WO2022166026A1 - 基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法 - Google Patents

基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法 Download PDF

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WO2022166026A1
WO2022166026A1 PCT/CN2021/093905 CN2021093905W WO2022166026A1 WO 2022166026 A1 WO2022166026 A1 WO 2022166026A1 CN 2021093905 W CN2021093905 W CN 2021093905W WO 2022166026 A1 WO2022166026 A1 WO 2022166026A1
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loxp
cre
sequence
plasmid
stop
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董文吉
赵忠亮
陈志川
董祖伊
刘子瑾
程谟斌
张艳君
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中吉智药(南京)生物技术有限公司
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Definitions

  • the invention relates to the technical field of genetic medicine, in particular to a large-scale lentiviral genetic medicine preparation system and method based on Cre recombinase induction.
  • Gene therapy drugs are an important part of biomedicine. They have the opportunity to fundamentally cure rare genetic diseases and cancers for the first time in human history. They also have the potential to greatly improve existing cardiovascular diseases, metabolic diseases, autoimmune diseases, and neurodegeneration. Inherent diseases such as traditional drugs such as sexually transmitted diseases are ineffective, and the treatment methods are cumbersome.
  • the production method of recombinant lentivirus is to divide the genome of lentivirus into 3 or 4 parts, which are respectively loaded into different plasmid vectors, and the plasmid vectors are used to transfect adherent or suspension cultured HEK293 cells to package the recombinant lentivirus.
  • four plasmid vector production systems are commonly used in clinical practice. As shown in Figure 1.
  • the tetracycline induction system is relatively mature in research and development, but the addition and removal of tetracycline is a difficult point in the production and purification of clinical-grade lentiviruses, and this method has not yet been applied on a large scale.
  • the present invention provides a large-scale lentiviral gene drug preparation system and method based on Cre recombinase induction, which solves the problems of high cost of plasmid transient transfection and difficulty in amplification;
  • the virus infection of Cre gene is induced to avoid the process problem that is difficult to remove after induction by antibiotics such as tetracycline.
  • the main technical scheme adopted in the present invention includes:
  • the present invention provides a large-scale lentiviral gene drug preparation method based on Cre recombinase induction, comprising:
  • S1 construct a suspension stable transfection cell line for producing lentivirus; the genome of the suspension stable transfection cell line is stably integrated with the lentiviral packaging protein expression sequence and the target gene sequence, and the lentiviral packaging protein expression sequence and driving its
  • the loxP-Stop-loxP sequence is inserted between the expressed promoters; the loxP-Stop-loxP sequence contains two loxP sites in the same direction and a Stop sequence connected between the two loxP sites;
  • S2 construct a virus expressing Cre recombinase
  • the virus expressing the Cre gene is used to infect the suspension stable cell line, and the Cre recombinase expressed by the virus is used to excise the Stop sequence of the loxP-Stop-loxP sequence to induce lentiviral packaging proteins.
  • the expression sequence is expressed, thereby packaging recombinant lentiviral particles expressing the target gene, and the recombinant lentiviral particles are viral gene drugs.
  • the virus expressing Cre gene is any virus that can infect suspended 293 cells and can express Cre protein.
  • baculovirus is a replicating virus, it is more convenient to produce, so it can be realized by using a baculovirus expressing the Cre gene.
  • step S1 when constructing a stably transfected cell line in suspension, first construct a plasmid expressing gag-pol and a plasmid expressing VSV.G, and the plasmid expressing gag-pol includes a promoter and gag -pol gene sequence, and insert the loxP-Stop-loxP sequence between the promoter and the gag-pol gene sequence, the VSV.G expressing plasmid contains the promoter and the VSV.G gene sequence, and between the promoter and VSV.G The loxP-Stop-loxP sequence was inserted between the gene sequences; the gag-pol expressing plasmid and the VSV.G expressing plasmid were then integrated into the suspension cell genome (via the PiggyBac vector).
  • step S1 when constructing a stably transfected cell line, insert the loxP-Stop-loxP sequence between the target gene sequence and the promoter driving its expression to construct the target gene plasmid, and then (through PiggyBac vector) to integrate the target gene plasmid into the genome of the suspension cell line.
