps are required later to remove the residual detergents.

[0005] 2. After host cell lysis, a large amount of free DNA of unassembled virus and host DNA will also be released. Long fragments of host DNA can easily promote the aggregation of virus particles, making downstream purification difficult. Although adenovirus particles carry an anionic charge on their surface, due to their sufficient surface area, most of the free virus DNA can still bind to the surface of the capsid. If not separated, it may be absorbed by target cells, resulting in abnormal replication; usually, nucleases are used to remove this exogenous DNA, but these residual nucleases need to be removed later.

[0006] 3. The cesium chloride (CsCl) density gradient centrifugation method can separate intact virus particles from nucleic acids, proteins, and empty capsid particles to obtain highly purified virus particles. However, this technique requires ultra-high-speed centrifugation equipment and two rounds of ultracentrifugation. Subsequently, toxic CsCl molecules need to be removed by dialysis, which greatly increases the cost and duration and is prone to loss of infectious particles. Additionally, this method cannot be scaled up for large-scale preparation temporarily, restricting its clinical development. Compared with CsCl, the iodixanol density gradient centrifugation method has a shorter centrifugation time and stronger biocompatibility, and can maintain the infectivity of virus particles during the purification process. However, this method also relies on high-speed centrifugation equipment, is complex to operate, and cannot be prepared on a large scale. Chromatography is the most commonly used method for virus purification currently, but it also has high requirements for instrument equipment and high cost consumption.

[0007] Studies have shown that intramuscular injection of adenovirus vector-based vaccines requires a dose of 10 11 -10 12 vp / mL (Viral particles per mL). The dose of oncolytic virus therapy varies depending on the administration route, tumor size, number of lesions, and treatment stage. For example, when administered locally, considering the spillage of the drug in the area around the lesion, the virus concentration required for high-dose therapy is as high as 10 14 vp / mL, while for intravenous injection, due to blood dilution and neutralization by serum antibodies, the required dose of the virus is higher. Therefore, how to simply, economically, and efficiently prepare purified recombinant adenovirus remains an important obstacle restricting its clinical application. Summary of the Invention

[0008] Based on this, the purpose of the present invention is to provide a simple, efficient, large-scale, and low-cost method for preparing recombinant adenovirus.

[0009] The specific technical solutions for achieving the above invention purpose are as follows.

[0010] A method for preparing recombinant adenovirus, comprising the following steps:

[0011] (1) Collect HEK 293 cells and culture medium packaged with adenovirus, add chloroform for lysis, centrifuge to obtain the supernatant, infect HEK 293 cells, change the culture medium after 22 h to 26 h, and culture until more than 75% of the HEK 293 cells float in the culture medium;

[0012] (2) Collect the HEK 293 cells and culture medium in step (1), add chloroform for lysis, centrifuge to obtain the supernatant, infect HEK 293 cells, and change the culture medium after 22 h to 26 h; repeat this step until 90% to 100% of the HEK 293 cells float in the culture medium;

[0013] (3) Add chloroform for extraction, take the supernatant, infect HEK 293 cells, change the medium after 22 h - 26 h, and culture until the HEK 293 cells become round and float in the medium completely. Then add chloroform for extraction and take the supernatant; then add PEG with a final concentration of 5% - 20%, mix well by shaking, and let it stand.

[0014] (4) Centrifuge, discard the supernatant, dissolve the precipitate, add chloroform for extraction, take the supernatant, continue to add chloroform for extraction, and take the supernatant; repeat this step until there is no precipitate.

[0015] (5) Add DNase I and RNase A for digestion, then add chloroform for extraction, take the supernatant, and that's it.

[0016] The inventors of the present invention found in the research on the preparation of recombinant adenovirus that: in the virus release and amplification stage, use chloroform for lysis; in the virus collection and purification stage, first use chloroform for extraction, then use polyethylene glycol with an appropriate final concentration for precipitation and concentration, and after adding nuclease for digestion, use chloroform for repeated extraction; each step is closely linked. Only by using common laboratory reagents such as chloroform and polyethylene glycol for routine tests can recombinant adenovirus be successfully prepared, and the yield of recombinant adenovirus is high. The titer in a 10 cm dish is greater than 5×10 10 vg, which can fully meet the needs of cell experiments and animal experiments.

