CN115786400A - Efficient adeno-associated virus preparation system and method - Google Patents
Efficient adeno-associated virus preparation system and method Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a high-efficiency adeno-associated virus preparation system and a method, wherein the system comprises a 293T cell line capable of stably expressing Cre recombinase, recombinant adenovirus carrying part of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus; elements necessary for generating the recombinant adeno-associated virus include Cap genes, rep genes and AAV core expression elements with exogenous genes; the genome of the recombinant adenovirus is deleted with E1 and E3 genes, and parts of elements necessary for generating the recombinant adeno-associated virus are inserted into the areas where the E1 gene and/or the E3 gene are located, and both ends of a packaging signal psi of the recombinant adenovirus are inserted with a loxp sequence in the same direction. Compared with the traditional three-plasmid system, the system of the invention can obviously improve the yield and purity of AAV and the stability and biological safety of the whole system.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a high-efficiency adeno-associated virus preparation system and method.
Background
The existing method for preparing adeno-associated virus (AAV) by transfecting 293 cells based on three-plasmid (AAV-ITR, AAV-RC and Helper) system has many problems, for example, firstly, the production of AAV by AAV suspension or adherent cells is generally 10 times 9 ～10 10 Between vg/mL, the yield is difficult to meet the amount required by gene therapy, so the existing AAV gene therapy is very expensive; secondly, when AAV is prepared on a large scale based on a three-plasmid transfection system, three plasmid DNAs are required to be prepared simultaneously, the large-scale plasmid preparation may have many problems, and the quality of plasmids prepared in different batches is different, which greatly affects the AAV yield and increases the AAV preparation cost. Although the currently used BAC-sf9 system can achieve higher yields in AAV production, due to the use of insect cells in the production process, genes from heterologous species may be introduced during AAV production, leading to potential risks of gene therapy, and the extremely unstable baculovirus genome also greatly increases the uncertainty of AAV production.
Therefore, there is a need to develop a new high-efficiency AAV production system, which can greatly increase AAV yield and improve overall system stability and biosafety.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a high-efficiency adeno-associated virus preparation system and method, which can improve the yield and purity of AAV, avoid the introduction of heterologous species genes, and improve the stability of the preparation system.
In order to achieve the above objects, the present invention provides a high efficiency recombinant adeno-associated virus production system, which comprises a 293T cell line capable of stably expressing Cre recombinase, a recombinant adenovirus carrying a part of elements necessary for producing recombinant adeno-associated virus, and an expression plasmid carrying other elements necessary for producing recombinant adeno-associated virus;
the elements necessary for generating the recombinant adeno-associated virus comprise an AAVCap gene, an AAVRep gene and an AAV core expression element ITR-GOI with a foreign gene;
the genome of the recombinant adenovirus is deleted with E1 gene and E3 gene, and the elements necessary for partially generating recombinant adeno-associated virus are inserted into the region where the E1 gene and/or the E3 gene are located, two ends of a packaging signal psi of the recombinant adenovirus are respectively inserted with a loxp sequence, and the two loxp sequences are in the same direction.
Preferably, the elements necessary for the partial production of the recombinant adeno-associated virus are the AAVRep gene and the AAVCap gene, and the other elements necessary for the production of the recombinant adeno-associated virus are the AAV core expression elements ITR-GOI carrying the foreign genes.
According to another aspect of the present invention, there is also provided a method for preparing a recombinant adeno-associated virus with high efficiency, comprising the steps of:
s1, respectively constructing a 293T cell line capable of stably expressing Cre recombinase, a recombinant adenovirus carrying part of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus;
the elements necessary for generating the recombinant adeno-associated virus comprise an AAVCap gene, an AAVRep gene and an AAV core expression element ITR-GOI with a foreign gene;
the genome of the recombinant adenovirus is deleted with E1 gene and E3 gene, and the elements necessary for partially generating recombinant adeno-associated virus are inserted into the region where the E1 gene and/or the E3 gene are located, two ends of a packaging signal psi of the recombinant adenovirus are respectively inserted with a loxp sequence, and the two loxp sequences are in the same direction;
s2, transfecting the 293T cell line capable of stably expressing Cre recombinase with the expression plasmid carrying other elements necessary for generating the recombinant adeno-associated virus, then infecting the 293T cell line capable of stably expressing Cre recombinase after transfection with the recombinant adenovirus, and culturing to obtain virus liquid containing the recombinant adeno-associated virus;
and S3, purifying the virus liquid containing the recombinant adeno-associated virus, and removing the adenovirus in the virus liquid to obtain the pure recombinant adeno-associated virus.
