CN112852746A - Large-scale lentivirus gene medicine preparation system and method based on Cre recombinase induction - Google Patents
Large-scale lentivirus gene medicine preparation system and method based on Cre recombinase induction Download PDFInfo
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Abstract
The invention relates to a large-scale lentivirus gene medicine preparation system based on Cre recombinase induction, which comprises: suspending 293 stable cell line and virus expressing Cre gene; the genome of the suspended 293 stable cell line integrates a lentivirus packaging protein expression sequence and a target gene sequence; and loxP-Stop-loxP sequence is inserted between the lentivirus packaging protein expression sequence and a promoter for driving the expression of the lentivirus packaging protein expression sequence; the loxP-Stop-loxP sequence comprises two loxP sites with the same direction and a Stop sequence connected between the two loxP sites; the virus for expressing the Cre gene is a virus which can infect the suspension 293 cell and can express Cre protein; the virus expressing the Cre gene is used for infecting the suspended 293 stable cell strain and activating a lentivirus packaging protein expression sequence to express so as to package and generate lentivirus. The invention solves the problems of high cost and difficult amplification of plasmid transient transfection; meanwhile, virus infection expressing Cre gene is used for induction, so that the technical problem that tetracycline and other antibiotics are difficult to remove after induction is avoided.
Description
Technical Field
The invention relates to the technical field of gene medicines, in particular to a large-scale lentivirus gene medicine preparation system and method based on Cre recombinase induction.
Background
The gene therapy medicine is an important component of biological medicine, has the opportunity to cure rare genetic diseases and cancers for the first time in human history, and also has the potential to greatly improve inherent diseases such as poor treatment effect, complicated treatment mode and the like of the traditional medicines of cardiovascular diseases, metabolic diseases, autoimmune diseases, neurodegenerative diseases and the like.
There are 7 new drugs for global cell and gene therapy on the market, and tens of new drugs will come on the market in the next decade, and the total value will break through billions of dollars. There are 3 currently marketed products based on lentiviral vector gene drugs: 2 of them are Car-T drugs for lymphoma and 1 is a genetic drug zynteglo (bluebird bio) for the treatment of beta thalassemia. At present, the price of gene drugs is high, and the production cost of virus vectors is a key factor.
The recombinant lentivirus is produced through dividing the lentivirus genome into 3 or 4 parts, loading the parts into different plasmid vectors, transfecting HEK293 cell with the plasmid vectors, and packing the recombinant lentivirus. Currently, 4 plasmid vector production systems are generally used clinically based on safety considerations. As shown in fig. 1.
The production of clinical-grade lentivirus still has no ideal solution internationally at present, and the transient transfection method requires a large amount of clinical-grade plasmid vectors for both adherent cells and suspension cells. The production of plasmid vectors requires a separate production line and poses significant challenges in terms of legislation and processing. In addition, the culture of adherent cells is difficult to scale up industrially and is extremely costly. Suspension cell culture is easier to scale up using bioreactors than adherent cell culture, but the transfection efficiency of plasmids is greatly reduced. In order to avoid the above problems, scientists have looked at inductive systems. The developed and mature tetracycline induction system is a difficulty in clinical-grade lentivirus production and purification, but tetracycline addition and removal are difficult problems, and the method is not applied on a large scale at present.
Disclosure of Invention
Technical problem to be solved
In view of the defects and shortcomings of the prior art, the invention provides a large-scale lentiviral gene drug preparation system and method based on Cre recombinase induction, which solves the problems of high cost and difficult amplification of plasmid transient transfection; meanwhile, virus infection expressing Cre gene is used for induction, so that the technical problem that tetracycline and other antibiotics are difficult to remove after induction is avoided.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
in a first aspect, the present invention provides a method for preparing a large-scale lentiviral gene drug based on Cre recombinase induction, comprising:
s1, constructing a suspension stable transfer cell strain for generating lentivirus; the genome of the suspension stable transfer cell strain is stably integrated with a lentivirus packaging protein expression sequence and a target gene sequence, and a loxP-Stop-loxP sequence is inserted between the lentivirus packaging protein expression sequence and a promoter for driving the expression of the lentivirus packaging protein expression sequence; the loxP-Stop-loxP sequence comprises two loxP sites with the same direction and a Stop sequence connected between the two loxP sites;
s2, constructing a virus for expressing Cre recombinase;
s3, amplifying and culturing the stable transfer cell strain in a large scale;
when the lentivirus particles need to be produced, the virus expressing Cre genes is used for infecting the suspension stable transfer cell strain, Cre recombinase expressed by the virus is used for excising the Stop sequence of the loxP-Stop-loxP sequence so as to induce the expression sequence of the lentivirus packaging protein to be expressed, thereby packaging the recombinant lentivirus particles expressing target genes, and the recombinant lentivirus particles are virus gene medicines.
The virus for expressing Cre gene is any virus which can infect the suspension 293 cell and can express Cre protein. In actual production, since baculovirus is replication-competent virus and is easy to produce, it can be realized by using baculovirus expressing Cre gene.
According to a preferred embodiment of the present invention, in step S1, in constructing a suspension-stable cell strain, a plasmid expressing gag-pol comprising a promoter and a gag-pol gene sequence with a loxP-Stop-loxP sequence inserted between the promoter and the gag-pol gene sequence and a plasmid expressing vsv.g comprising a promoter and a vsv.g gene sequence with a loxP-Stop-loxP sequence inserted between the promoter and the vsv.g gene sequence are separately constructed; the gag-pol expressing plasmid and the vsv.g expressing plasmid were then integrated onto the suspension cell genome (via PiggyBac vector).
According to a preferred embodiment of the present invention, in step S1, in constructing a stable transgenic cell line, loxP-Stop-loxP sequence is inserted between the target gene sequence and the promoter driving its expression to construct a target gene plasmid, which is then integrated into the genome of the suspension cell line (via PiggyBac vector).
According to a preferred embodiment of the present invention, in step S1, the step of constructing the 293 cell line stably transfected in suspension comprises: s11, constructing the following three plasmids:
gag-pol plasmid: piggybac-CBA-loxp-stop-loxp-gag-pol,
Vsv. g plasmid: piggybac-CBA-loxp-stop-loxp-VSV.G and
the target plasmid: piggybac-CBA-loxp-stop-loxp-Target Gene plasmid;
s12, constructing a suspension 293 stable transfer cell strain: the three plasmids obtained are transfected into suspension 239 cells and are screened by using a culture medium containing antibiotics to obtain resistant cells, namely 239 suspension stable cells for generating lentiviruses.
According to a preferred embodiment of the present invention, the construction of the plasmid in step S11 includes the following steps:
carrying out double digestion on the piggybac-dual-promoter plasmid by using restriction enzymes SnaBI and HindIII, and cutting gel after agarose electrophoresis to recover the piggybac-dual-promoter vector fragment;
(II) designing a CBA (promoter) sequence fragment (shown in SEQ ID NO: 1), a loxp-stop-loxp sequence fragment (shown in SEQ ID NO: 2), a gag-pol sequence (shown in SEQ ID NO: 4) or a VSV.G (shown in SEQ ID NO: 5) or a Target Gene sequence fragment for seamless cloning;
thirdly, connecting the piggybac-dual-promoter vector segment, the CBA sequence segment and the loxp-stop-loxp segment with the gag-pol or VSV.G or Target Gene sequence segment respectively by adopting a seamless cloning kit;
fourthly, transforming the ligation products into competence trans1-t1, uniformly mixing, carrying out ice bath, carrying out heat shock, immediately carrying out ice bath again, firstly culturing by using an antibiotic-free culture solution, and then coating the bacterial solution on a plate culture medium containing ampicillin for continuous culture; and (3) selecting a monoclonal colony, inoculating the colony into a liquid culture solution containing ampicillin, performing shake culture, extracting a plasmid, performing double enzyme digestion identification by XbaI and HpaI, and performing sequencing identification to obtain a target plasmid.
According to a preferred embodiment of the present invention, in step S1, the Target Gene is codon-optimized COL7a1 Gene, and the sequence thereof is as shown in SEQ ID NO: shown at 7.
According to a preferred embodiment of the present invention, in S2, the virus expressing Cre recombinase is baculovirus, and the construction method thereof comprises:
firstly, a Cre gene expression sequence (shown as SEQ ID NO: 6) is seamlessly cloned into a pFast Bac1 plasmid (invitrogen, Cat.No.10359-016) to construct a baculovirus plasmid pFast-Bac-Cre with a Cre expression cassette; and then transforming the pFast-Bac-Cre plasmid into a DH10Bac competent cell to obtain Bacmid-Cre, transfecting Sf9 cells by Bacmid, and collecting supernatant after transfecting for a preset time to obtain Baculovirus Baculovir-Cre for expressing Cre recombinase.