[0017] In addition, the inventors of the present invention also found that in the virus collection and purification stage, using chloroform and a certain concentration of NaCl for extraction simultaneously can increase the titer of the prepared recombinant adenovirus.

[0018] The preparation method of the recombinant adenovirus of the present invention does not require cumbersome virus lysis and purification steps, does not need complex equipment, is simple, fast, and low-cost. It can be used for large-scale production and purification of recombinant adenovirus, and has important basic research and clinical application values. Brief Description of the Drawings

[0019] Figure 1 and Figure 2 is the preparation flow chart of the recombinant adenovirus in Example 1 of the present invention.

[0020] Figure 3 is the standard working curve for detecting virus titer in Example 2 of the present invention.

[0021] Figure 4 is the detection result of the Ct value and virus titer of the recombinant adenovirus in Example 2 of the present invention.

[0022] Figure 5 is the SDS-PAGE electrophoresis result of the recombinant adenovirus in Example 2 of the present invention.

[0023] Figure 6 This is the transmission electron micrograph of the recombinant adenovirus in Example 2 of the present invention.

[0024] Figure 7 This is the result of the effect of chloroform and 0.1% Triton X-100 on the virus particle titer during virus collection in Comparative Example 1 of the present invention.

[0025] Figure 8 This is the result of the effect of chloroform and repeated freeze-thaw on the virus particle titer during virus collection in Comparative Example 2 of the present invention.

[0026] Figure 9 This is the result of the effect of different PEG8000 concentrations on the virus particle titer in Comparative Example 3 of the present invention.

[0027] Figure 10 This is the effect of different NaCl concentrations on the virus particle titer in Comparative Example 4 of the present invention. Detailed implementation manners

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0030] If not otherwise specified, the embodiments are carried out under conventional experimental conditions, such as in the Molecular Cloning Experimental Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2013), or under the conditions recommended by the manufacturer's instructions.

[0031] In some embodiments of the present invention, a method for preparing a recombinant adenovirus is disclosed, including the following steps:

[0032] (1) Collect HEK 293 cells and culture medium packaged with adenovirus, add chloroform for lysis, centrifuge to obtain the supernatant, infect HEK 293 cells, change the culture medium after 22 h to 26 h, and culture until more than 75% of the HEK 293 cells float in the culture medium;

[0033] (2) Collect the HEK 293 cells and the culture medium described in step (1), add chloroform for lysis, centrifuge and take the supernatant, infect the HEK 293 cells, and change the culture medium after 22 h to 26 h; repeat this step until 90% to 100% of the HEK 293 cells float in the culture medium;

[0034] (3) Add chloroform for extraction, take the supernatant, infect the HEK 293 cells, change the culture medium after 22 h to 26 h, and after culturing until the HEK 293 cells become round and all float in the culture medium, add chloroform for extraction and take the supernatant; then add PEG with a final concentration of 5% to 20%, shake well and let stand;

[0035] (4) Centrifuge, discard the supernatant, dissolve the precipitate, add chloroform for extraction, take the supernatant, and continue to add chloroform for extraction and take the supernatant; repeat this step until there is no precipitate;

[0036] (5) Add DNase I and RNase A for digestion, then add chloroform for extraction, take the supernatant, and that's it.