  • step S1 the steps of constructing a suspension 293 stable transfection cell line include: 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
  • Target plasmid piggybac-CBA-loxp-stop-loxp-Target Gene plasmid
  • step S11 when constructing the plasmid in step S11, the following steps are included:
  • the piggybac-dual-promoter plasmid was double-digested with restriction enzymes SnaBI and HindIII, and the piggybac-dual-promoter vector fragment was recovered by cutting the gel after agarose electrophoresis;
  • 2CBA promoter sequence fragment (sequence shown in SEQ ID NO: 1), loxp-stop-loxp sequence fragment (sequence shown in SEQ ID NO: 2), and gag-pol designed for seamless cloning Sequence (sequence shown in SEQ ID NO: 4) or VSV.G (sequence shown in SEQ ID NO: 5) or Target Gene sequence fragment;
  • the piggybac-dual-promoter vector fragment, CBA sequence fragment and loxp-stop-loxp fragment are respectively connected with gag-pol or VSV.G or Target Gene sequence fragment using seamless cloning kit;
  • the Target Gene is a codon-optimized COL7A1 gene, and its sequence is shown in SEQ ID NO: 7.
  • the virus expressing Cre recombinase is baculovirus, and its construction method is:
  • the Cre gene expression sequence (as shown in SEQ ID NO: 6) was seamlessly cloned into the pFast Bac1 plasmid (invitrogen, Cat. No. 10359-016), and the baculovirus plasmid pFast- Bac-Cre; then the pFast-Bac-Cre plasmid was transformed into DH10bac competent cells to obtain Bacmid-Cre, and then Sf9 cells were transfected with Bacmid. Virus Baculovirus-Cre.
  • the construction method of pFast-Bac-Cre plasmid is:
  • the preparation method of Baculovirus-Cre expressing Cre recombinase is:
  • Sf9 cells were transfected with 20 ⁇ 1 ⁇ l Bacmid-Cre in each well at 1 ⁇ 10 ⁇ 6 cells/well, the medium was changed 6-8 hours after transfection, and the supernatant was collected 96 hours after transfection to obtain the Baculovirus with Cre expression cassette.
  • step S3 includes: amplifying and culturing the suspension 293 stable transfected cell line to a predetermined density, infecting the suspended 293 stable transgenic cell line with Baculovirus-Cre according to MOI1, and collecting the cell supernatant after 72 hours of infection, Chromatographic purification to obtain a lentivirus expressing the target gene.
  • the present invention provides a large-scale lentiviral gene drug preparation system based on Cre recombinase induction, comprising:
  • the genome of the suspension 293 stably transfected cell line is integrated with the lentiviral packaging protein expression sequence and the target gene sequence; and the loxP-Stop-loxP sequence is inserted between the lentiviral packaging protein expression sequence and the promoter driving its expression;
  • the loxP-Stop-loxP sequence contains two loxP sites in the same direction and a Stop sequence connected between the two loxP sites;
  • the virus expressing the Cre gene is a virus that can infect the suspension 293 cells and can express the Cre protein; the virus expressing the Cre gene is used to infect the suspension 293 stably transfected cell line, and activate the lentiviral packaging protein expression sequence for expression, Lentiviruses are produced by packaging.
  • baculovirus As the virus expressing the Cre gene, baculovirus can be used, and baculovirus is a replicating virus and is easy to mass-produce.
  • the loxP-Stop-loxP sequence is inserted between the target gene sequence and the promoter driving its expression.
  • the target gene is a codon-optimized COL7A1 gene, the sequence of which is shown in SEQ ID NO: 7.
  • the prepared lentiviral particle is a lentiviral gene drug for the treatment of dystrophic epidermolysis bullosa.
  • the present invention utilizes an inducible lentivirus production system mediated by the Cre-loxP recombination system. Get rid of the high cost of plasmid transient transfection and the difficulty of amplification.
  • the present invention utilizes virus infection for induction, so as to avoid the technical problem that antibiotics such as tetracycline are difficult to remove after induction.
  • the loxP-Stop-loxP sequence is also inserted between the target gene sequence and the promoter driving its expression to construct a target gene plasmid, and then the target gene plasmid is integrated into the genome of the suspension cell line. superior. It can avoid the pressure brought by the continuous large-scale expression of exogenous proteins to the cells during the expansion and culture process of the constructed suspension stable transfection cell line, and inhibit the growth and culture density of the suspension stable transfection cells.
  • Fig. 1 is a schematic diagram of the production method of recombinant lentivirus in the prior art.