According to the preferred embodiment of the invention, in S2, the construction method of the pFast-Bac-Cre plasmid comprises the following steps:
(1) carrying out double enzyme digestion on a pFast Bac1 plasmid for 50-70min at 37 +/-0.5 ℃ by using restriction endonucleases SnaBI and HpaI, cutting gel after agarose electrophoresis to recover a pFast Bac1 vector fragment, carrying out dephosphorizing treatment on the recovered pFast Bac1 vector fragment for 50-70min at 50 +/-2 ℃ by using alkaline phosphatase CIAP, and carrying out inactivation treatment for 10-25min at 65 +/-2 ℃;
(2) connecting the pFast Bac1 vector fragment treated by alkaline phosphatase and the gene fragment containing the Cre expression cassette by adopting a seamless cloning recombinase, and reacting for 8-15min at 50 +/-2 ℃ to obtain a seamless connection recombinant product;
(3) taking a seamless connection recombinant product transformation competence DH5a, uniformly mixing, carrying out ice bath and heat shock, immediately carrying out ice bath again, carrying out shake culture by using an antibiotic-free culture solution, uniformly coating the bacterial solution on a plate culture medium containing ampicillin, and carrying out inverted culture at 37 +/-0.5 for 12-18 h;
(4) selecting a monoclonal colony, inoculating the colony in a liquid culture solution containing ampicillin, and oscillating for 12-18h at 37 +/-0.5 ℃; extracting pFast-Bac-Cre plasmid, performing double enzyme digestion identification on SnaBI and HpaI, performing sequencing identification, and constructing to obtain baculovirus plasmid with Cre expression cassette.
According to the preferred embodiment of the present invention, in S2, the method for preparing baculoviral-Cre expressing Cre recombinase comprises:
(1) the pFast-Bac-Cre plasmid was transformed into DH10Bac competent cells: transforming pFast-Bac-Cre plasmid into competent DH10Bac, mixing the plasmids gently and performing ice bath for 25 to 35 min; performing heat shock at 42 ℃ +/-0.5 for 40-50s, immediately performing ice bath for 2-5min, adding LB culture solution without antibiotics at 37 ℃ +/-0.5, oscillating for 40-80min, uniformly coating the bacterial solution on an LB agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG by using a sterile glass coater, and performing inverted culture at 37 ℃ +/-0.5 for 24-32 h;
(2) selecting white monoclonal to inoculate in liquid culture solution containing kanamycin, gentamicin and tetracycline, oscillating for 14-18h at 37 +/-0.5 ℃, extracting Bacmid-Cre by a phenol-chloroform extraction method, and obtaining Bacmid-Cre which is identified by a PCR method;
(3) and (3) transfecting Sf9 cells by using a pore plate cell plate according to the proportion of 1x10^6 cells/pore by using 20 +/-1 mu l Bacmid-Cre per pore, changing the liquid 6-8h after transfection, and collecting supernatant 96h after transfection to obtain the baculovirus carrying the Cre expression cassette.
According to the preferred embodiment of the present invention, step S3 includes: amplifying and culturing the suspended 293 stable transgenic cell line to a preset density, infecting the suspended 293 stable transgenic cell line by Baculovir-Cre according to MOI1, collecting cell supernatant after infecting for 72h, and carrying out chromatography purification to obtain the lentivirus expressing the target gene.
In a second aspect, the present invention provides a large-scale lentiviral gene drug preparation system based on Cre recombinase induction, comprising:
suspending 293 stable cell line and virus expressing Cre gene;
the genome of the suspended 293 stable cell line integrates a lentivirus packaging protein expression sequence and a target gene sequence; and loxP-Stop-loxP sequence is inserted between the lentivirus packaging protein expression sequence and a promoter for driving the expression of the lentivirus packaging protein expression sequence; the loxP-Stop-loxP sequence comprises two loxP sites with the same direction and a Stop sequence connected between the two loxP sites;
the virus for expressing the Cre gene is a virus which can infect the suspension 293 cell and can express Cre protein; the virus expressing the Cre gene is used for infecting the suspended 293 stable cell strain and activating a lentivirus packaging protein expression sequence to express so as to package and generate lentivirus.
Baculovirus can be used for expressing the Cre gene, and the baculovirus is replication-type virus and is easy to produce in large quantities.
Preferably, in the suspension 293 stable transgenic cell line, a loxP-Stop-loxP sequence is inserted between the target gene sequence and a promoter for driving the expression of the target gene sequence.
Preferably, the target gene is a codon-optimized COL7a1 gene, and the sequence thereof is shown in SEQ ID NO: shown at 7. At this time, the produced lentiviral particles are lentiviral gene drugs for the treatment of dystrophic epidermolysis bullosa.
(III) advantageous effects
The invention has the main technical effects that:
the invention utilizes an inducible lentivirus generation system mediated by a Cre-loxP recombination system. The problems of high cost and difficult amplification of plasmid transient transfection are solved. In addition, the invention utilizes virus infection to induce, thereby avoiding the difficult process problem that tetracycline and other antibiotics are difficult to remove after induction.
Preferably, in constructing a stable transformant cell line, a loxP-Stop-loxP site is similarly inserted between a target gene sequence and a promoter for driving the expression thereof to construct a target gene plasmid, and the target gene plasmid is integrated into the genome of a suspension cell line. The pressure brought to the cells by continuously expressing a large amount of exogenous protein in the process of amplification culture of the constructed suspension stable transgenic cell strain can be avoided, and the growth and culture density of the suspension stable transgenic cells are inhibited.
Drawings
FIG. 1 is a schematic diagram of a prior art method for producing recombinant lentiviruses.
FIG. 2 shows the results of comparison of the expression levels of the lentiviral packaging proteins gag-pol and VSV.G in cell lysates of the 293 stable transformants in suspension, before and after infection of the 293 stable transformants with baculovirus carrying the Cre gene, using Western Blot, wherein-indicates before infection and + indicates after infection.
FIG. 3 shows the elution of purified lentivirus from the supernatant of cells of 293 stable transformants in suspension, before and after infection of 293 stable transformants in suspension with baculovirus carrying Cre gene, to infect target cells (239T) with lentivirus and to observe the change in fluorescence of the target cells (239T) with a fluorescence microscope; where Cre (-) indicates before infection and Cre (+) indicates after infection.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
The main ideas and principles of the technical scheme of the invention are as follows:
the Cre recombinase (Cyclization Recombination Enzyme) is a 38kD protein consisting of 343 amino acids encoded by the Cre gene of Escherichia coli bacteriophage P1. It not only has catalytic activity, but also can specifically recognize loxP sites similarly to restriction enzymes. LoxP (loxs of X-overP1) site is 34bp long, including two 13bp inverted repeats and an 8bp spacer region. Wherein the inverted repeat is a specific recognition site for Cre recombinase, and the spacer region determines the orientation of the loxP site. When loxP sites are present in the genome, Cre recombinase, upon encountering Cre recombinase, binds to the inverted repeat regions at both ends of the loxP sites to form a dimer. This dimer binds to dimers of other loxP sites, thereby forming a tetramer. Subsequently, the DNA between the two loxP sites is excised by Cre recombinase, and the nicks are religated by DNA ligase.
Based on the principle, the invention arranges the Stop sequence between two loxP sites with the same direction to form a loxP-Stop-loxP sequence, to form a switch sequence which can be induced and activated by Cre recombinase, then arranges the loxP-Stop-loxP sequence in front of the expression sequence of lentivirus packaging protein (gag-pol or VSV.G, etc.) (behind the promoter which drives the expression of the expression sequence of the lentivirus packaging protein (gag-pol or VSV.G, etc.), thereby obtaining a plasmid structure which can be induced and activated by Cre recombinase, and then integrates the plasmid into the genome of the germ plasm 293 cell through a PiggyBac vector system. The obtained suspension 293 cell does not express lentivirus packaging protein under the condition of no Cre recombinase, avoids the cytotoxicity of the lentivirus packaging protein from inhibiting the growth of the suspension 293 cell, and is beneficial to the large-scale amplification and culture of the suspension 293 cell. In addition, a virus expressing Cre recombinase is constructed, stably integrated suspension 293 cells (i.e., a lentivirus packaging protein expression sequence in which a loxP-Stop-loxP sequence is integrated in the genome) are infected with the Cre recombinase-expressing virus, and a lentivirus packaging protein expression sequence (gag-pol, VSV.G, or the like) following the loxP-Stop-loxP sequence is activated and expressed by utilizing the Sotp sequence in the loxP-Stop-loxP sequence of the Cre recombinase, thereby packaging the recombinant lentivirus.
The verification of the scheme, the realizability and the technical effect of the invention will be described by taking G2P (GFP-T2A-Puro) as an example of a target gene. The implementation method comprises the following steps:
construction of suspension 293 Stable transgenic cell line
Before constructing a suspended 293 stable cell strain, three plasmids, namely a target gene plasmid, a lentiviral packaging protein gag-pol plasmid, a lentiviral packaging protein VSV.G plasmid and the like, need to be constructed, and then enter a suspended 293 cell through transfection, and a cell line integrated into the genome of the suspended 293 cell is screened out by using antibiotics. The specific method and steps are as follows:
firstly, constructing a piggybac-CBA-loxp-stop-loxp-G2P plasmid, wherein the process comprises the following steps:
(1) the piggybac-dual-promoter plasmid (Ubaoz, VT1663) was double digested with restriction enzymes SnaBI and HindIII at 37 ℃ for 30min, and the piggybac-dual-promoter vector fragment was recovered by cutting the gel after agarose electrophoresis.