Preferably, in step S1, the 293T cell line capable of stably expressing Cre recombinase is constructed by means of plasmid transfection or lentivirus.
Further preferably, the method for constructing the 293T cell line capable of stably expressing Cre recombinase comprises the following steps: transfecting 293T cells with recombinant lentivirus plasmids carrying Cre genes, and screening by using purine to obtain a positive monoclonal cell line;
in the recombinant lentiviral plasmid, the Cre gene contains a nuclear localization sequence upstream.
Further preferably, the method for constructing the 293T cell line capable of stably expressing Cre recombinase further comprises: and respectively infecting the positive monoclonal cell lines obtained by screening by the same amount of the recombinant adenovirus with the infection complex number ranging from 1 to 100, and selecting the cell lines which are well maintained after infecting for 48 hours to be used as 293T cell lines for preparing the recombinant adeno-associated virus.
Preferably, in step S1, the method for constructing the recombinant adenovirus comprises: inserting a section of loxp sequence into each of two ends of a packaging signal psi in an adenovirus shuttle plasmid, and inserting elements necessary for partially generating recombinant adeno-associated virus into the adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; electrically transforming the recombinant shuttle plasmid and the adenovirus skeleton plasmid into BJ5183 competent cells, and obtaining a recombinant adenovirus plasmid through homologous recombination; and (3) transfecting 293 cells with the recombinant adenovirus plasmid to save the recombinant adenovirus capable of expressing elements necessary for generating the recombinant adeno-associated virus.
Preferably, in step S2, the recombinant adenovirus has a multiplicity of infection of 10 to 20.
Preferably, in step S3, the method for purifying the virus solution containing the recombinant adeno-associated virus comprises chloroform extraction and iodixanol density gradient centrifugation.
Preferably, the recombinant adeno-associated virus production method is used for producing AAV serotype 1, 2, 5, 8, 9 or PHPeB.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) Compared with the AAV prepared by pure plasmid transfection (such as a three-plasmid system), the adeno-associated virus preparation system provided by the invention has the advantages that the adenovirus can infect 293T cells by 100%, the transfection efficiency of suspension cells is generally about 50-70%, namely, 30-50% of cells can not be transfected by the plasmid carrying elements necessary for generating recombinant adeno-associated virus and do not produce AAV, and simultaneously, because the cells are not loaded with the plasmid, the cells can keep higher activity in a culture medium, can be rapidly amplified, consume a large amount of nutrients, can adsorb a large amount of newly generated AAV particles, and further reduce the yield of the final AAV. The method for preparing the adeno-associated virus uses adenovirus infection, 100% of 293T cells are infected, and newly generated AAV particles enter cells which are not transfected with expression plasmids, and can start recombinant adeno-associated virus amplification under the assistance of adenovirus, so that the final AAV yield is improved. Meanwhile, the Cre/loxp system is utilized in the recombinant adenovirus, so that the amplification of the adenovirus can be inhibited in the process of producing the rAAV, and the purity of AAV is further improved. AAV produced by the system of the present invention has yield as high as 10 12 The Vg/mL is higher than that of the traditional three-plasmid system, and the Vg/mL can be improved by more than 100 times.
(2) The RC plasmid and the helper plasmid of the three-plasmid system are equivalent to be directly provided by the recombinant adenovirus in the invention, and the adenovirus has the characteristic of efficiently infecting 293 series cells, so that the cells can be infected by 100 percent, and therefore, the Rep-Cap gene of AAV carried by the adenovirus can be highly expressed, and simultaneously, all genes of the helper plasmid exist in the adenovirus.