  • Figure 2 shows the comparison results of the expression levels of lentiviral packaging proteins gag-pol and VSV.G in the suspension 293 stable transfection cell lysate before and after infection with the baculovirus carrying the Cre gene before and after the suspension 293 stable transfection strain was detected by Western Blot , where - means before infection and + means after infection.
  • Figure 3 shows before and after infection of the suspended 293 stable transfected strain with the baculovirus carrying the Cre gene, chromatographic purification of the lentivirus from the supernatant of the suspended 293 stable transgenic strain, infect the target cells (239T) with the lentivirus, and observe with a fluorescence microscope Fluorescence changes of target cells (239T); where Cre(-) means before infection, Cre(+) means after infection.
  • Cre recombinase (Cyclization Recombination Enzyme) is a 38kD protein composed of 343 amino acids encoded by the Cre gene of Escherichia coli phage P1. It not only has catalytic activity, but also, similar to restriction enzymes, can specifically recognize the loxP site.
  • the LoxP (locus ofX-overP1) site is 34 bp long, including two 13 bp inverted repeats and an 8 bp spacer region. Among them, the inverted repeat sequence is the specific recognition site of Cre recombinase, and the spacer region determines the orientation of the loxP site.
  • Cre recombinase When there is a loxP site in the genome, once the Cre recombinase is encountered, the Cre recombinase will bind to the inverted repeat sequence regions at both ends of the loxP site to form a dimer. This dimer binds to dimers at other loxP sites to form tetramers. Subsequently, the DNA between the two loxP sites is cleaved by Cre recombinase, and the nicks are reconnected under the action of DNA ligase.
  • the present invention is to place the Stop sequence between two loxP sites in the same direction to form a loxP-Stop-loxP sequence to form a switch sequence that can be induced and activated by Cre recombinase, and then loxP-Stop-loxP
  • the sequence is placed in front of the lentiviral packaging protein expression sequence (gag-pol or VSV.G, etc.) (behind the promoter driving the expression of the lentiviral packaging protein expression sequence (gag-pol or VSV.G, etc.)), thereby obtaining A plasmid construct inducible by Cre recombinase activation was then integrated into the suspension 293 cell genome via the PiggyBac vector system.
  • the suspension 293 cells thus obtained do not express the lentiviral packaging protein in the absence of Cre recombinase, which avoids the cytotoxicity of the lentiviral packaging protein from inhibiting the growth of the suspension 293 cells, and is beneficial to the large-scale expansion and culture of the suspension 293 cells.
  • a virus expressing Cre recombinase was constructed, and the stably integrated suspension 293 cells (that is, the lentiviral packaging protein expression sequence with the pre-loxP-Stop-loxP sequence integrated on its genome) were infected with the virus expressing Cre recombinase, using The Sotp sequence in the Cre recombinase loxP-Stop-loxP sequence can activate the lentiviral packaging protein expression sequence (gag-pol or VSV.G, etc.) connected to the loxP-Stop-loxP sequence to express, and package the recombinant lentivirus .
  • G2P-T2A-Puro G2P
  • the implementation method is as follows:
  • Plasmids were transfected into suspension 293 cells, and cell lines that integrated into the genome of suspension 293 cells were screened with antibiotics. The specific methods and steps are as follows:
  • the piggybac-dual-promoter plasmid (Youbao Bio, VT1663) was double-digested with restriction enzymes SnaBI and HindIII at 37°C for 30 min, and the piggybac-dual-promoter vector fragment was recovered by gel cutting after agarose electrophoresis.
  • piggybac-CBA-loxp-stop-loxp-G2P plasmid constructed above was double-digested with restriction enzymes HpaI (KspAI) and HindIII at 37°C for 30 min, and the gel was recovered after agarose electrophoresis. piggybac-CBA-loxp-stop-loxp vector fragment.
  • gag-pol fragment (SEQ ID NO: 4) was amplified by PCR from the psPAX2 plasmid vector (addgene plasmad#12260), and the gag was recovered after agarose electrophoresis. -pol fragment.
  • the piggybac-CBA-loxp-stop-loxp-gag-pol plasmid was extracted with the axygen plasmid extraction kit, identified by double digestion with NdeI and HpaI (KspAI), and then sequenced and identified to obtain piggybac-CBA-loxp-stop- loxp-gag-pol plasmid.
  • piggybac-CBA-loxp-stop-loxp-G2P plasmid constructed above was double-digested with restriction enzymes HpaI (KspAI) and HindIII at 37°C for 30 min, and the gel was recovered after agarose electrophoresis. piggybac-CBA-loxp-stop-loxp vector fragment.