(2) The CBA sequence fragment (SEQ ID NO: 1, loxp-stop-loxp sequence fragment (SEQ ID NO: 2) and G2P (GFP-T2A-Puro) sequence fragment (SEQ ID NO: 3) were designed for seamless cloning.
(3) The piggybac-dual-promoter vector fragment, the CBA fragment and the loxp-stop-loxp fragment which are respectively recovered by the gel recovery kit are connected by adopting a seamless cloning kit of CloneSmarter, and a 10ul system reacts for 15min at 50 ℃.
(4) 5ul of ligation was transformed into E.coli: the ligation product is transformed into competence trans1-t1, mixed evenly and cooled in ice for 20 min; performing heat shock at 42 ℃ for 80s, immediately performing ice bath for 5min, adding LB culture solution without antibiotics, shaking at 37 ℃ for about 60min, uniformly coating the bacteria solution on an LB agar plate containing ampicillin by using a sterile glass coater, and performing inverted culture at 37 ℃ for 14 h.
(5) Selecting a monoclonal colony, inoculating the colony in an ampicillin-containing LB liquid culture solution, and oscillating for 16h at 37 ℃; extracting the piggybac-CBA-loxp-stop-loxp-G2P plasmid by using an axygen plasmid extraction kit, carrying out XbaI and HpaI (KspAI) double enzyme digestion identification, and then carrying out sequencing identification to obtain the piggybac-CBA-loxp-stop-loxp-G2P plasmid.
(II) constructing piggybac-CBA-loxp-stop-loxp-gag-pol plasmid, wherein the process comprises the following steps:
(1) for simplicity, the plasmid of piggybac-CBA-loxp-stop-loxp-G2P constructed above is subjected to double digestion for 30min at 37 ℃ by using restriction enzymes HpaI (KspAI) and HindIII, and the vector fragment of piggybac-CBA-loxp-stop-loxp is recovered by cutting gel after agarose electrophoresis.
(2) Designing a seamless cloning primer gag-pol-F
(GCTGGCGCGAGCTGGTCGACAAGCTgaattcgggccgcgtt) and gag-pol-R (Taaccatatagctgcaaaaacaagttaactgagagagacaaaaaattcc), the gag-pol fragment (sequence SEQ ID NO: 4) was PCR amplified from psPAX2 plasmid vector (adddge plasmid #12260), and the gag-pol fragment was recovered by cutting gel after agarose electrophoresis.
(3) The piggybac-CBA-loxp-stop-loxp-G2P vector fragment and the gag-pol fragment respectively recovered by the gel recovery kit are connected by adopting a seamless cloning kit of CloneSmarter, and a 10ul system reacts for 15min at 50 ℃.
(4) 5ul of ligation was transformed into E.coli: the ligation product is transformed into competence trans1-t1, mixed evenly and cooled in ice for 20 min; performing heat shock at 42 ℃ for 80s, immediately performing ice bath for 5min, adding LB culture solution without antibiotics, shaking at 37 ℃ for about 60min, uniformly coating the bacteria solution on an LB agar plate containing ampicillin by using a sterile glass coater, and performing inverted culture at 37 ℃ for 14 h.
(5) The single colony was inoculated in LB liquid medium containing ampicillin, and shaken at 37 ℃ for 16 hours.
(6) Extracting piggybac-CBA-loxp-stop-loxp-gag-pol plasmid by using an axygen plasmid extraction kit, performing NdeI and HpaI (KspAI) double-enzyme digestion identification, and then performing sequencing identification to obtain the piggybac-CBA-loxp-stop-loxp-gag-pol plasmid.
(III) constructing a piggybac-CBA-loxp-stop-loxp-VSV.G plasmid, wherein the process comprises the following steps:
(1) for simplicity, the plasmid of piggybac-CBA-loxp-stop-loxp-G2P constructed above is subjected to double digestion for 30min at 37 ℃ by using restriction enzymes HpaI (KspAI) and HindIII, and the vector fragment of piggybac-CBA-loxp-stop-loxp is recovered by cutting gel after agarose electrophoresis.
(2) Design of seamless cloning primer VSV.G-F
(GCTGGCGCGAGCTGGTCGACAAGCTgaattctgacactatgaagtgcct) and VSV.G-R (aaccatataagctgcaaacaaaacaagttaaactaaaataacaagcatagaaaac), PCR-amplifying the VSV.G fragment (sequence SEQ ID NO: 5) from pMD2.G plasmid vector (adddge plasmid #12259), cutting the gel after agarose electrophoresis, and recovering the VSV.G fragment.
(3) The piggybac-CBA-loxp-stop-loxp vector fragment and the VSV.G fragment respectively recovered by the gel recovery kit are connected by adopting a seamless cloning kit of CloneSmarter, and a 10ul system reacts for 15min at 50 ℃.
(4) 5ul of ligation was transformed into E.coli: the ligation product is transformed into competence trans1-t1, mixed evenly and cooled in ice for 20 min; performing heat shock at 42 ℃ for 80s, immediately performing ice bath for 5min, adding LB culture solution without antibiotics, shaking at 37 ℃ for about 60min, uniformly coating the bacteria solution on an LB agar plate containing ampicillin by using a sterile glass coater, and performing inverted culture at 37 ℃ for 14 h.
(5) Selecting monoclonal colony, inoculating in LB liquid culture medium containing ampicillin, and shaking at 37 deg.C for 16h
(6) Extracting piggybac-CBA-loxp-stop-loxp-VSV.G plasmid by using an axygen plasmid extraction kit, performing XbaI and HpaI (KspAI) double enzyme digestion identification, and then performing sequencing identification to obtain the piggybac-CBA-loxp-stop-loxp-VSV.G plasmid.
(II) transfecting the three plasmids into a suspension 293 cell, and screening a cell strain meeting the requirement as a stable transformant for producing the lentivirus
The stable Cell line screening is to enter plasmids into a suspension 293 Cell by transfection, and screen Cell pool integrated into the genome of the suspension 293 Cell, and comprises the following specific steps:
(1) spreading 293 cells on a 35mm plate at 2x10^ 6/hole, and culturing at 37 ℃ for 1 h;
(2) and taking piggybac-CBA-loxp-stop-loxp-G2P plasmid, 2 mu G of each of the piggybac-CBA-loxp-stop-loxp-gag-pol plasmid and the piggybac-CBA-loxp-stop-loxp-vsV.G plasmid to be diluted to 100 mu l by using Grace culture medium, mixing the plasmids in a flick manner, standing the mixture at room temperature for 5min, taking 8 mu l of Cellffectin II transfection reagent to be diluted to 100 mu l by using Grace culture medium, mixing the transfection reagent in a flick manner, standing the mixture at room temperature for 5min, adding the diluted transfection reagent into the diluted plasmid, mixing the transfection reagent in a flick manner, and standing the mixture at room temperature for 15 min.
(3) The transfection reagent plasmid mixture was added to 293 cells and replaced with fresh medium after 6h incubation at 37 ℃.
(4) At 24h after transfection, 293 cells were replaced with medium containing 8. mu.g/ml Puromycin, and the culture was continued at 37 ℃ and every three days with fresh medium containing 8. mu.g/ml Puromycin until the cells no longer died, and the surviving cells were all anti-Puromycin cells, thus obtaining an inducible lentivirus-producing suspension 293 cell line.
In this example, the three plasmids were not successfully transfected one by one, but the cells resistant to Puromycin necessarily contained the suspension 293 cell line into which the three plasmids had been stably integrated, and thus the selected cells resistant to Puromycin were used as lentivirus-producing suspension cells (see validation procedure below).
In other embodiments and actual production, the three plasmids can be transfected one by one and screened one by one according to actual conditions to screen out the suspension 293 cell line stably integrated with the three plasmids.
Second, construct baculovirus expressing Cre recombinase
In fact, any virus that can infect 293 cells and is capable of expressing Cre recombinase can be used as the infecting virus. In practice, replication-competent baculovirus is selected as a vector for expressing Cre recombinase in consideration of cost and manufacturability. The construction process of the baculovirus expressing the Cre recombinase comprises the construction and recombination of pFast-Bac-Cre plasmids to obtain the baculovirus expressing the Cre recombinase. The process is as follows:
(I) construction of pFast-Bac-Cre plasmid
Seamlessly cloning the synthesized Cre gene expression sequence (the sequence is SEQ ID NO: 6) into a pFast Bac1 plasmid (invitrogen, Cat.No.10359-016) to obtain a pFast-Bac-Cre plasmid; the cloning process comprises the following steps:
(1) the plasmid pFast Bac1 is subjected to double enzyme digestion for 1h at 37 ℃ by using restriction enzymes SnaBI and HpaI, a pFast Bac1 vector fragment is recovered by cutting gel after agarose electrophoresis, the recovered pFast Bac1 vector fragment is subjected to dephosphorizing treatment for 60min at 50 ℃ by using alkaline phosphatase CIAP, and is subjected to inactivation treatment for 15min at 65 ℃.