(3) Compared with the traditional three-plasmid preparation system, the preparation method of the recombinant adeno-associated virus provided by the invention does not need to prepare three plasmids at the same time, only one plasmid needs to be prepared, so that the plasmid extraction cost is greatly reduced, and meanwhile, when a 293T cell is transfected, only one plasmid needs to be transfected, compared with the three plasmids which are transfected by a three-plasmid system at the same time, more plasmids can be transfected in a unit volume, so that the transfection efficiency is improved, or less transfection reagents are used, the toxicity of the transfection reagents is reduced, and the activity of the cell is improved.
(4) In the AAV preparation method, the adopted adenovirus amplification process is very simple, the amplification can be realized only by adding cell lysate into 293 cells, and the subsequent infection can be realized without purification after the virus is obtained.
(5) The preparation method can be used for preparing all serotype AAV currently existing, and only needs to select corresponding Cap genes in the process of constructing recombinant adenovirus.
Drawings
FIG. 1 is a schematic diagram of the process for preparing AAV by using the modified Ad-easy system in the present invention.
FIG. 2 is a graph comparing production of AAV of different serotypes prepared in examples of the present invention and comparative examples.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a high-efficiency recombinant adeno-associated virus preparation system, which comprises a 293T cell line 293T-Cre capable of stably expressing Cre recombinase, a recombinant adenovirus carrying part of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus.
The 293T-Cre cell line can stably express Cre recombinase and is used for preparing recombinant adeno-associated virus (rAAV). An E1 gene and an E3 gene are deleted in a genome of a recombinant adenovirus Ad-loxp-psi-loxp-Rep-Cap, elements necessary for partially generating recombinant adeno-associated virus are inserted into a region where the E1 gene and/or the E3 gene are located, a loxp sequence is inserted into each of two ends of a packaging signal psi of the recombinant adenovirus, and the two loxp sequences are in the same direction. Deletion of both genes E1 and E3 in the adenovirus genome can provide insertion sites for larger foreign DNA and eliminate the ability to self-replicate. Deletion of the E1 gene results in defective viral replication and the inability to produce infectious viral particles in the host cell; the E3 gene encodes a protein involved in host immune escape, if any. In the invention, the E1 locus is used for inserting elements which are required by a foreign part to generate the recombinant adeno-associated virus, so that the operation is more convenient. And inserting a section of homodromous loxp sequence at two ends of a packaging signal Ψ in the recombinant adenovirus genome respectively, wherein the loxp sequences can be recognized by Cre recombinase, and then cutting off the packaging signal Ψ sequence between two loxp sites, so that the amplification of adenovirus is inhibited, and the finally prepared rAAV virion with higher purity in virus liquid is obtained.
As is well known, the elements necessary for generating the recombinant adeno-associated virus comprise AAV Cap genes, AAV Rep genes and AAV core expression elements ITR-GOI with exogenous genes, the invention uses recombinant adenovirus to express the elements necessary for generating the recombinant adeno-associated virus in a 293T cell line, and uses an expression plasmid to transfect the 293T cell line to express the rest of the elements necessary for generating the recombinant adeno-associated virus. Preferably, the recombinant adenovirus in the embodiment of the invention carries AAVRep gene and AAVCap gene in the genome, and the AAV core expression element ITR-GOI carrying the foreign gene is carried on the expression plasmid.
In another aspect, the present invention provides a method for preparing a recombinant adeno-associated virus with high efficiency, comprising the steps of:
s1, respectively constructing a 293T cell line capable of stably expressing Cre recombinase, a recombinant adenovirus carrying part of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus;
the elements necessary for generating the recombinant adeno-associated virus comprise an AAVCap gene, an AAVRep gene and an AAV core expression element ITR-GOI with a foreign gene;
the genome of the recombinant adenovirus is deleted with E1 gene and E3 gene, and the elements necessary for partially generating recombinant adeno-associated virus are inserted into the region where the E1 gene and/or the E3 gene are located, two ends of a packaging signal psi of the recombinant adenovirus are respectively inserted with a loxp sequence, and the two loxp sequences are in the same direction;
s2, transfecting the 293T cell line capable of stably expressing Cre recombinase with the expression plasmid carrying other elements necessary for generating the recombinant adeno-associated virus, then infecting the 293T cell line capable of stably expressing Cre recombinase after transfection with the recombinant adenovirus, and culturing to obtain virus liquid containing the recombinant adeno-associated virus;
and S3, purifying the virus liquid containing the recombinant adeno-associated virus, and removing the adenovirus in the virus liquid to obtain the pure recombinant adeno-associated virus.