  • VSV.G-R (aaccattataagctgcaataaacaagttaactaaaatacagcatagcaaaac)
  • VSV.G fragment (SEQ ID NO: 5) was amplified by PCR from the pMD2.G plasmid vector (addgene plasmid#12259), and the VSV was recovered by gel cutting after agarose electrophoresis. .G fragment.
  • the piggybac-CBA-loxp-stop-loxp-VSV.G plasmid was extracted with the axygen plasmid extraction kit, identified by XbaI and HpaI (KspAI) double enzyme digestion, and then sequenced and identified to obtain piggybac-CBA-loxp-stop- loxp-VSV.G plasmid.
  • Stable cell line screening is to transfect the plasmid into suspension 293 cells, and screen out the Cell pool integrated into the suspension 293 cell genome. The specific steps are as follows:
  • transfection reagent plasmid mixture was added to the 293 cells, cultured at 37°C for 6 hours, and then replaced with fresh medium.
  • 293 cells were changed to medium containing 8 ⁇ g/ml Puromycin, and continued to be cultured at 37°C, and fresh medium of 8 ⁇ g/ml Puromycin was changed every three days until the cells no longer died and survived.
  • the cells were all anti-Puromycin cells, and thus the inducible lentivirus-producing suspension 293 cell line was obtained.
  • the three plasmids were not successfully transfected and screened one by one, but the Puromycin-resistant cells must contain the suspension 293 cell line that has stably integrated the above three plasmids. Therefore, the selected Puromycin-resistant cells Can be used as suspension cells for lentivirus production (see below for validation procedure).
  • the three plasmids can be transfected one by one and confirmed by screening one by one according to the actual situation, so as to screen out the suspension 293 cell line that stably integrates the three plasmids.
  • any virus that can infect 293 cells and can express Cre recombinase can be used as an infectious virus.
  • a replicative baculovirus was selected as a carrier for expressing Cre recombinase.
  • the construction process of baculovirus expressing Cre recombinase includes the construction of pFast-Bac-Cre plasmid and recombination to obtain baculovirus expressing Cre recombinase. The process is as follows:
  • the synthetic Cre gene expression sequence (SEQ ID NO: 6) was seamlessly cloned into the pFast Bac1 plasmid (invitrogen, Cat. No. 10359-016) to obtain the pFast-Bac-Cre plasmid; the cloning process includes the following steps :
  • the pFast Bac1 plasmid was double-digested with restriction enzymes SnaBI and HpaI at 37°C for 1 h. After agarose electrophoresis, the pFast Bac1 vector fragment was recovered by cutting the gel, and the recovered pFast Bac1 vector fragment was treated with alkaline phosphatase CIAP. The dephosphorization treatment was carried out at 50°C for 60min, and the inactivation treatment was carried out at 65°C for 15min.
  • DH10bac competent cells were transformed with pFast-Bac-Cre plasmid to obtain Bacmid-Cre, and then Sf9 cells were transfected with Bacmid. After transfection for a predetermined time, the supernatant was collected to obtain Baculovirus-Cre.
  • the specific process includes the following steps:
  • the verification consists of two parts, one part is the expression verification of lentiviral packaging protein in the cells after baculovirus infection of suspended 293 cells; the other part is the lentiviral infection produced by chromatographic purification of the supernatant after baculovirus infection of suspended 293 cells Validation of target gene expression after targeting cells.
  • NC membrane was incubated with 5% skim milk at room temperature for 1 h, and the membrane was washed three times with TBST for 5 min each time.
  • the process includes the following steps:
  • the suspension 293 cell line integrating gag-pol, VSV.G and GFP was expanded to a cell density of 1 ⁇ 10 7 cells/ml.
  • the target cells were infected with MOI1 of 0.1, and flow analysis and fluorescence microscopy were performed.
  • the positive rate of GFP was detected by flow cytometry, there were no positive cells before infection, and the positive rate after infection was 46%. Observed by fluorescence microscope, the results are shown in Figure 3. After Cre recombinase infects suspended 293 cells, the lentivirus collected from the supernatant and purified by chromatography infects the target cells, and fluorescence appears, indicating that the target gene G2P carried in the lentivirus passes through. Infect target cells and express in target cells. This proves that the recombinant lentivirus prepared by the method of the present invention has the ability to infect the target cells and express the target gene in the target cells.