(2) And (3) connecting the pFast Bac1 vector fragment treated by alkaline phosphatase with the gene fragment containing the Cre expression cassette by adopting a seamless cloning recombinase, and reacting for 10min at 50 ℃ to obtain a seamless connection recombinant product.
(3) Taking the seamless connection recombinant product transformation competence DH5a, gently mixing uniformly, and carrying out ice bath for 30 min; performing heat shock at 42 deg.C for 80s, immediately ice-bathing for 4min, adding LB culture solution without antibiotic, shaking at 37 deg.C for 60min, uniformly coating the bacterial solution on LB agar plate containing ampicillin with sterile glass coater, and performing inverted culture at 37 deg.C for 14 h.
(4) Selecting a monoclonal colony, inoculating the colony in an ampicillin-containing LB liquid culture solution, and oscillating for 16h at 37 ℃; plasmid pFast-Bac-Cre plasmid is extracted by using a plasmid extraction kit, and sequencing identification is carried out after double enzyme digestion identification of SnaBI and HpaI, so that the construction of the baculovirus plasmid with the Cre expression cassette is successful.
(II) recombining to obtain baculovirus expressing Cre recombinase
And transforming DH10Bac competent cells by using pFast-Bac-Cre plasmid to obtain Bacmid-Cre, then transfecting Sf9 cells by using Bacmid, and collecting supernatant after a preset time of transfection to obtain Baculovir-Cre. The specific process comprises the following steps:
(1) the pFast-Bac-Cre plasmid was transformed into DH10Bac competent cells: transforming pFast-Bac-Cre plasmid into competent DH10Bac, mixing the mixture evenly and gently, and carrying out ice bath for 30 min; performing heat shock at 42 ℃ for 45s, immediately performing ice bath for 4min, adding LB culture solution without antibiotics, shaking at 37 ℃ for 60min, uniformly coating the bacterial solution on an LB agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG by using a sterile glass coater, and performing inverted culture at 37 ℃ for 28 h.
(2) Selecting white monoclonal antibody, inoculating the white monoclonal antibody into LB liquid culture solution containing kanamycin, gentamicin and tetracycline, oscillating the white monoclonal antibody for 16 hours at 37 ℃, extracting Bacmid-Cre by using a phenol chloroform extraction method, and obtaining Bacmid-Cre to identify by using a PCR method.
(3) According to 2X106And (3) spreading one cell/well, spreading a 6-well plate, transfecting Sf9 cells by using 20 mu L Bacmid-Cre, changing the liquid 8h after transfection, and collecting supernatant 96h after transfection to obtain baculovirus carrying a Cre expression cassette.
Third, verification of baculovirus infected suspension 293 cell lentivirus-producing inducible system
The verification comprises two parts, wherein one part is the expression verification of lentivirus packaging protein in the cell after the baculovirus infects the suspension 293 cell; the other part is the verification of the expression of the target gene after the target cells are infected by lentivirus generated by chromatographic purification of supernatant after the suspension 293 cells are infected by the baculovirus.
And (I) infecting the suspended 293 cells stably integrated with the lentivirus packaging system by using baculovirus carrying a Cre recombinase expression cassette, and detecting the expression of the lentivirus packaging components before and after infection by Western Blot. Results are shown in FIG. 2, before infection neither gag-pol nor VSV.G was expressed due to the presence of loxp-stop-loxp. Is obviously expressed after infection. The process comprises the following steps:
(1) according to 2X106Spreading one 6-pore plate per cell/pore, infecting the suspension 293 cells by Baculovir-Cre according to MOI1000, and collecting the cells after infection for 72h to obtain lysate.
(2) Preparing 10% SDS-PAGE gel, measuring the protein concentration of the lysate by using a BCA method, adding the cell lysate with the same protein amount into the 6% SDS-PAGE gel, using 80V voltage to run to the junction of the compression gel and the separation gel, and using 120V voltage to run to the bottom of the gel.
(3) SDS-PAGE gels containing proteins were transferred to NC membranes using a constant current of 400 mA.
(4) The protein-transferred NC membrane was incubated with 5% skim milk at room temperature for 1h, and the membrane was washed 3 times with TBST for 5min each.
(5) Incubate with gag-pol and primary antibody to VSV.G protein overnight at 4 ℃.
(6) The following day the membranes were washed 3 times with TBST for 10min each, incubated with secondary HRP containing antibody for 1h at room temperature, and washed 3 times with TBST for 5min each.
(7) The results of comparison of the expression levels of gag-pol and VSV.G before and after infection were obtained by development using the ECL method. The results are shown in FIG. 2, where the expression of gag-pol and VSV.G was clearly seen after infection.
Demonstrating that the lentiviral packaging proteins gag-pol and VSV.G sequences can be activated for expression by infecting suspension 293 cells stably integrated with the lentiviral packaging system with a baculovirus carrying the Cre recombinase expression cassette.
And (II) infecting the suspension 293 cells of the stably integrated lentivirus packaging system by using baculovirus carrying the Cre recombinase expression cassette, collecting the culture medium supernatant of the suspension 293 cells before and after infection, purifying the recombinant lentivirus, and infecting target cells (293T cells). The process comprises the following steps:
(1) expansion of gag-pol, VSV.G and GFP integrated suspension 293 cell lines to a cell density of 1X107One per ml.
(2) The cell line was infected with the Cre recombinase baculovirus with the genome titer already determined at MOI1, and the cell culture supernatant was collected 72 hours after infection.
(3) Purifying by adopting an AKTA avant chromatographic system of GE and DEAE chromatography, tangential flow filtration and core700 chromatography purification processes to obtain the lentivirus expressing GFP.
(4) Target cells were infected at an MOI1 of 0.1 and subjected to flow analysis and fluorescence microscopy.
The positive rate of GFP was determined by flow cytometry, with no positive cells before infection and a positive rate of 46% after infection. As a result of observation by a fluorescence microscope, as shown in FIG. 3, the Cre recombinase, after infecting the suspension 293 cells, was collected from the supernatant and purified by chromatography to infect the target cells, and then fluorescence was observed, indicating that the target gene G2P carried by the lentivirus was expressed in the target cells by infecting the target cells. Thus, it was demonstrated that the recombinant lentivirus prepared by the method of the present invention has the ability to infect target cells and express a gene of interest in the target cells.