[0037] In the present invention, in the lysis and amplification stage of the adenovirus, by adding chloroform to the collected infected cells and the culture medium, on the one hand, chloroform can quickly lyse the cells and release the virus particles, which can be immediately used for virus amplification after centrifugation, and on the other hand, chloroform can denature and precipitate most of the proteins, and the crude virus extract can be obtained by centrifugation; in the collection and purification stage of the adenovirus, first, it is preferably to use chloroform in combination with a high-concentration sodium chloride solution for extraction. Chloroform and the hypertonic condition can quickly lyse the cells and release the virus particles, and can also precipitate most of the miscellaneous proteins, which can be removed by centrifuging and taking the supernatant; then, adding a certain concentration of high-molecular-weight PEG (such as PEG8000) to the supernatant can promote the aggregation and precipitation of virus particles, and the high-molecular-weight polyethylene glycol occupies a certain volume in the solution, increasing the effective concentration of virus particles; finally, after precipitation with PEG, the precipitate is dissolved with PBS to obtain a concentrated crude extract of the recombinant adenovirus, and then nuclease is added to the crude extract to digest and remove the remaining DNA and RNA, and then an equal volume of chloroform is added for extraction 2 to 3 times. On the one hand, the nuclease is removed, and on the other hand, the remaining miscellaneous proteins can be further removed, and finally purified recombinant adenovirus particles are obtained.

[0038] In some embodiments, in step (3), after the HEK 293 cells become round and all float in the culture medium, add chloroform and sodium chloride for extraction.

[0039] In some embodiments, the final concentration of the sodium chloride is 0.5 M to 2.0 M.

[0040] In some of these embodiments, the final concentration of the sodium chloride is 1.0 M to 2.0 M.

[0041] In some of these embodiments, the final concentration of the sodium chloride is 1.8 M to 2.0 M.

[0042] In some of these embodiments, the PEG in step (3) is PEG8000.

[0043] In some of these embodiments, the final concentration of the PEG8000 is 10% to 20%.

[0044] In some of these embodiments, the final concentration of the PEG8000 is 18% to 20%.

[0045] In some of these embodiments, the volume ratio of the chloroform to the HEK 293 cells and the culture medium in step (1) or (2) is 1:5 to 20.

[0046] In some of these embodiments, the volume ratio of the chloroform to the HEK 293 cells and the culture medium is 1:9 to 11.

[0047] In some of these embodiments, the volume ratio of the chloroform to the HEK 293 cells and the culture medium is 1:9.5 to 10.5.

[0048] In some of these embodiments, the lysis in steps (1) to (2) includes the steps of: after oscillating for 4 min to 6 min, centrifuging at 3000 rpm to 5000 rpm for 4 min to 6 min.

[0049] In some of these embodiments, the extraction in steps (3) to (5) includes the steps of: after oscillating for 4 min to 6 min, centrifuging at 3000 rpm to 5000 rpm for 4 min to 6 min.

[0050] In some of these embodiments, the temperature of the centrifugation in step (4) is 3°C to 5°C, the rotation speed of the centrifugation is 3000 rpm to 5000 rpm, and the time of the centrifugation is 30 min to 90 min.

[0051] In some of these embodiments, the final concentration of the DNase I in step (5) is 8 μg / mL to 12 μg / mL, and the final concentration of the RNase A is 0.8 μg / mL to 1.2 μg / mL.

[0052] In some of these embodiments, MgCl2 and CaCl2 are also added during the digestion in step (5).

[0053] In some of these embodiments, the final concentration of MgCl2 is 2.0 mM to 3.0 mM, and the final concentration of CaCl2 is 0.08 mM to 0.12 mM.

[0054] In some of these embodiments, the HEK 293 cells packaging the adenovirus in step (1) are HEK 293 cells that have been cultured for 7 d to 14 d after packaging the adenovirus.

[0055] In some of these embodiments, the HEK 293 cells are HEK 293T cells.

[0056] In some of these embodiments, the culture medium in steps (1) to (3) is DMEM medium containing 0.5% FBS.

[0057] In some of these embodiments, the static standing in step (3) is static standing on ice for 1 h to 4 h, or static standing overnight at 3°C to 5°C.

[0058] In some of these embodiments, PBS is used to dissolve the precipitate in step (4).

[0059] In some of these embodiments, in step (4), the volume ratio of chloroform to PBS is 0.8 to 1.2:2.

[0060] In some of these embodiments, the temperature of digestion in step (5) is 37 ± 2°C, and the time of digestion is 30 min to 90 min.

[0061] In some of these embodiments, after step (5), there is also a step of concentrating the recombinant adenovirus using an ultrafiltration tube.