  • the target gene plasmid can be represented by the general formula piggybac-CBA-loxp-stop-loxp-Target Gene, wherein, Target Gene can be various target genes, such as Target Gene is COL7A1 gene ( The sequence is SEQ ID NO: 7), from which a slow gene drug for treating dystrophic epidermolysis bullosa can be produced.

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Abstract

一种基于Cre重组酶诱导的大规模慢病毒基因药物制备系统，其包括：悬浮293稳转细胞株和表达Cre基因的病毒；悬浮293稳转细胞株的基因组上整合了慢病毒包装蛋白表达序列和目的基因序列；且所述慢病毒包装蛋白表达序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列；loxP-Stop-loxP序列包含两个方向相同的loxP位点以及连接在两个loxP位点之间的Stop序列；表达Cre基因的病毒为可感染悬浮293细胞且能够表达Cre蛋白的病毒；表达Cre基因的病毒用于感染所述悬浮293稳转细胞株，激活慢病毒包装蛋白表达序列进行表达，以包装产生慢病毒。

Description

基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法 技术领域
本发明涉及基因药物技术领域，尤其涉及一种基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法。
背景技术
基因治疗药物是生物医药的重要组成部分，有机会在人类历史上首次实现从根本上治愈罕见遗传病和癌症，也有潜力大幅度改善现有心血管疾病、代谢性疾病、自身免疫性疾病、神经退行性疾病等传统药物治疗效果不佳、治疗方式繁琐等固有疾病。
目前全球细胞和基因治疗的新药已有7款上市，未来十年将有数十个新药上市，总产值将突破千亿美元。目前已上市的产品中有3款是基于慢病毒载体的基因药物：其中2款针对淋巴瘤的Car-T药物和1款治疗β地中海贫血的基因药物Zynteglo(Bluebird Bio)。目前基因药物价格昂贵，病毒载体的生产成本是关键因素。
重组慢病毒的产生方式是将慢病毒的基因组分成3个或4个部分，分别装到不同的质粒载体中，利用质粒载体转染贴壁或悬浮培养的HEK293细胞，包装重组慢病毒。目前基于安全性考虑，临床上普遍应用4个质粒载体的生产系统。如图1所示。
临床级慢病毒的生产，目前国际上仍然没有理想的解决方案，瞬时转染的方法无论是贴壁细胞还是悬浮细胞都需要大量的临床级质粒载体。质粒载体的生产需要单独的生产线，同时在法规和工艺等方面都面临着巨大的挑战。此外，贴壁细胞的培养很难工业化放大，且成本极高。悬浮细胞培养相比贴壁细胞培养更容易利用生物反应器进行放大，但质粒的转染效率会大大降低。为了避免上述问题，科学家们把目光转向诱导性系统。其中研发比较成熟的是四环素诱导体系，但四环素的添加和去除是临床级慢 病毒生产和纯化的难点，此方法目前仍然没有被大规模应用。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，本发明提供一种基于Cre重组酶诱导的大规模慢病毒基因药物制备系统及方法，其解决了质粒瞬时转染成本高，放大困难的问题；同时利用表达Cre基因的病毒感染进行诱导，避免四环素等抗生素诱导后难以去除的工艺难题。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
第一方面，本发明提供一种基于Cre重组酶诱导的大规模慢病毒基因药物制备方法，其包括：
S1:构建用于产慢病毒的悬浮稳转细胞株；该悬浮稳转细胞株的基因组上稳定整合了慢病毒包装蛋白表达序列和目的基因序列，且所述慢病毒包装蛋白表达序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列；loxP-Stop-loxP序列包含两个方向相同的loxP位点以及连接在两个loxP位点之间的Stop序列；
S2:构建表达Cre重组酶的病毒；