In some embodiments, the 293T cell line capable of stably expressing Cre recombinase is constructed by plasmid transfection or lentivirus. The 293T cell line used in the present invention includes, but is not limited to, HEK-293T cell line. In order to rapidly construct 293T-Cre cell line, it is preferable to construct cell line by means of lentivirus. In consideration of the risk of subsequent gene therapy, the invention preferably constructs a recombinant lentiviral plasmid carrying Cre gene, then transfects the recombinant lentiviral plasmid into 293T cells, cultures the transfected recombinant lentiviral plasmid for a period of time, and finally obtains a positive monoclonal cell line by purine screening. Furthermore, in order to select cell lines which are stable and highly express Cre, the positive monoclonal cell lines obtained by selection are infected with the same amount of the recombinant adenovirus with the multiplicity of infection ranging from 1 to 100, and after 48 hours of infection, the cell lines which are maintained in a good state are selected and used as 293T cell lines for preparing recombinant adeno-associated viruses. In order to allow the Cre recombinase to be better expressed in the nucleus, the plasmid vector is designed by adding a nuclear localization sequence, such as SV40NLS, upstream of the Cre gene to guide the Cre recombinase into the nucleus. The cell line can be cultured by adherent culture or suspension culture, and a suspension culture mode is preferred, so that the later large-scale preparation is facilitated.
In some embodiments, the recombinant adenovirus is constructed by a method comprising: inserting a section of loxp sequence into each of two ends of a packaging signal psi in an adenovirus shuttle plasmid, and inserting elements necessary for partially generating recombinant adeno-associated virus into the adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; electrically transforming the recombinant shuttle plasmid and the adenovirus skeleton plasmid into BJ5183 competent cells, and obtaining a recombinant adenovirus plasmid through homologous recombination; and (3) transfecting 293 cells with the recombinant adenovirus plasmid to save the recombinant adenovirus capable of expressing elements necessary for generating the recombinant adeno-associated virus. The recombinant adenovirus plasmid constructed by the invention can be transformed on Ad-easy system plasmid, and also can be transformed on other commercial adenovirus preparation systems (such as Admax system). In order to facilitate the acquisition of recombinant adenovirus, the Ad-easy system is preferably optimized in the embodiments of the present invention. Generally, 293 cells and 293T cells can be used for adenovirus amplification, and the invention adopts 293 cells to amplify adenovirus in order to obtain higher yield of recombinant adenovirus.
In the present invention, experimental studies are performed on the optimal multiplicity of infection of 293T cells by the recombinant adenovirus to prepare the recombinant adeno-associated virus, and in step S2, the multiplicity of infection of the recombinant adenovirus is preferably 10 to 20, and more preferably, MOI =10.
In step S3 of some embodiments, the method for purifying the virus solution containing the recombinant adeno-associated virus may include extracting with chloroform and performing iodixanol density gradient centrifugation, and may also employ a purification column to effectively remove the residual adenovirus in the virus solution, thereby further improving the purity of rAAV.
The method for preparing the recombinant adeno-associated virus can be used for preparing AAV serotype 1, 2, 5, 8, 9 or PHPeB. Correspondingly, the AAVCap gene can be selected from the Cap gene sequence of AAV serotype 1, 2, 5, 8, 9 or PHPeB, and the AAVRep gene can be selected from the Rep gene sequence of AAV serotype 2. Preferably, AAV serotype 2, 8 or 9 is prepared by the recombinant adeno-associated virus preparation method, and the rAAV yield is obviously improved compared with a three-plasmid preparation system.