[0062] In some of these embodiments, the molecular weight cut-off of the ultrafiltration tube is 80 kDa to 120 kDa.

[0063] In some of these embodiments, the molecular weight cut-off of the ultrafiltration tube is 90 kDa to 110 kDa.

[0064] In some of these embodiments, the method for preparing the recombinant adenovirus includes the following steps:

[0065] (1) Collect the HEK 293T cells packaging the adenovirus and the culture medium, add chloroform for lysis, take the supernatant, and infect HEK 293T cells for 6 d to 8 d; collect the HEK 293T cells and the culture medium, add chloroform for lysis, take the supernatant, and infect HEK 293T cells for 2 d to 4 d; collect the HEK 293T cells and the culture medium, add chloroform for lysis, take the supernatant, and infect HEK 293T cells for 2 d to 4 d; add chloroform for lysis, and take the supernatant;

[0066] (2) Infect HEK 293T cells with the supernatant of step (1) until the HEK 293T cells become round and float completely. Add chloroform and sodium chloride with a final concentration of 1.8 M - 2.0 M to the HEK 293T cells and the culture medium for extraction, and take the supernatant; then add PEG8000 with a final concentration of 18% - 20%, mix well by shaking, and let it stand overnight at 3°C - 5°C.

[0067] (3) At 3°C - 5°C, centrifuge at 3000 rpm - 5000 rpm for 50 min - 70 min, discard the supernatant. After dissolving the precipitate with PBS, add chloroform for extraction, take the supernatant, and continue to add chloroform for extraction. Repeat this step until there is no precipitate.

[0068] (4) Add MgCl2 with a final concentration of 2.0 mM - 3.0 mM, CaCl2 with a final concentration of 0.08 mM - 0.12 mM, DNase I with a final concentration of 8 μg / mL - 12 μg / mL, and RNase A with a final concentration of 0.8 μg / mL - 1.2 μg / mL, and digest at 37 ± 2°C for 60 ± 5 min. Then add chloroform for extraction and take the supernatant.

[0069] (5) Use an ultrafiltration tube with a cut-off molecular weight of 90 kDa - 110 kDa to centrifuge and concentrate the supernatant of step (4) to obtain the product.

[0070] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0071] Example 1 A method for preparing recombinant adenovirus

[0072] Please refer to Figure 1 and Figure 2 , in this example, the fluorescent protein mCherry is used as the target gene, and a method for preparing recombinant adenovirus is provided, which specifically includes the following steps:

[0073] 1. Recombinant adenovirus packaging

[0074] Digest HEK 293T cells with trypsin, seed them into a 6-cm dish at a density of 5×10 5 cells / dish, and culture for 12 h, then perform transfection using the Ad-MAX packaging system.

[0075] Add 4 μg of the core plasmid pDC316-EF1-mCherry (i.e., the recombinant adenovirus vector) and 8 μg of the helper packaging factor pBHGlox(delta)E1,E3Cre (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) to 300 μL of serum-free MEM medium. After mixing, add the PEI transfection reagent at a ratio of 1:3 (1 μg of core plasmid: 3 μL of PEI). After mixing, let it stand at room temperature for 30 min. Then, add the mixture dropwise to a dish seeded with HEK 293T cells. After gently shaking, continue culturing at 37 °C and 5% CO2. After 12 h, replace the medium with DMEM medium containing 0.5% FBS, and then continue culturing for 8 days.

[0076] 2. Amplification of recombinant adenovirus

[0077] Transfer the HEK 293T cells from step 1 together with the supernatant medium (using a pipette to blow and suck) into a 15 mL centrifuge tube. Add 1 / 10 volume of chloroform, vortex for 5 min, then centrifuge at 4000 rpm for 5 min, and collect the supernatant into a new centrifuge tube.

[0078] Take 1 / 10 volume of the supernatant and infect HEK 293T cells (1×10 6 cells, 6 cm dish). After 24 h, replace the medium with DMEM medium containing 0.5% FBS, observe the fluorescence expression of the cells and the cell adhesion situation. Culture for about 7 days until most cells float in the medium (i.e., the first amplification). Collect the cells and the supernatant medium into a 15 mL centrifuge tube, add 1 / 10 volume of chloroform, vortex for 5 min, then centrifuge at 4000 rpm for 5 min, and collect the supernatant into a new centrifuge tube.