S3:大规模扩增、培养所述稳转细胞株；
在需要生产慢病毒颗粒时，利用该表达Cre基因的病毒感染所述悬浮稳转细胞株，利用该病毒表达的Cre重组酶切除loxP-Stop-loxP序列的Stop序列，以诱导慢病毒包装蛋白表达序列得以表达，从而包装表达目的基因的重组慢病毒颗粒，该重组慢病毒颗粒即为病毒基因药物。
其中，所述表达Cre基因的病毒为任何一种可感染悬浮293细胞且能够表达Cre蛋白的病毒。实际生产中，由于杆状病毒是复制型病毒，生产起来比较方便，因而可使用表达Cre基因的杆状病毒来实现。
根据本发明较佳实施例，步骤S1中，在构建悬浮稳转细胞株时，先分别构建表达gag-pol的质粒和表达VSV.G的质粒，该表达gag-pol的质 粒包含启动子和gag-pol基因序列，且在启动子和gag-pol基因序列之间插入loxP-Stop-loxP序列，该表达VSV.G的质粒包含启动子和VSV.G基因序列，且在启动子和VSV.G基因序列之间插入loxP-Stop-loxP序列；然后(通过PiggyBac载体)将表达gag-pol的质粒和表达VSV.G的质粒整合到该悬浮细胞基因组上。
根据本发明较佳实施例，步骤S1中，在构建稳转细胞株时，在目的基因序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列以构建目的基因质粒，再(通过PiggyBac载体)将该目的基因质粒整合到悬浮细胞株的基因组上。
根据本发明较佳实施例，步骤S1中，构建悬浮293稳转细胞株的步骤包括：S11、构建如下三种质粒：
gag-pol质粒：piggybac-CBA-loxp-stop-loxp-gag-pol、
VSV.G质粒：piggybac-CBA-loxp-stop-loxp-VSV.G和
目的质粒：piggybac-CBA-loxp-stop-loxp-Target Gene质粒；
S12、构建悬浮293稳转细胞株：将获得的上述三种质粒，通过转染进入悬浮239细胞，并使用含有抗生素的培养基进行筛选，获得抗性细胞，该抗性细胞中即包含用于产慢病毒的239悬浮稳转细胞。
根据本发明的较佳实施例，步骤S11中在构建所述质粒时，包括如下步骤：
①将piggybac-dual-promoter质粒用限制性内切酶SnaBI和HindIII进行双酶切，琼脂糖电泳后切胶回收piggybac-dual-promoter载体片段；
②设计用于无缝克隆的CBA(启动子)序列片段(序列如SEQ ID NO：1所示)、loxp-stop-loxp序列片段(序列如SEQ ID NO：2所示)、和gag-pol序列(序列如SEQ ID NO：4所示)或VSV.G(序列如SEQ ID NO：5所示)或Target Gene序列片段；
③然后将piggybac-dual-promoter载体片段、CBA序列片段及loxp-stop-loxp片段，分别与gag-pol或VSV.G或Target Gene序列片段采用无缝克隆试剂盒进行连接；
④将连接产物转化感受态trans1-t1，混匀、冰浴、热休克、立即再次冰浴，先用无抗生素培养液培养，再将菌液涂布至含有氨苄青霉素的平板培养基中继续培养；挑取单克隆菌落接种到含有氨苄青霉素的液体培养液中振荡培养，提取质粒，并用XbaI和HpaI双酶切鉴定后进行测序鉴定，获得目标质粒。
根据本发明较佳实施例，步骤S1中，所述Target Gene为密码子优化的COL7A1基因，其序列如SEQ ID NO：7所示。
根据本发明较佳实施例，S2中，所述表达Cre重组酶的病毒为杆状病毒，其构建方法为：
先将Cre基因表达序列(如SEQ ID NO：6所示)无缝克隆至pFast Bac1质粒(invitrogen，Cat.No.10359-016)中，构建构带有Cre表达盒的杆状病毒质粒pFast-Bac-Cre；然后将该pFast-Bac-Cre质粒转化DH10bac感受态细胞，获取Bacmid-Cre，再用Bacmid转染Sf9细胞，转染预定时间后，收集上清，获得表达Cre重组酶的杆状病毒Baculovirus-Cre。
根据本发明较佳实施例，S2中，pFast-Bac-Cre质粒的构建方法为：
(1)将pFast Bac1质粒用限制性内切酶SnaBI和HpaI于37℃±0.5进行双酶切50-70min，琼脂糖电泳后切胶回收pFast Bac1载体片段，将回收的pFast Bac1载体片段用碱性磷酸酶CIAP于50℃±2进行去磷处理50-70min，用65℃±2进行灭活处理10-25min；