The above examples are based on the object factor G2P. When the lentivirus gene medicament is prepared in actual production and application, different target genes can be carried according to treatment requirements so as to achieve the specific treatment purpose. Thus, based on the above examples, the desired Gene plasmid can be represented by the general formula piggybac-CBA-loxp-stop-loxp-Target Gene, wherein Target Gene can be a variety of desired genes, for example, Target Gene is COL7A1 Gene (SEQ ID NO: 7), from which a lentiviral drug for the treatment of dystrophic epidermolysis bullosa can be produced.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Zhongji Zhi medicine (Nanjing) Biotech Co., Ltd
<120> large-scale slow virus gene medicine preparation system and method based on Cre recombinase induction
<141> 2021-02-03
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atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 180
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atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 120
cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 180
cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag 240
cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 300
ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 360
ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 420
cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 480
gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 540
tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg 600
tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 660
gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 720
cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 780
gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 840
gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 900
tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 960
cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg 1020
gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat 1080
aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc 1140
ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt 1200
ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa 1260
cgtcggggcg gcaggccctg ccaataactt cgtatagcat acattatacg aagttatcgt 1320
cggatcagct ggcgcgagct ggtcgacaag cttgccacca gcttcgaggg gctcgcatct 1380
ctc 1383
<210> 3
<211> 1392
<212> DNA/RNA
<213> Artificial Sequence
<400> 3
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720
ggactcagat ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag 780
aatcctggac cgatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 840
cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 900
gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 960
gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 1020
accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 1080
gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 1140
cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 1200
aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 1260
cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 1320
accgtcaccg ccgacgtcga gtgcccgaag gaccgcgcga cctggtgcat gacccgcaag 1380
cccggtgcct ga 1392
<210> 4
<211> 6182
<212> DNA/RNA
<213> Artificial Sequence
<400> 4
gctggcgcga gctggtcgac aagctgaatt cgggccgcgt tgacgcgcac ggcaagaggc 60
gaggggcggc gactggtgag agatgggtgc gagagcgtca gtattaagcg ggggagaatt 120
agatcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca 180
tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac 240
atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga 300
agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga 360
gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagaa 420
aaaagcacag caagcagcag ctgacacagg acacagcaat caggtcagcc aaaattaccc 480
tatagtgcag aacatccagg ggcaaatggt acatcaggcc atatcaccta gaactttaaa 540
tgcatgggta aaagtagtag aagagaaggc tttcagccca gaagtgatac ccatgttttc 600
agcattatca gaaggagcca ccccacaaga tttaaacacc atgctaaaca cagtgggggg 660
acatcaagca gccatgcaaa tgttaaaaga gaccatcaat gaggaagctg cagaatggga 720
tagagtgcat ccagtgcatg cagggcctat tgcaccaggc cagatgagag aaccaagggg 780
aagtgacata gcaggaacta ctagtaccct tcaggaacaa ataggatgga tgacacataa 840
tccacctatc ccagtaggag aaatctataa aagatggata atcctgggat taaataaaat 900
agtaagaatg tatagcccta ccagcattct ggacataaga caaggaccaa aggaaccctt 960
tagagactat gtagaccgat tctataaaac tctaagagcc gagcaagctt cacaagaggt 1020
aaaaaattgg atgacagaaa ccttgttggt ccaaaatgcg aacccagatt gtaagactat 1080
tttaaaagca ttgggaccag gagcgacact agaagaaatg atgacagcat gtcagggagt 1140
ggggggaccc ggccataaag caagagtttt ggctgaagca atgagccaag taacaaatcc 1200
agctaccata atgatacaga aaggcaattt taggaaccaa agaaagactg ttaagtgttt 1260
caattgtggc aaagaagggc acatagccaa aaattgcagg gcccctagga aaaagggctg 1320
ttggaaatgt ggaaaggaag gacaccaaat gaaagattgt actgagagac aggctaattt 1380
tttagggaag atctggcctt cccacaaggg aaggccaggg aattttcttc agagcagacc 1440
agagccaaca gccccaccag aagagagctt caggtttggg gaagagacaa caactccctc 1500
tcagaagcag gagccgatag acaaggaact gtatccttta gcttccctca gatcactctt 1560
tggcagcgac ccctcgtcac aataaagata ggggggcaat taaaggaagc tctattagat 1620
acaggagcag atgatacagt attagaagaa atgaatttgc caggaagatg gaaaccaaaa 1680
atgatagggg gaattggagg ttttatcaaa gtaagacagt atgatcagat actcatagaa 1740
atctgcggac ataaagctat aggtacagta ttagtaggac ctacacctgt caacataatt 1800
ggaagaaatc tgttgactca gattggctgc actttaaatt ttcccattag tcctattgag 1860
actgtaccag taaaattaaa gccaggaatg gatggcccaa aagttaaaca atggccattg 1920
acagaagaaa aaataaaagc attagtagaa atttgtacag aaatggaaaa ggaaggaaaa 1980
atttcaaaaa ttgggcctga aaatccatac aatactccag tatttgccat aaagaaaaaa 2040
gacagtacta aatggagaaa attagtagat ttcagagaac ttaataagag aactcaagat 2100
ttctgggaag ttcaattagg aataccacat cctgcagggt taaaacagaa aaaatcagta 2160
acagtactgg atgtgggcga tgcatatttt tcagttccct tagataaaga cttcaggaag 2220
tatactgcat ttaccatacc tagtataaac aatgagacac cagggattag atatcagtac 2280
aatgtgcttc cacagggatg gaaaggatca ccagcaatat tccagtgtag catgacaaaa 2340
atcttagagc cttttagaaa acaaaatcca gacatagtca tctatcaata catggatgat 2400
ttgtatgtag gatctgactt agaaataggg cagcatagaa caaaaataga ggaactgaga 2460
caacatctgt tgaggtgggg atttaccaca ccagacaaaa aacatcagaa agaacctcca 2520
ttcctttgga tgggttatga actccatcct gataaatgga cagtacagcc tatagtgctg 2580
ccagaaaagg acagctggac tgtcaatgac atacagaaat tagtgggaaa attgaattgg 2640
gcaagtcaga tttatgcagg gattaaagta aggcaattat gtaaacttct taggggaacc 2700
aaagcactaa cagaagtagt accactaaca gaagaagcag agctagaact ggcagaaaac 2760
agggagattc taaaagaacc ggtacatgga gtgtattatg acccatcaaa agacttaata 2820
gcagaaatac agaagcaggg gcaaggccaa tggacatatc aaatttatca agagccattt 2880
aaaaatctga aaacaggaaa gtatgcaaga atgaagggtg cccacactaa tgatgtgaaa 2940
caattaacag aggcagtaca aaaaatagcc acagaaagca tagtaatatg gggaaagact 3000
cctaaattta aattacccat acaaaaggaa acatgggaag catggtggac agagtattgg 3060
caagccacct ggattcctga gtgggagttt gtcaataccc ctcccttagt gaagttatgg 3120
taccagttag agaaagaacc cataatagga gcagaaactt tctatgtaga tggggcagcc 3180
aatagggaaa ctaaattagg aaaagcagga tatgtaactg acagaggaag acaaaaagtt 3240
gtccccctaa cggacacaac aaatcagaag actgagttac aagcaattca tctagctttg 3300
caggattcgg gattagaagt aaacatagtg acagactcac aatatgcatt gggaatcatt 3360
caagcacaac cagataagag tgaatcagag ttagtcagtc aaataataga gcagttaata 3420
aaaaaggaaa aagtctacct ggcatgggta ccagcacaca aaggaattgg aggaaatgaa 3480
caagtagata aattggtcag tgctggaatc aggaaagtac tatttttaga tggaatagat 3540
aaggcccaag aagaacatga gaaatatcac agtaattgga gagcaatggc tagtgatttt 3600
aacctaccac ctgtagtagc aaaagaaata gtagccagct gtgataaatg tcagctaaaa 3660
ggggaagcca tgcatggaca agtagactgt agcccaggaa tatggcagct agattgtaca 3720
catttagaag gaaaagttat cttggtagca gttcatgtag ccagtggata tatagaagca 3780
gaagtaattc cagcagagac agggcaagaa acagcatact tcctcttaaa attagcagga 3840
agatggccag taaaaacagt acatacagac aatggcagca atttcaccag tactacagtt 3900
aaggccgcct gttggtgggc ggggatcaag caggaatttg gcattcccta caatccccaa 3960
agtcaaggag taatagaatc tatgaataaa gaattaaaga aaattatagg acaggtaaga 4020
gatcaggctg aacatcttaa gacagcagta caaatggcag tattcatcca caattttaaa 4080
agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 4140
gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat 4200