[0079] Take 1 / 10 volume of the supernatant and infect HEK 293T cells (1×10 6 cells, 6 cm dish). After 24 h, replace the medium with DMEM medium containing 0.5% FBS, observe the fluorescence expression of the cells and the cell adhesion situation. After culturing for 3 days (i.e., the second amplification), collect the cells and the supernatant medium into a 15 mL centrifuge tube, add 1 / 10 volume of chloroform, vortex for 5 min, then centrifuge at 4000 rpm for 5 min, and collect the supernatant into a new centrifuge tube.

[0080] Take 1 / 10 volume of the supernatant and infect HEK 293T cells (1×10 6cells, 6 cm dish), replaced with DMEM medium containing 0.5% FBS after 24 h, observed cell fluorescence expression and cell adhesion, and cultured for 3 days (i.e., the third amplification, at which time the cell infection efficiency reached 100%, and all HEK 293T cells floated in the culture medium), collected the cells and supernatant medium into a 15 mL centrifuge tube, added 1 / 10 volume of chloroform, vortexed for 5 min, centrifuged at 4000 rpm for 5 min, and collected the supernatant into a new centrifuge tube.

[0081] 3. Lyse cells, collect recombinant adenovirus, and purify recombinant adenovirus

[0082] (1) The adenovirus supernatant (2 mL) extracted with chloroform was used to infect HEK 293T cells (approximately 1.5 × 10 7 cells), and after 12 hours, replace it with 40 mL of DMEM medium containing 0.5% FBS, and wait until the cells become round and all float in the medium (about 48 to 72 hours); transfer the cells and the supernatant medium to a 50 mL centrifuge tube, add sodium chloride with a final concentration of 1 M and 1 / 10 volume of chloroform, vortex for 5 minutes, and centrifuge at 4000 rpm for 5 minutes; collect the supernatant, add PEG8000 with a final concentration of 10% (w / v) (purchased from Beijing Solebow Technology Co., Ltd., Cat: P8260), shake vigorously to mix, and let it stand on ice for more than 1 hour or at 4°C overnight;

[0083] (2) Centrifuge at 4000 rpm for 60 min at 4°C, discard the supernatant, drain the residual liquid at the bottom as much as possible, and use 1 mL of PBS to dissolve the precipitate; add 500 μL of chloroform, vortex for 5 min, centrifuge at 4000 rpm for 5 min, and collect the supernatant; continue to add an equal volume of chloroform for extraction until there is no obvious protein precipitation;

[0084] (3) Add MgCl2 at a final concentration of 2.5 mM, CaCl2 at a final concentration of 0.1 mM, DNase I at a final concentration of 10 μg / mL, and RNase A at a final concentration of 1 μg / mL, and digest at 37°C for 60 min; then add an equal volume of chloroform for extraction to remove nucleases.

[0085] 4. Recombinant adenovirus concentration

[0086] The obtained recombinant adenovirus supernatant was concentrated by centrifugation at 4000 rpm and 4° C. using an ultrafiltration tube with a molecular weight cutoff of 100 kDa to obtain the final recombinant adenovirus product.

[0087] Example 2 Detection of virus titer of recombinant adenovirus

[0088] In this example, the qPCR method was used to determine the titer of the recombinant adenovirus prepared in Example 1, including the following steps:

[0089] 1. Preparation of the standard curve

[0090] Take 1 pmol of plasmid pBHGlox(delta)E1,E3Cre and add it to 1 mL of ddH2O. The copy number is 6.02×10 11 Copies / mL. Then, perform a 10-fold serial dilution of the plasmid. Combine it with the 6.02×10 10 Copies / mL, 6.02×10 9 Copies / mL, 6.02×10 8 Copies / mL, 6.02×10 7 Copies / mL, 6.02×10 6 Copies / mL, 6.02×10 5 Copies / mL, a total of 7 gradient templates for qPCR (Forward Primer: CTCCAACTGTGCCTTTTC, SEQ ID NO:1; Reverse Primer: GGCTCACAGTGGTTACATT, SEQ ID NO:2. The qPCR reaction system and conditions are shown in Tables 1 and 2); Use the log10 of the DNA copy number as the abscissa and the corresponding Ct value as the ordinate to plot the fluorescence quantitative curve, as Figure 3 shown.