The technical solution described above is explained in detail below with reference to specific examples.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Examples
The method for preparing the high-efficiency adeno-associated virus provided by the embodiment comprises the following specific steps:
(1) Construction of recombinant adenovirus plasmid Ad 5-loxp-psi-loxp-Rep-Cap
In order to obtain recombinant adenovirus, this example uses Ad-easy adenovirus vector system as the basis for preparing recombinant adenovirus plasmid. Referring to fig. 1, in an Ad-easy adenovirus system, according to a conventional cloning method, homodromous loxp sequences (wherein a nucleotide sequence of an adenovirus packaging signal Ψ is shown in SEQ ID No.1, and a nucleotide sequence of loxp is shown in SEQ ID No. 2) are inserted at two ends of a packaging signal Ψ in a shuttle plasmid pshuttle, and a Rep-Cap gene sequence is inserted on the shuttle plasmid pshuttle, the Rep gene selects a Rep gene sequence of AAV serotype 2, and the Cap gene is selected according to the serotype of AAV to be prepared, in this example, AAV1, AAV2, AAV8 and AAV9 are taken as examples to perform experiments, so as to obtain a recombinant plasmid pshuttle-loxp- Ψ -loxp-Rep-Cap, and confirm successful construction of the plasmid sequencing. Then the constructed recombinant shuttle plasmid pshuttle-loxp-psi-loxp-Rep-Cap and the adenovirus skeleton plasmid pAd-easy-1 are transformed into BJ5183 competent cells together, pAd 5-loxp-psi-loxp-Rep-Cap recombinant adenovirus plasmid is obtained through homologous recombination, and the correctness of the plasmid is verified through sequencing.
(2) Rescue of recombinant adenovirus Ad5-loxp- Ψ -loxp-Rep-Cap
HEK-293 cells (ATCC source, low generation preservation) were seeded on 6cm dishes and transfection was initiated by changing fresh medium until the confluency of cells was about 80%. The pAd5-loxp- Ψ -loxp-Rep-Cap plasmid before transfection was linearized, transfected using fugene-HD at a plasmid dosage of 4 μ g, transfection reagent of 12 μ L, dissolved in 200 μ L serum-free DMEM, mixed well, left at room temperature for 15min, added to a petri dish at 37 ℃ 5% CO 2 The culture was continued for 12 days. The cells are diseased, partial areas generate cavities, the cells and the supernatant are collected, repeated freeze thawing is carried out for 3 times at the temperature of liquid nitrogen/37 ℃, the supernatant is collected by centrifugation to obtain the P0 generation virus seeds, and the titer of the general P0 generation virus seeds is 10 7～8 Left and right. Can enlarge and culture adenopathy according to follow-up requirementsAnd obtaining a large number of recombinant adenovirus virus seeds for AAV preparation.
(3) HEK-293T-Cre cell construction
In order to quickly construct an HEK-293T-Cre cell line, a lentivirus method is selected to construct the cell line in the embodiment, firstly, lv-CAG-SV40NLS-Cre-PGK-Puro-WPRE plasmids are constructed, and in order to enable Cre to be better expressed in cell nuclei, SV40NLS nuclear localization sequences (a coding sequence of Cre recombinase is shown as SEQ ID No.3, a nucleotide sequence of SV40NLS is shown as SEQ ID No. 4) are added in front of Cre sequences during vector design to guide Cre recombinase to enter cell nuclei. Transfecting the constructed recombinant lentivirus plasmid into 293T cells to package lentiviruses, collecting supernatant after transfecting for 48h, infecting suspension HEK-293T cells by using different infection complex numbers, adding puromycin 1 mu g/mL after 48h, continuously culturing for 14 days, selecting a small number of cells after 14 days to carry out relative quantification, and detecting cre expression conditions. While performing limiting dilution sorting of the monoclonals. The obtained monoclones are quantitatively detected, and the expression content of cre in each monoclonal cell line is confirmed. Because Cre-loxp regulation has leakage, cre cell lines with different expression levels have to be tested for the control effect of the recombinant adenovirus Ad 5-loxp-psip-loxp-Rep-Cap. Simultaneously using equivalent recombinant adenovirus Ad 5-loxp-psi-loxp-Rep-Cap to respectively infect monoclonal cell lines, observing the replication condition of the recombinant adenovirus in HEK-293T-Cre monoclonal cell strains, selecting the monoclonal cell line in which the cells can maintain a better state after infecting Ad 5-loxp-psi-loxp-Rep-Cap adenovirus (MOI = 1-100) for 48h, using the monoclonal cell line as a cell line for preparing recombinant adeno-associated virus, testing the stability of the monoclonal cell line, and preserving strains which have not changed the property of inhibiting the adenovirus amplification after continuously passaging about 50 times.