tacagggaca gcagagatcc agtttggaaa ggaccagcaa agctcctctg gaaaggtgaa 4260
ggggcagtag taatacaaga taatagtgac ataaaagtag tgccaagaag aaaagcaaag 4320
atcatcaggg attatggaaa acagatggca ggtgatgatt gtgtggcaag tagacaggat 4380
gaggattaac acatggaatt ctgcaacaac tgctgtttat ccatttcaga attgggtgtc 4440
gacatagcag aataggcgtt actcgacaga ggagagcaag aaatggagcc agtagatcct 4500
agactagagc cctggaagca tccaggaagt cagcctaaaa ctgcttgtac caattgctat 4560
tgtaaaaagt gttgctttca ttgccaagtt tgtttcatga caaaagcctt aggcatctcc 4620
tatggcagga agaagcggag acagcgacga agagctcatc agaacagtca gactcatcaa 4680
gcttctctat caaagcagta agtagtacat gtaatgcaac ctataatagt agcaatagta 4740
gcattagtag tagcaataat aatagcaata gttgtgtggt ccatagtaat catagaatat 4800
aggaaaatgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 4860
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 4920
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 4980
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 5040
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 5100
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 5160
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 5220
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 5280
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 5340
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 5400
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 5460
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 5520
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 5580
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 5640
cagagacaga tccattcgat tagtgaacgg atccttggca cttatctggg acgatctgcg 5700
gagcctgtgc ctcttcagct accaccgctt gagagactta ctcttgattg taacgaggat 5760
tgtggaactt ctgggacgca gggggtggga agccctcaaa tattggtgga atctcctaca 5820
atattggagt caggagctaa agaatagtgc tgttagcttg ctcaatgcca cagccatagc 5880
agtagctgag gggacagata gggttataga agtagtacaa ggagcttgta gagctattcg 5940
ccacatacct agaagaataa gacagggctt ggaaaggatt ttgctataag ctcgaaacaa 6000
ccggtacctc tagaactata gctagcagat ctttttccct ctgccaaaaa ttatggggac 6060
atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca 6120
atagtgtgtt ggaatttttt gtgtctctca gttaacttgt ttattgcagc ttataatggt 6180
ta 6182
<210> 5
<211> 2200
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
gctggcgcga gctggtcgac aagctgaatt ctgacactat gaagtgcctt ttgtacttag 60
cctttttatt cattggggtg aattgcaagt tcaccatagt ttttccacac aaccaaaaag 120
gaaactggaa aaatgttcct tctaattacc attattgccc gtcaagctca gatttaaatt 180
ggcataatga cttaataggc acagccttac aagtcaaaat gcccaagagt cacaaggcta 240
ttcaagcaga cggttggatg tgtcatgctt ccaaatgggt cactacttgt gatttccgct 300
ggtatggacc gaagtatata acacattcca tccgatcctt cactccatct gtagaacaat 360
gcaaggaaag cattgaacaa acgaaacaag gaacttggct gaatccaggc ttccctcctc 420
aaagttgtgg atatgcaact gtgacggatg ccgaagcagt gattgtccag gtgactcctc 480
accatgtgct ggttgatgaa tacacaggag aatgggttga ttcacagttc atcaacggaa 540
aatgcagcaa ttacatatgc cccactgtcc ataactctac aacctggcat tctgactata 600
aggtcaaagg gctatgtgat tctaacctca tttccatgga catcaccttc ttctcagagg 660
acggagagct atcatccctg ggaaaggagg gcacagggtt cagaagtaac tactttgctt 720
atgaaactgg aggcaaggcc tgcaaaatgc aatactgcaa gcattgggga gtcagactcc 780
catcaggtgt ctggttcgag atggctgata aggatctctt tgctgcagcc agattccctg 840
aatgcccaga agggtcaagt atctctgctc catctcagac ctcagtggat gtaagtctaa 900
ttcaggacgt tgagaggatc ttggattatt ccctctgcca agaaacctgg agcaaaatca 960
gagcgggtct tccaatctct ccagtggatc tcagctatct tgctcctaaa aacccaggaa 1020
ccggtcctgc tttcaccata atcaatggta ccctaaaata ctttgagacc agatacatca 1080
gagtcgatat tgctgctcca atcctctcaa gaatggtcgg aatgatcagt ggaactacca 1140
cagaaaggga actgtgggat gactgggcac catatgaaga cgtggaaatt ggacccaatg 1200
gagttctgag gaccagttca ggatataagt ttcctttata catgattgga catggtatgt 1260
tggactccga tcttcatctt agctcaaagg ctcaggtgtt cgaacatcct cacattcaag 1320
acgctgcttc gcaacttcct gatgatgaga gtttattttt tggtgatact gggctatcca 1380
aaaatccaat cgagcttgta gaaggttggt tcagtagttg gaaaagctct attgcctctt 1440
ttttctttat catagggtta atcattggac tattcttggt tctccgagtt ggtatccatc 1500
tttgcattaa attaaagcac accaagaaaa gacagattta tacagacata gagatgaacc 1560
gacttggaaa gtaactcaaa tcctgcacaa cagattcttc atgtttggac caaatcaact 1620
tgtgatacca tgctcaaaga ggcctcaatt atatttgagt ttttaatttt tatgaaaaaa 1680
aaaaaaaaaa acggaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg 1740
tgtggctaat gccctggccc acaagtatca ctaagctcgc tttcttgctg tccaatttct 1800
attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt atgaagggcc 1860
ttgagcatct ggattctgcc taataaaaaa catttatttt cattgcaatg atgtatttaa 1920
attatttctg aatattttac taaaaaggga atgtgggagg tcagtgcatt taaaacataa 1980
agaaatgaag agctagttca aaccttggga aaatacacta tatcttaaac tccatgaaag 2040
aaggtgaggc tgcaaacagc taatgcacat tggcaacagc ccctgatgcc tatgccttat 2100
tcatccctca gaaaaggatt caagtagagg cttgatttgg aggttaaagt tttgctatgc 2160
tgtattttag ttaacttgtt tattgcagct tataatggtt 2200
<210> 6
<211> 1207
<212> DNA/RNA
<213> Artificial Sequence
<400> 6
ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa 60
cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa tttcgagggg cagagccgat 120
cctgtacact ttacttaaaa ccattatctg agtgtgaaat gtccaattta ctgaccgtac 180
accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga 240
tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg 300
tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac 360
ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta 420
tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa 480
gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg 540
gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac 600
tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt 660
ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta 720
ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag 780
gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct 840
ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg 900
ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa 960
ctcatcgatt gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg 1020
gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg 1080
agatcatgca agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc 1140
tggatagtga aacaggggca atggtgcgcc tgctggaaga tggcgattag ccattaacgc 1200
gaattca 1207
<210> 7
<211> 8835
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
atgaccctga ggctgttggt cgcagcactg tgcgctggaa tccttgctga ggctccaaga 60
gtgagggccc agcacagaga gagagtgacc tgtacaagac tgtacgctgc agacattgtg 120
tttctgttag acggcagtag ctctattggg agatctaact tccgggaggt cagaagcttc 180
cttgagggcc tggtgctgcc ttttagcggg gccgcttctg cccagggcgt gaggttcgcc 240
actgtgcagt atagtgacga cccaaggact gagttcgggc tggatgcctt ggggagcggc 300
ggagatgtga ttagggcaat cagagagttg agctacaaag gcggcaatac tagaaccggc 360
gccgccatcc tgcacgtggc tgaccacgtg tttctgcccc aactggccag gcccggcgtc 420
cccaaagtgt gtatcctgat tactgacggc aaatcccagg acctggtaga caccgccgca 480
cagcgcctaa agggacaggg agtgaagttg ttcgcagtgg ggatcaagaa tgctgacccc 540
gaggaactga agcgggtggc cagtcagccc acctccgact tctttttctt cgtgaatgat 600
ttctcaatct tgaggaccct gctgcctctg gtgagccgta gagtgtgcac aacagcaggc 660
ggcgtacctg tgactagacc ccctgacgat tccacctcag ctcctagaga cctggtactc 720
tccgagccgt cctcccagag tctcagagtg cagtggactg ccgccagcgg gcccgtgaca 780
gggtacaagg tgcagtatac acctctgaca ggattgggac agcccctacc aagtgagcgg 840
caagaggtga acgtgccagc aggagagaca tcagtgagac tcagaggtct gaggcctctc 900
actgagtatc aggtcactgt gatcgcactc tatgccaatt ctataggaga ggccgttagt 960
ggaaccgccc gcaccacagc ccttgagggg cccgaactga ccattcagaa caccaccgcc 1020
cactcactgc tcgtggcctg gcggtctgtg ccaggtgcta ctgggtacag agtgacttgg 1080
agagtgctta gcggaggccc tacccagcag caggagttag gtcctggcca ggggagcgtg 1140
ctactcagag atctggagcc cggcaccgat tacgaagtga cagtaagtac cctctttggc 1200
agatccgttg ggcccgctac atcactcatg gctcgcaccg atgcctcagt ggagcaaaca 1260
ctgcggcccg tgatcttggg acctacctct attttgttaa gttggaacct ggtgcctgaa 1320
gccagggggt accggctgga atggcgcaga gagaccggac tggagcctcc acaaaaagtt 1380
gtgctgcctt ccgacgttac cagatatcag ttagacggat tacagcccgg aaccgaatac 1440
aggctgacgc tctacaccct cttggagggg cacgaggtcg ccacccccgc aactgttgtc 1500
ccaaccggac ctgaactgcc agtttctccc gtgaccgatc tgcaggcaac agagctgccc 1560