[0091] Table 1 qPCR reaction system

[0092]

[0093]

[0094] Note: 2×SGExcelFastSYBR Mixture is purchased from Sangon Biotech (Shanghai) Co., Ltd. (NO.B532955).

[0095] Table 2 qPCR reaction conditions

[0096]

[0097] 2. Determination of adenovirus titer

[0098] Take the purified recombinant adenovirus stock solution of Example 1 and dilute it 20-fold with ddH2O; add the diluted virus solution to proteinase K (5 mg / mL) and digest at 55 °C for 4 h; heat at 95 °C for 10 min to inactivate proteinase K; centrifuge at 12,000 rpm for 5 min, and take the supernatant samples (the virus stock solution diluted 20-fold), the sample after diluting 10-fold with water (the virus stock solution diluted 200-fold), and the sample after diluting 100-fold with water (the virus stock solution diluted 2,000-fold), and then perform qPCR to detect their Ct values, and then calculate the virus titer according to the standard curve, and take the average value of 3 measurements as the final titer value.

[0099] The results are as Figure 4 shown. Approximately 1 mL of virus solution with a titer of 5×10 10 vg / mL can be obtained from a 10-cm dish, which can fully meet the requirements of cell experiments and animal experiments.

[0100] Add the purified adenovirus particles to the protein loading buffer, heat and denature at 95 °C, and then perform SDS-PAGE electrophoresis. Obvious adenovirus capsid protein bands can be found by silver staining ( Figure 5 ); and obvious adenovirus particle morphology can also be observed by transmission electron microscopy ( Figure 6 ); indicating that the preparation method of the present invention can obtain adenovirus particles with relatively high purity.

[0101] Comparative Example 1

[0102] This comparative example provides a method for preparing recombinant adenovirus, which specifically includes the following steps:

[0103] 1. Recombinant adenovirus packaging

[0104] Same as Example 1.

[0105] 2. Recombinant adenovirus amplification

[0106] Same as Example 1.

[0107] 3. Lyse cells, collect recombinant adenovirus, and purify recombinant adenovirus

[0108] (1) Infect the adenovirus supernatant (2 mL) extracted with chloroform into HEK 293T cells (about 1.5×10 7(0 cells), after 12 h, the medium was replaced with 40 mL of DMEM medium containing 0.5% FBS, and the cells were allowed to become round until all floating in the medium (about 48 - 72 h); the cells and the supernatant medium were transferred to a 50 mL centrifuge tube, and TritonX-100 with a final concentration of 0.1% (purchased from Sangon Biotech (Shanghai) Co., Ltd., Cat: A110694-0500) was added, mixed well and lysed for 20 min, and centrifuged at 4000 rpm for 5 min; the supernatant was collected, and PEG8000 with a final concentration of 10% (w / v) (purchased from Solarbio Science & Technology Co., Ltd., Beijing, Cat: P8260) was added. After vigorous shaking and mixing, it was left standing on ice for more than 1 hour or overnight at 4°C;

[0109] Other steps were the same as in Example 1.

[0110] 4. Recombinant adenovirus concentration

[0111] Same as Example 1.

[0112] The titer of the recombinant adenovirus prepared in this comparative example was measured, and the titer was compared with that of the recombinant adenovirus prepared by the method of Example 1. The results are as Figure 7 shown. It can be seen from Figure 7 that when TritonX-100 was used to replace sodium chloride-chloroform in the virus collection and purification stages, the titer of the finally prepared recombinant adenovirus would decrease significantly.

[0113] Comparative Example 2

[0114] This comparative example provides a method for preparing a recombinant adenovirus, which specifically includes the following steps:

[0115] 1. Recombinant adenovirus packaging

[0116] Same as Example 1.