(4) Recombinant adeno-associated virus preparation
After the HEK-293T-Cre cell is successfully constructed, 100mL of cell culture is taken as an example to prepare the adeno-associated virus, the cell culture medium is cultured by using a serum-free medium, the cell density reaches 8.0E +06cell/mL, and then the cells are divided into bottles according to the density of 4.0E +06cell/mL, wherein each bottle is 100mL. After the cells are bottled, transfection is started, and PEI-Max is used for transfection, and the transfection is carried out according to the proportion of 0.2 mu g DNA/1.0E +The AAV-ITR core plasmid pAAV-CMV-EGFP-WPRE-pA is added in the standard of 06cells, the cell amount is 100mL,4.0E +06cells/mL, and the dosage of the AAV-ITR core plasmid is 80 mu g. Preparing transfection complex, adding 80 μ g AAV-ITR into 1mL Excell293 serum-free medium, mixing, adding 160 μ L PEI-Max (1 mg/mL), mixing, standing at room temperature for 15min, adding into 100mL cells, adding CO at 8% at 37 deg.C 2 Culturing for 6 h-12 h, adding adenovirus Ad 5-loxp-psi-loxp-Rep-Cap, adding into cells transfected with AAV-ITR according to MOI =1, 10, 20 and 50 respectively, collecting cells and supernatant 72h after transfection, and detecting the virus titer in the cell and supernatant mixture as shown in Table 1. The optimal adenovirus multiplicity of infection MOI =10 was explored.
TABLE 1 preparation of viral titers of different serotypes of AAV at different recombinant adenovirus multiplicity of infection
(5) Purification of recombinant adeno-associated virus
Collecting supernatant and cells 72h after transfection, respectively, precipitating the supernatant with PEG8000-NaCl with final concentrations of 8% (W/V) and 500mM respectively, ice-cooling for 1 h-2 h or overnight at 4 deg.C, centrifuging (8000g, 20min,4 deg.C), removing supernatant, and retaining precipitate; after the cells were resuspended in PBS, the cells were repeatedly frozen and thawed 3 times at 37 ℃ with the addition of 50U/mL nuclease, digested at 37 ℃ for 1 hour, centrifuged (1200 g,5min,4 ℃), and the supernatant was collected and used to resuspend the precipitate produced by PEG-NaCl. Then adding nuclease with the final concentration of 25U/mL, digesting at 37 ℃, adding 1/10 volume of trichloromethane, violently shaking at 37 ℃ and 250rpm for 30min to denature protein and adenovirus, centrifuging (6000g, 5min,4 ℃) to collect an upper-layer water phase, adding isometric trichloromethane, violently shaking for 30s, centrifuging (6000g, 5min,4 ℃) to collect an upper-layer water phase, repeating the steps once to completely inactivate and remove adenovirus and denatured impurity protein, and centrifuging the obtained supernatant by using iodixanol density gradient. The 40% layer of iodixanol was collected, dialyzed overnight and concentrated by ultrafiltration to complete virus purification.