ggccagcggg tgcgcgtcag ctggtcccca gtgcctgggg ccacacagta cagaatcatc 1620
gtgcgatcta ctcagggcgt ggagcggact cttgtgctcc caggctccca gacagccttt 1680
gatctcgacg acgttcaggc cgggctctcc tatacagtga gagtcagtgc cagagtgggc 1740
cctagggagg ggtccgccag tgtgcttact gtgagaagag agccagagac ccctctggct 1800
gttcccggct tgcgcgtggt cgtgtctgac gccacaagag tgagggtggc ctggggaccc 1860
gtcccaggag cttctgggtt tcgcatatct tggagtaccg gatcaggccc cgaaagttct 1920
cagaccttgc cacctgactc aaccgctacc gacattactg gcctgcagcc cggtaccacc 1980
tatcaggtgg cagtctcagt gctgcgtgga agagaagaag gacctgcagc cgttattgtg 2040
gctcggaccg acccactcgg acctgtcagg accgtgcatg tcacgcaggc aagcagctct 2100
agcgtaacaa ttacatggac tcgagttccc ggagccacag gctaccgagt gtcctggcac 2160
agcgcccatg ggcctgagaa gtctcagctg gtcagcgggg aggctactgt agcagaactg 2220
gatggactcg agccggatac tgagtatacg gttcacgttc gtgctcacgt ggccggtgta 2280
gacggccctc ccgccagcgt tgtggtcaga actgcccctg agcccgtcgg cagggtgtca 2340
aggttacaaa ttttaaacgc aagctccgac gtcttgcgta tcacctgggt tggcgtgact 2400
ggggccaccg cctaccggtt agcttggggt cgatctgagg gaggacctat gaggcatcag 2460
atcctgcctg gtaatactga ttctgccgag atacgcggcc tggaaggtgg tgtgagctat 2520
tctgtgcgcg tcactgccct ggtgggtgac agagagggta cccctgtcag catcgtggtc 2580
accaccccac cagaagcacc acctgcactg ggaacactgc atgtcgtcca gagaggcgag 2640
cacagtctga gactgaggtg ggagcctgtc cccagggccc agggctttct gctccactgg 2700
cagcccgaag gaggccagga gcagagccgg gtcttgggtc ctgaactgtc ctcttaccac 2760
ctggacggcc tggagcctgc aacccagtac cgggtgaggc tttctgttct aggacccgcc 2820
ggggaaggac catcagccga agtcaccgcg aggacagaaa gtcctagggt accaagcatt 2880
gagctgcggg tggttgatac ttccatcgac agcgtgactc tcgcttggac ccctgtctct 2940
cgggcctcca gctatatcct ctcatggcgt cctctgcgag gacctggaca ggaagtccca 3000
ggctcccccc aaactctgcc aggtattagc tcttctcagc gggtaacagg cctggaaccc 3060
ggagttagct acatattcag cttgacccct gttctggatg gagtgagggg acctgaagct 3120
tcagtgacac agacacccgt gtgccctcgc ggtctcgcgg acgtcgtgtt tctgccccac 3180
gccactcagg acaacgcaca ccgagccgag gcaactagac gcgtgctcga gagactggtc 3240
ctggcccttg ggcctttggg cccccaggct gttcaggtag gactgttgag ttattcacac 3300
cggccatccc ccctatttcc cctgaacgga agccatgatc tcggaatcat cctacagcga 3360
atccgtgata tgccttacat ggacccttct ggaaacaatc tggggactgc agtggtcaca 3420
gcccacaggt acatgcttgc acctgatgca ccaggccgca ggcagcacgt gcccggagtt 3480
atggtgctcc tcgtcgatga acccctgcgg ggcgatattt tctctcccat ccgagaggca 3540
caagcaagtg gactcaacgt cgtgatgctg ggcatggccg gggctgaccc agagcagctt 3600
agacgccttg ctccaggaat ggattctgtt cagaccttct ttgctgtgga tgatggcccc 3660
tcactagacc aggcagtgag cggcctggcc actgctctct gccaggccag tttcacaacg 3720
caaccacgcc ccgagccatg tcctgtgtat tgccctaaag gccagaaggg cgaaccaggg 3780
gagatgggcc tgcgcggaca agtggggcca ccaggagacc ccggactccc tgggcgaacc 3840
ggggctccgg gcccacaagg cccacctgga agcgctaccg caaagggcga aagggggttt 3900
ccaggagccg atggcaggcc aggttctccc ggaagagctg gtaatcctgg tactcctggc 3960
gcgcccggac taaaaggtag tcctggactg ccaggtccta ggggagatcc aggagaaaga 4020
ggcccaagag gtcctaaggg agagcctggg gcaccagggc aagtgattgg aggagaaggg 4080
ccaggcctgc caggaaggaa aggcgatcct ggaccttctg gccctccagg gccacgcggg 4140
cctcttggag atcctggccc tcgcggccct cctggcctgc caggtacagc catgaaggga 4200
gataagggag accggggaga gaggggcccc cccggaccag gcgagggagg catcgcccca 4260
ggggaaccag gtctcccagg cctgcctggt tccccaggtc cccagggtcc agtgggacct 4320
ccaggcaaga agggggagaa gggagattcg gaagatggcg ctcctggcct acctggccag 4380
cccggtagtc ctggggagca gggtccaaga ggaccgccag gcgccattgg ccccaaagga 4440
gaccgcggct tccctggccc attaggcgaa gccggcgaga aaggtgagcg gggacctccc 4500
ggccccgccg gctccagagg cctgcccggt gtggccgggc gtcccggtgc gaaaggacct 4560
gaagggcccc ctggccctac cggcaggcag ggagagaagg gggagccagg acggcctgga 4620
gaccccgctg tggtgggacc tgccgtcgcc ggtccaaagg gcgagaaagg cgatgttgga 4680
cctgctggac cgaggggcgc tacaggcgtg cagggagagc gagggccacc cggattggtt 4740
ctgccagggg atccaggtcc aaagggagac cctggcgata ggggcccaat cggtttaaca 4800
ggcagagccg gaccccctgg ggattccgga ccaccaggcg aaaaagggga ccctggtagg 4860
cctggccctc ctggtccagt gggcccacgc ggccgagatg gcgaagtagg cgaaaaggga 4920
gatgaaggcc ctccaggcga tccagggttg cccggaaagg ctggggaacg cggcctacga 4980
ggagcacctg gggtgcgcgg ccccgtcggg gagaagggcg accaaggcga cccaggggaa 5040
gatgggcgga atgggtcccc tggctccagt ggtcccaaag gggacagagg ggaaccaggc 5100
ccacccggac cacccggcag gctggttgat acaggacctg gggccaggga gaaaggcgaa 5160
ccgggagaca gagggcagga gggaccacgc ggacctaaag gagacccagg acttcctgga 5220
gcacctgggg aacgcggtat cgaggggttc agaggacccc ctggacctca aggagaccct 5280
ggcgtccgcg gccctgcagg tgagaaggga gatagaggcc cccccggatt ggatgggaga 5340
tccggactgg atggcaaacc cggtgctgcc gggccaagtg gaccaaacgg agccgccgga 5400
aaagcaggag acccaggtcg cgatggtctg cctggcctta gaggcgaaca gggcctccca 5460
ggcccaagcg gacctcccgg actgcccggt aaaccaggtg aagatggcaa gcctggtctg 5520
aacgggaaga atggcgaacc aggcgaccct ggagaggatg gacggaaggg cgagaaaggc 5580
gactccggcg cctccggacg cgagggacgg gacggtccca agggcgagag aggcgctccc 5640
ggcattctgg gtccccaagg accacccgga ctccctggcc ctgtcggccc acctggacaa 5700
ggcttcccag gcgtgcccgg tgggactgga cctaagggcg acaggggtga gacaggttcc 5760
aaaggcgagc agggcctacc tggagagcgg ggactgcgtg gcgaacctgg cagcgtgccc 5820
aatgttgaca ggctgcttga aaccgctgga attaaggcct ctgcccttcg cgaaatcgtt 5880
gagacatggg atgaaagtag cggcagcttt cttcctgtcc ctgagcggag gcgggggcct 5940
aagggagact ccggcgaaca aggacctcct ggcaaggagg ggcccatcgg ttttccaggc 6000
gaacgggggc tgaaaggaga ccgaggggac cctggccccc agggcccccc cggtctggcc 6060
ctcggcgaac gtggcccacc agggcctagc ggcctggctg gagagcctgg caagccgggg 6120
atcccagggt tacctggccg agccggcggc gtcggcgagg ctggccgtcc aggcgagcga 6180
ggggaacggg gagagaaagg agagaggggg gagcagggca gggacggccc ccccggcctg 6240
ccaggaacac ccggcccacc tggtcctccc ggacctaaag taagcgtgga tgagcccgga 6300
cctggactga gcggagagca agggcctcct ggactgaagg gagctaaggg cgagccgggt 6360
agcaatggcg atcaggggcc caaaggcgac cgaggcgtcc ccggtatcaa gggcgaccgc 6420
ggggagccag ggcctcgggg ccaggatggt aatcctggcc tgcccggcga gagaggaatg 6480
gccgggcccg aaggaaagcc aggccttcag ggacctaggg gcccaccggg gcccgtgggt 6540
ggacacgggg atccaggccc acctggcgcc cctggcttgg ctggccccgc tggcccccag 6600
ggcccgtcag gcctgaaagg cgagccaggg gagaccggac ccccaggaag aggactaaca 6660
ggccccacag gcgctgtggg cctgcccgga ccgccaggac catcaggcct ggtgggccct 6720
cagggaagtc ctggcttgcc tggacaggta ggggagactg gaaagccagg ggctcctgga 6780
agagatgggg cttcaggtaa agacggcgac aggggaagtc caggggtgcc tgggtcccct 6840
ggccttccag gccctgtggg cccaaagggc gaacccggcc caactggcgc accaggacaa 6900
gccgtggtgg ggctgccagg cgccaagggc gaaaaggggg cccctggagg cctggcgggc 6960
gacctggtgg gagaaccagg cgctaaaggg gacagaggtc tccctgggcc tcgcggagag 7020
aagggggagg ctgggagagc tggggagccc ggcgacccag gcgaggatgg ccagaagggg 7080
gcaccaggcc ctaaggggtt taagggcgac ccaggggttg gggtgccagg ctcaccaggg 7140
cccccaggtc cccctggcgt gaaaggcgat ttggggcttc ccggactgcc tggggcccct 7200
ggcgtggtgg gttttcccgg acagacagga cccagaggag agatggggca accaggcccg 7260
tcaggcgaga gaggattagc aggccctcca ggaagggaag gcatccccgg cccactgggg 7320
cccccaggac ctccaggtag cgtgggtccc cctggcgctt ccggcttgaa gggagataag 7380
ggcgatcctg gcgtcggact gcctgggcca agaggcgagc ggggagaacc cggaatcagg 7440
ggggaagatg ggcgcccagg acaggaaggc cccaggggac tgacaggtcc tcctggcagc 7500
agaggggagc gtggtgaaaa gggcgatgtg ggctcagctg gccttaaggg cgataaaggg 7560
gattccgcag taattctggg cccaccaggt cccagagggg caaaaggaga catgggggaa 7620
aggggccctc ggggactgga tggcgacaaa ggccctcgcg gcgacaacgg ggaccccggc 7680
gacaagggga gcaagggaga acccggagat aagggatctg ccgggcttcc aggattgaga 7740
gggctcctgg gcccccaagg acagcccggc gccgctggga ttcctggtga ccccggaagc 7800
cccggtaaag atggcgtgcc cggtatccgc ggcgagaagg gcgacgtggg ctttatgggc 7860
ccacgcggcc tgaagggtga gcgcggcgtg aagggcgcat gtgggctgga cggcgagaag 7920
ggcgataagg gtgaggcagg tcccccaggc cggcctggcc tggccgggca caagggcgaa 7980
atgggcgagc caggcgtgcc aggtcaatcc ggagccccag gcaaggaggg gctgattggg 8040
cccaagggcg accggggctt tgacggtcag ccggggccaa agggcgacca gggggagaaa 8100
ggggagcgtg gtacacctgg aatcggcggc ttccccggac ccagtgggaa tgacgggtct 8160
gccggccccc ctggccctcc cggctccgtg ggacctaggg gacccgaagg actgcaggga 8220
cagaaggggg aacgcgggcc tccaggcgag cgcgtggtcg gcgctcccgg cgtgcctggg 8280
gccccaggtg agagagggga gcagggccgg cctggccctg ccggcccaag aggagagaag 8340
ggagaggcag cactcactga ggatgatatc cgcggattcg ttaggcagga aatgagtcaa 8400
cattgtgcct gccagggcca gtttatcgcc tcaggatccc gccctctccc ctcctatgcc 8460