[0117] 2. Recombinant adenovirus amplification

[0118] Same as Example 1.

[0119] 3. Lyse cells, collect recombinant adenovirus, and purify recombinant adenovirus

[0120] (1) Infect the adenovirus supernatant (2 mL) extracted with chloroform on HEK 293T cells (about 1.5×10 7Cells), after 12 h, the medium was replaced with 40 mL of DMEM medium containing 0.5% FBS, and the cells were allowed to become round until all of them floated in the medium (about 48 - 72 h); the cells and the supernatant medium were transferred to a 50 mL centrifuge tube, and in a liquid nitrogen environment, they were repeatedly frozen and thawed 3 times at 37 °C, and centrifuged at 4000 rpm for 5 min; the supernatant was collected, and PEG8000 with a final concentration of 10% (w / v) (purchased from Beijing Solarbio Science & Technology Co., Ltd., Cat: P8260) was added. After thorough mixing by vigorous shaking, it was left standing on ice for more than 1 hour or overnight at 4 °C;

[0121] Other steps were the same as those in Example 1.

[0122] 4. Concentration of recombinant adenovirus

[0123] Same as Example 1.

[0124] The titer of the recombinant adenovirus prepared in this comparative example was measured and compared with the titer of the recombinant adenovirus prepared by the method of Example 1. The results are as Figure 8 shown. From Figure 8 it can be seen that in the virus collection stage, when the commonly used method of repeated freezing and thawing is used to collect the virus, compared with the method of collecting the virus by sodium chloride-chloroform extraction and lysis, the titer of the finally prepared recombinant adenovirus will decrease significantly.

[0125] Comparative Example 3

[0126] This comparative example provides a method for preparing a recombinant adenovirus, which specifically includes the following steps:

[0127] 1. Packaging of recombinant adenovirus

[0128] Same as Example 1.

[0129] 2. Amplification of recombinant adenovirus

[0130] Same as Example 1.

[0131] 3. Lysing cells, collecting recombinant adenovirus, and purifying recombinant adenovirus

[0132] Except that in step (1), PEG8000 with final concentrations of 0%, 1.25%, 2.5%, 5%, 10%, and 20% (w / v) were used respectively; other steps were the same as those in Example 1.

[0133] 4. Concentration of recombinant adenovirus

[0134] Same as Example 1.

[0135] The adenovirus titer results are as Figure 9 shown. From Figure 9It can be seen that after extraction with sodium chloride and chloroform and then treatment with different concentrations of PEG8000, when the concentration of PEG8000 is greater than 2.5%, the titer of the finally precipitated virus particles increases significantly, and the effect is best when the concentration of PEG8000 is greater than 10%.

[0136] Comparative Example 4

[0137] This comparative example provides a method for preparing recombinant adenovirus, which specifically includes the following steps:

[0138] 1. Packaging of recombinant adenovirus

[0139] Same as Example 1.

[0140] 2. Amplification of recombinant adenovirus

[0141] Same as Example 1.

[0142] 3. Lysing cells, collecting recombinant adenovirus, and purifying recombinant adenovirus

[0143] Except that sodium chloride with final concentrations of 0M, 0.5M, 1M, and 2M and 1 / 10 volume of chloroform were added separately in step (1) and vortexed; other steps were the same as in Example 1.

[0144] 4. Concentration of recombinant adenovirus

[0145] Same as Example 1.

[0146] The adenovirus titer results are as Figure 10 shown, and from Figure 10 it can be seen that as the final concentration of sodium chloride increases, the titer of the finally precipitated virus particles will increase to some extent.