Comparative example
In the comparative example, the production process for preparing AAV by using suspension cells is adopted, 100mL of cell culture is taken as an example for preparing adeno-associated virus, a serum-free culture medium is adopted as the cell culture medium, the cell density reaches 8.0E +06cell/mL, and the cells are separated into 100mL of bottles according to the density of 4.0E + 06cell/mL. After the cells are separated into bottles, transfection is started, PEI-Max is used for transfection, the cell amount is 100mL,4.0E +06cells/mL, the AAV-ITR core plasmid dosage is 80 mu g, the RC plasmid (carrying Rep and Cap genes) dosage is 160 mu g, and the Helper plasmid (AAV packaging auxiliary plasmid) dosage is 160 mu g; PEI-Max is typically used in an amount of 2 to 3 times the total DNA mass. Firstly preparing a transfection complex, taking 1mL of excell293 serum-free culture medium, adding 80 mu g of AAV-ITR,160 mu g of gRC plasmid and 160 mu g of helper plasmid, mixing uniformly, then adding 800 mu L of PEI-Max (1 mg/mL), mixing uniformly, and standing at room temperature for 15min. Then added to the cell culture at 37 ℃ and 8% CO 2 Culturing for 48-72 h, collecting cells and supernatant, and purifying AAV to obtain recombinant adeno-associated virus.
According to the optimal adenovirus infection complex number found in the examples and the yield of rAAV prepared in the comparative examples and comparative examples, as can be seen from figure 2, AAV of various serotypes can be prepared by the method provided by the invention, the yield of recombinant adeno-associated virus is obviously improved, and especially the effect on AAV serotype 2, 8 and 9 is more obvious.
Compared with a three-plasmid transfection mode, the method disclosed by the invention can effectively improve the virus yield, and simultaneously avoids the heavy labor and kit cost for extracting a large amount of RC plasmids and Helper plasmids. The recombinant adenovirus is easily obtained in the common HEK-293 cell, and the titer is 10 11 PFU/mL, virus does not need to be purified, only cell lysis, cell lysate supernatant, can be used for AAV production.
Compared with a three-plasmid transfection mode, the method reduces the using amount of the plasmids and the using amount of PEI, the PEI has certain toxicity to cells, the using amount is too large, the transfection efficiency is improved to a certain extent, but the cell state is influenced, and the cell toxicity production efficiency is reduced, so that the using amount of PEI transfection is 2-3 times of the DNA amount. And the efficiency of PEI transfection suspension cell is about 50% -70% generally, while the adenovirus infection 293 series cell can reach 100% efficiency, and those cells which do not transfer AAV-ITR are infected with adenovirus, the cell growth is inhibited, and the AAV of filial generation can be further replicated after entering, and can produce new AAV, so that it can raise yield, and for 8 type and 9 type AAV, the new AAV is secreted into culture medium in large quantity, so that it can infect partial cell which does not transfer AAV-ITR plasmid originally. After the three-plasmid system is transfected once, only 50-70% of cells produce AAV, the rest cells grow more vigorously due to the fact that no load plasmid exists, nutrient substances in a culture medium are consumed more, meanwhile, a part of AAV viruses can be adsorbed, and after the adsorbed AAV enters the cells, no rep-cap exists, the AAV cannot be amplified continuously, and accordingly the AAV is consumed.
It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.
Claims (10)
1. A high-efficiency recombinant adeno-associated virus production system is characterized in that: comprises a 293T cell line which can stably express Cre recombinase, recombinant adenovirus carrying parts of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus;
the elements necessary for generating the recombinant adeno-associated virus comprise AAVCap genes, AAVRep genes and AAV core expression elements ITR-GOI with exogenous genes;
the genome of the recombinant adenovirus is deleted with E1 gene and E3 gene, and the elements necessary for partially generating recombinant adeno-associated virus are inserted into the region where the E1 gene and/or the E3 gene are located, two ends of a packaging signal psi of the recombinant adenovirus are respectively inserted with a loxp sequence, and the two loxp sequences are in the same direction.
2. The highly efficient recombinant adeno-associated virus production system according to claim 1, wherein: the elements required for partially producing the recombinant adeno-associated virus are AAVRep gene and AAVCap gene, and the other elements required for producing the recombinant adeno-associated virus are AAV core expression element ITR-GOI with exogenous genes.