gccgacacag cagggagcca gctgcacgca gtccccgtgc tgagagtgtc tcatgcagag 8520
gaggaggaaa gggtgccacc tgaagatgat gagtacagcg agtatagtga gtacagtgtc 8580
gaggagtacc aggaccctga ggctccctgg gacagcgacg acccatgcag cctgcctctg 8640
gacgagggct catgcactgc ctacacactc cgctggtacc atagggccgt gactggcagc 8700
actgaggcct gccacccctt tgtgtacggc gggtgcggag gcaatgctaa tagattcggg 8760
acaagagaag cctgcgaaag gcgctgtcct ccgagggtgg tgcagtccca gggcacaggc 8820
acagcccagg attga 8835
Claims (10)
1. A large-scale slow virus gene medicine preparation system based on Cre recombinase induction is characterized by comprising: suspending 293 stable cell line and virus expressing Cre gene;
the genome of the suspended 293 stable cell line integrates a lentivirus packaging protein expression sequence and a target gene sequence; and loxP-Stop-loxP sequence is inserted between the lentivirus packaging protein expression sequence and a promoter for driving the expression of the lentivirus packaging protein expression sequence; the loxP-Stop-loxP sequence comprises two loxP sites with the same direction and a Stop sequence connected between the two loxP sites;
the virus for expressing the Cre gene is a virus which can infect the suspension 293 cell and can express Cre protein;
the virus expressing the Cre gene is used for infecting the suspended 293 stable cell strain and activating a lentivirus packaging protein expression sequence to express so as to package and generate lentivirus.
2. The large scale lentiviral gene drug preparation system of claim 1, wherein a loxP-Stop-loxP sequence is inserted between the gene sequence of interest and the promoter driving its expression in the suspended 293 stably transfected cell line.
3. A preparation method of a large-scale lentivirus gene medicine based on Cre recombinase induction is characterized by comprising the following steps:
s1, constructing a suspension stable transfer cell strain for generating lentivirus; the genome of the stable transgenic cell strain is stably integrated with a lentivirus packaging protein expression sequence and a target gene sequence, and a loxP-Stop-loxP sequence is inserted between the lentivirus packaging protein expression sequence and a promoter for driving the expression of the lentivirus packaging protein expression sequence; the loxP-Stop-loxP sequence comprises two loxP sites with the same direction and a Stop sequence connected between the two loxP sites;
s2, constructing a virus for expressing Cre recombinase;
s3, amplifying and culturing the stable transfer cell strain in a large scale;
when the lentivirus particles need to be produced, the virus expressing Cre genes is used for infecting the suspension stable transfer cell strain, Cre recombinase expressed by the virus is used for excising the Stop sequence of the loxP-Stop-loxP sequence so as to induce the expression sequence of the lentivirus packaging protein to be expressed, thereby packaging the recombinant lentivirus particles expressing target genes, and the recombinant lentivirus particles are virus gene medicines.
4. The method of claim 3, wherein in the step S1, in the construction of the stable transformant cell line, loxP-Stop-loxP site is inserted between the target gene sequence and the promoter for driving its expression to construct a target gene plasmid, and the plasmid is integrated into the genome of the host cell.
5. The method according to claim 3, wherein the step of constructing 293 stably transfected cell line in suspension in step S1 comprises: s11, constructing the following three plasmids:
gag-pol plasmid: piggybac-CBA-loxp-stop-loxp-gag-pol,
Vsv. g plasmid: piggybac-CBA-loxp-stop-loxp-VSV.G and
the target plasmid: piggybac-CBA-loxp-stop-loxp-Target Gene plasmid;
s12, constructing a suspension 293 stable transfer cell strain: the three plasmids obtained are transfected into suspension 239 cells and are screened by using a culture medium containing antibiotics to obtain resistant cells, namely 239 suspension stable cells for generating lentiviruses.
6. The method of claim 5, wherein in step S1, the Target Gene is codon-optimized COL7A1 Gene, and its sequence is as shown in SEQ ID NO: shown at 7.
7. The method according to claim 3, wherein the Cre recombinase-expressing virus is a baculovirus in step S2, and the method comprises:
firstly, a Cre gene expression sequence is seamlessly cloned into a pFast Bac1 plasmid, and a baculovirus plasmid pFast-Bac-Cre with a Cre expression box is constructed; then, the pFast-Bac-Cre plasmid is transformed into a DH10Bac competent cell to obtain Bacmid-Cre, then Bacmid is used for transfecting Sf9 cells, and after the transfection is carried out for a preset time, the supernatant is collected to obtain Baculovirus Baculovirus-Cre for expressing Cre recombinase.
8. The method according to claim 7, wherein the pFast-Bac-Cre plasmid is constructed in S2 by:
(1) carrying out double enzyme digestion on a pFast Bac1 plasmid for 50-70min at 37 +/-0.5 ℃ by using restriction endonucleases SnaBI and HpaI, cutting gel after agarose electrophoresis to recover a pFast Bac1 vector fragment, carrying out dephosphorizing treatment on the recovered pFast Bac1 vector fragment for 50-70min at 50 +/-2 ℃ by using alkaline phosphatase CIAP, and carrying out inactivation treatment for 10-25min at 65 +/-2 ℃;
(2) connecting the pFast Bac1 vector fragment treated by alkaline phosphatase and the gene fragment containing the Cre expression cassette by adopting a seamless cloning recombinase, and reacting for 8-15min at 50 +/-2 ℃ to obtain a seamless connection recombinant product;
(3) taking a seamless connection recombinant product transformation competence DH5a, uniformly mixing, carrying out ice bath and heat shock, immediately carrying out ice bath again, carrying out shake culture by using an antibiotic-free culture solution, uniformly coating the bacterial solution on a plate culture medium containing ampicillin, and carrying out inverted culture at 37 +/-0.5 for 12-18 h;
(4) selecting a monoclonal colony, inoculating the colony in a liquid culture solution containing ampicillin, and oscillating for 12-18h at 37 +/-0.5 ℃; extracting pFast-Bac-Cre plasmid, performing double enzyme digestion identification on SnaBI and HpaI, performing sequencing identification, and constructing to obtain baculovirus plasmid with Cre expression cassette.
9. The method according to claim 7, wherein baculoviral-Cre expressing Cre recombinase in S2 is prepared by:
(1) the pFast-Bac-Cre plasmid was transformed into DH10Bac competent cells: transforming pFast-Bac-Cre plasmid into competent DH10Bac, mixing the plasmids gently and performing ice bath for 25 to 35 min; performing heat shock at 42 ℃ +/-0.5 for 40-50s, immediately performing ice bath for 2-5min, adding LB culture solution without antibiotics at 37 ℃ +/-0.5, oscillating for 40-80min, uniformly coating the bacterial solution on an LB agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG by using a sterile glass coater, and performing inverted culture at 37 ℃ +/-0.5 for 24-32 h;
(2) selecting white monoclonal to inoculate in liquid culture solution containing kanamycin, gentamicin and tetracycline, oscillating for 14-18h at 37 +/-0.5 ℃, extracting Bacmid-Cre by a phenol-chloroform extraction method, and obtaining Bacmid-Cre which is identified by a PCR method;
(3) and (3) transfecting Sf9 cells by using a pore plate cell plate according to the proportion of 1x10^6 cells/pore by using 20 +/-1 mu l Bacmid-Cre per pore, changing the liquid 6-8h after transfection, and collecting supernatant 96h after transfection to obtain the baculovirus carrying the Cre expression cassette.
10. The method according to claim 5, wherein step S3 includes: amplifying and culturing the suspended 293 stable transgenic cell line to a preset density, infecting the suspended 293 stable transgenic cell line by Baculovir-Cre according to MOI1, collecting cell supernatant after infecting for 72h, and carrying out chromatography and purification to obtain the lentivirus expressing the target gene.
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