(2)将碱性磷酸酶处理的pFast Bac1载体片段和含有Cre表达盒基因片段采用无缝克隆重组酶连接，50℃±2反应8-15min，得到无缝连接重组产物；
(3)取无缝连接重组产物转化感受态DH5a，混匀，冰浴、热休克，立刻再次冰浴，使用无抗生素的培养液振荡培养后，将菌液均匀涂布至含有氨苄青霉素的平板培养基上，37℃±0.5倒置培养12-18h；
(4)挑取单克隆菌落接种于含有氨苄青霉素的液体培养液中，37℃±0.5振荡12-18h；提取pFast-Bac-Cre质粒，进行SnaBI和HpaI双酶切鉴定后进行测序鉴定，构建得到带有Cre表达盒的杆状病毒质粒。
根据本发明较佳实施例，S2中，表达Cre重组酶的杆状病毒Baculovirus-Cre的制备方法为：
(1)将pFast-Bac-Cre质粒转化至DH10Bac感受态细胞：取pFast-Bac-Cre质粒转化感受态DH10Bac，轻轻混匀，冰浴25-35min；42℃±0.5热休克40-50s，立刻冰浴2-5min，加入无抗生素的LB培养液37℃±0.5振荡40-80min，用无菌玻璃涂布器将菌液均匀涂布至含有卡那霉素、庆大霉素、四环素、X-gal和IPTG的LB琼脂平板上，37℃±0.5倒置培养24-32h；
(2)挑取白色单克隆接种于含有卡那霉素、庆大霉素和四环素的液体培养液中，37℃±0.5振荡14-18h，用酚氯仿抽提法提取Bacmid-Cre，获取Bacmid-Cre的用PCR方法进行鉴定；
(3)用孔板细胞板，按照1x10^6细胞/孔，每孔用20±1μl Bacmid-Cre转染Sf9细胞，转染后6-8h换液，转染后96h收集上清，获得携带Cre表达盒的杆状病毒。
根据本发明较佳实施例，步骤S3包括：扩增培养悬浮293稳转株细胞系至预定密度，用Baculovirus-Cre按照MOI1感染悬浮293稳转株细胞系，感染72h后，收集细胞上清，层析纯化，得到表达目的基因的慢病毒。
第二方面，本发明提供一种基于Cre重组酶诱导的大规模慢病毒基因药物制备系统，其包括：
悬浮293稳转细胞株和表达Cre基因的病毒；
所述悬浮293稳转细胞株的基因组上整合了慢病毒包装蛋白表达序列和目的基因序列；且所述慢病毒包装蛋白表达序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列；loxP-Stop-loxP序列包含两个方向相同的loxP位点以及连接在两个loxP位点之间的Stop序列；
所述表达Cre基因的病毒为可感染悬浮293细胞且能够表达Cre蛋白的病毒；所述表达Cre基因的病毒用于感染所述悬浮293稳转细胞株，激活慢病毒包装蛋白表达序列进行表达，以包装产生慢病毒。
所述表达Cre基因的病毒可使用杆状病毒，杆状病毒为复制型病毒， 易于大量生产。
优选地，所述悬浮293稳转细胞株中，所述目的基因序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列。
优选地，所述目的基因为密码子优化的COL7A1基因，其序列如SEQ ID NO：7所示。此时，制备的慢病毒颗粒为治疗营养不良型大疱性表皮松解症的慢病毒基因药物。
(三)有益效果
本发明的技术效果主要为：
本发明利用Cre-loxP重组系统介导的诱导型慢病毒产生系统。摆脱质粒瞬时转染成本高，放大困难的问题。此外，本发明利用病毒感染进行诱导，避免四环素等抗生素诱导后难以去除的工艺难题。
优选在构建稳转细胞株时，在目的基因序列与驱动其表达的启动子之间同样也插入loxP-Stop-loxP序列以构建目的基因质粒，再将该目的基因质粒整合到悬浮细胞株的基因组上。可避免构建的悬浮稳转细胞株在扩增培养过程中持续大量表达外源蛋白给细胞带来的压力，抑制悬浮稳转细胞的增长和培养密度。
附图说明
图1为现有技术中重组慢病毒的产生方法示意图。
图2为采用携带Cre基因的杆状病毒感染悬浮293稳转株前后，用Western Blot检测悬浮293稳转株细胞裂解液中慢病毒包装蛋白gag-pol和VSV.G的表达水平的比对结果，其中-表示感染前，+表示感染后。
图3为使用携带Cre基因的杆状病毒感染悬浮293稳转株前后，从悬浮293稳转株细胞上清液中层析纯化慢病毒，以慢病毒感染靶细胞(239T)，用荧光显微镜观察靶细胞(239T)的荧光变化；其中Cre(-)表示感染前，Cre(+)表示感染后。
具体实施方式
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施 方式，对本发明作详细描述。