3. A method for preparing a high-efficiency recombinant adeno-associated virus, which is characterized by comprising the following steps:
s1, respectively constructing a 293T cell line capable of stably expressing Cre recombinase, a recombinant adenovirus carrying part of elements necessary for generating recombinant adeno-associated virus and expression plasmids carrying other elements necessary for generating recombinant adeno-associated virus;
the elements necessary for generating the recombinant adeno-associated virus comprise AAVCap genes, AAVRep genes and AAV core expression elements ITR-GOI with exogenous genes;
the genome of the recombinant adenovirus is deleted with E1 gene and E3 gene, and the elements necessary for partially generating recombinant adeno-associated virus are inserted into the region where the E1 gene and/or the E3 gene are located, two ends of a packaging signal psi of the recombinant adenovirus are respectively inserted with a loxp sequence, and the two loxp sequences are in the same direction;
s2, transfecting the 293T cell line capable of stably expressing Cre recombinase with the expression plasmid carrying other elements necessary for generating the recombinant adeno-associated virus, then infecting the 293T cell line capable of stably expressing Cre recombinase after transfection with the recombinant adenovirus, and culturing to obtain virus liquid containing the recombinant adeno-associated virus;
and S3, purifying the virus liquid containing the recombinant adeno-associated virus, and removing the adenovirus in the virus liquid to obtain the pure recombinant adeno-associated virus.
4. The method of claim 3, wherein the recombinant adeno-associated virus is prepared by: in step S1, the 293T cell line capable of stably expressing Cre recombinase is constructed by adopting a plasmid transfection or lentivirus mode.
5. The method for preparing a high efficiency recombinant adeno-associated virus according to claim 4, wherein the 293T cell line capable of stably expressing Cre recombinase comprises: transfecting 293T cells with recombinant lentivirus plasmids carrying Cre genes, and screening by using purine to obtain a positive monoclonal cell line;
in the recombinant lentiviral plasmid, the Cre gene contains a nuclear localization sequence upstream.
6. The method of claim 5, wherein the 293T cell line capable of stably expressing Cre recombinase further comprises: and respectively infecting the positive monoclonal cell lines obtained by screening by the same amount of the recombinant adenovirus with the infection complex number ranging from 1 to 100, and selecting the cell lines which are maintained in a good state after infecting for 48 hours to be used as 293T cell lines for preparing the recombinant adeno-associated virus.
7. The method for preparing a highly potent recombinant adeno-associated virus according to claim 3, wherein the recombinant adenovirus is constructed in step S1 by: inserting a loxp sequence into each of two ends of a packaging signal psi in an adenovirus shuttle plasmid, and inserting elements necessary for generating a recombinant adeno-associated virus into the adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; electrically transforming the recombinant shuttle plasmid and the adenovirus skeleton plasmid into BJ5183 competent cells, and obtaining a recombinant adenovirus plasmid through homologous recombination; and transfecting the recombinant adenovirus plasmid into 293 cells to save the recombinant adenovirus capable of expressing the elements necessary for generating the recombinant adeno-associated virus.
8. The method for producing a highly potent recombinant adeno-associated virus according to claim 3, wherein: in step S2, the multiplicity of infection of the recombinant adenovirus is 10-20.
9. The method for producing a recombinant adeno-associated virus according to claim 3, wherein in step S3, the method for purifying the virus solution containing the recombinant adeno-associated virus comprises chloroform extraction and iodixanol density gradient centrifugation.
10. The method of claim 3, wherein the recombinant adeno-associated virus is prepared by: the recombinant adeno-associated virus preparation method is used for preparing AAV serotype 1, 2, 5, 8, 9 or PHPeB.
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| CN117778431A (en) * | 2023-12-04 | 2024-03-29 | 广州派真生物技术有限公司 | Plasmid system for packaging recombinant adeno-associated virus and application thereof |
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| CN117683797A (en) * | 2023-12-04 | 2024-03-12 | 广州派真生物技术有限公司 | Plasmid system for packaging recombinant adeno-associated virus and application thereof |
| CN117778431A (en) * | 2023-12-04 | 2024-03-29 | 广州派真生物技术有限公司 | Plasmid system for packaging recombinant adeno-associated virus and application thereof |
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