[0147] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0148] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for preparing a recombinant adenovirus, characterized in that, It includes the following steps: (1) Collect HEK 293 cells and culture medium packaged with adenovirus, add chloroform for lysis, centrifuge and take the supernatant, infect HEK 293 cells, change the culture medium after 22 h to 26 h, and culture until more than 75% of the HEK 293 cells float in the culture medium; (2) Collect the HEK 293 cells and culture medium in step (1), add chloroform for lysis, centrifuge and take the supernatant, infect HEK 293 cells, change the culture medium after 22 h to 26 h; repeat this step until 90% to 100% of the HEK 293 cells float in the culture medium; (3) Add chloroform for extraction, take the supernatant, infect HEK 293 cells, change the culture medium after 22 h to 26 h, culture until the HEK 293 cells become round and all float in the culture medium, then add chloroform for extraction, take the supernatant; then add PEG with a final concentration of 5% to 20%, shake well, and let stand; (4) Centrifuge, discard the supernatant, dissolve the precipitate, add chloroform for extraction, take the supernatant, and continue to add chloroform for extraction, take the supernatant; Repeat this step until there is no precipitate; (5) Add DNase I and RNase A for digestion, then add chloroform for extraction, take the supernatant, and that's it.

2. The preparation method of the recombinant adenovirus according to claim 1, wherein, The PEG in step (3) is PEG8000; preferably, the final concentration of the PEG8000 is 10% to 20%, preferably 18% to 20%.

3. The preparation method of the recombinant adenovirus according to claim 1, wherein In step (3), after the HEK 293 cells become round and all float in the culture medium, add chloroform and sodium chloride for extraction.

4. The method for preparing the recombinant adenovirus according to claim 3, wherein, The final concentration of the sodium chloride is 0.5 M to 2.0 M, preferably 1.0 M to 2.0 M, more preferably 1.8 M to 2.0 M.

5. The preparation method of the recombinant adenovirus according to claim 1, wherein In step (1) or (2), the volume ratio of the chloroform to the HEK 293 cells and the culture medium is 1:5 to 20, preferably 1:9 to 11, more preferably 1:9.5 to 10.

5.

6. The method for preparing a recombinant adenovirus according to claim 1, wherein the lysis in steps (1) to (2) includes the steps: after oscillating for 4 min to 6 min, centrifuge at 3000 rpm to 5000 rpm for 4 min to 6 min; and / or, the extraction in steps (3) to (5) includes the steps: after oscillating for 4 min to 6 min, centrifuge at 3000 rpm to 5000 rpm for 4 min to 6 min.

7. The preparation method of the recombinant adenovirus according to claim 1, characterized in that, In step (4), the temperature of the centrifuge is 3°C to 5°C, the rotation speed of the centrifuge is 3000 rpm to 5000 rpm, and the time of the centrifuge is 30 min to 90 min.

8. The method for preparing a recombinant adenovirus according to claim 1, characterized in that, In step (5), the final concentration of the DNase I is 8 μg / mL to 12 μg / mL, and the final concentration of the RNase A is 0.8 μg / mL to 1.2 μg / mL; and / or, in step (5), MgCl2 and CaCl2 are also added during the digestion; preferably, the final concentration of the MgCl2 is 2.0 mM to 3.0 mM, and the final concentration of the CaCl2 is 0.08 mM to 0.12 mM.

9. The preparation method of the recombinant adenovirus according to any one of claims 1 to 8, characterized in that, The HEK 293 cells packaged with adenovirus in step (1) are HEK 293 cells cultured for 7 to 14 days after adenovirus packaging; preferably, the HEK 293 cells are HEK 293T cells; And / or, the culture medium in steps (1) to (3) is DMEM medium containing 0.5% FBS; And / or, the static standing in step (3) is static standing on ice for 1 to 4 hours, or static standing overnight at 3°C to 5°C; And / or, in step (4), PBS is used to dissolve the precipitate; preferably, in step (4), the volume ratio of chloroform to PBS is 0.8 to 1.2:2; And / or, the temperature of digestion in step (5) is 37 ± 2°C, and the digestion time is 30 minutes to 90 minutes.

10. The preparation method of the recombinant adenovirus according to any one of claims 1 to 8, characterized in that, After step (5), there is also a step of concentrating the recombinant adenovirus using an ultrafiltration tube; preferably, the molecular weight cut-off of the ultrafiltration tube is 80 kDa to 120 kDa, preferably 90 kDa to 110 kDa.

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