本发明的技术方案的主要思路和原理如下：
Cre重组酶(Cyclization Recombination Enzyme)是由大肠杆菌噬菌体P1的Cre基因所编码、由343个氨基酸组成的38kD的蛋白质。它不仅具有催化活性，而且与限制酶相似，能够特异性识别loxP位点。LoxP(locus ofX-overP1)位点长为34bp，包括两个13bp的反向重复序列和一个8bp的间隔区域。其中，反向重复序列是Cre重组酶的特异识别位点，而间隔区域决定了loxP位点的方向。当基因组内存在loxP位点时，一旦遇到Cre重组酶，Cre重组酶便会结合到loxP位点两端的反向重复序列区形成二聚体。此二聚体与其他loxP位点的二聚体结合，进而形成四聚体。随后，介于两个loxP位点之间的DNA被Cre重组酶切下，切口在DNA连接酶的作用下重新连接。
基于上述原理，本发明是将Stop序列置于两个方向相同的loxP位点之间组成loxP-Stop-loxP序列，组成一个可被Cre重组酶诱导激活的开关序列，然后将loxP-Stop-loxP序列置于慢病毒包装蛋白表达序列(gag-pol或VSV.G等)的前面(在驱动慢病毒包装蛋白表达序列(gag-pol或VSV.G等)表达的启动子后面)，由此得到一种可被Cre重组酶激活诱导的质粒结构，然后将这种质粒通过PiggyBac载体系统整合到悬浮293细胞基因组上。如此得到的悬浮293细胞在无Cre重组酶的情况下不表达慢病毒包装蛋白，避免慢病毒包装蛋白的细胞毒性抑制悬浮293细胞的生长，有利于悬浮293细胞的规模化扩增和培养。此外，构建表达Cre重组酶的病毒，以该表达Cre重组酶的病毒感染稳定整合的悬浮293细胞(即其基因组上整合了前置loxP-Stop-loxP序列的慢病毒包装蛋白表达序列)，利用Cre重组酶loxP-Stop-loxP序列中的Sotp序列，以激活连在loxP-Stop-loxP序列后面的慢病毒包装蛋白表达序列(gag-pol或VSV.G等)得以表达，包装出重组慢病毒。
现以目的基因为G2P(GFP-T2A-Puro)为例，对本发明方案、可实现性和技术效果验证进行说明。实施方法如下：
一、构建悬浮293稳转细胞株
在构建悬浮293稳转细胞株之前，需要先构建三种质粒，分别是目的基因质粒、慢病毒包装蛋白gag-pol质粒、慢病毒包装蛋白VSV.G质粒等三种质粒，然后以这三种质粒通过转染进入悬浮293细胞中，用抗生素筛选出整合到悬浮293细胞基因组的细胞系。具体方法和步骤如下：
(一)构建piggybac-CBA-loxp-stop-loxp-G2P质粒，其过程为：
(1)将piggybac-dual-promoter质粒(优宝生物，VT1663)用限制性内切酶SnaBI和HindIII于37℃进行双酶切30min，琼脂糖电泳后切胶回收piggybac-dual-promoter载体片段。
(2)设计用于无缝克隆的CBA序列片段(序列为SEQ ID NO：1),loxp-stop-loxp序列片段(序列为SEQ ID NO：2)和G2P(GFP-T2A-Puro)序列片段(序列为SEQ ID NO：3)。
(3)将胶回收试剂盒分别回收的piggybac-dual-promoter载体片段和CBA片段以及loxp-stop-loxp片段，采用CloneSmarter的无缝克隆试剂盒进行连接，10ul体系50℃反应15min。
(4)将5ul连接产物转化至大肠杆菌：连接产物转化感受态trans1-t1，轻轻混匀，冰浴20min；42℃热休克80s，立刻冰浴5min，加入无抗生素的LB培养液37℃振荡约60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(5)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用axygen质粒提取试剂盒提取piggybac-CBA-loxp-stop-loxp-G2P质粒，进行XbaI和HpaI(KspAI)双酶切鉴定后进行测序鉴定，获得piggybac-CBA-loxp-stop-loxp-G2P质粒。
(二)构建piggybac-CBA-loxp-stop-loxp-gag-pol质粒，其过程为：
(1)为了简便，取上述构建的piggybac-CBA-loxp-stop-loxp-G2P质粒用限制性内切酶HpaI(KspAI)和HindIII于37℃进行双酶切30min，琼脂糖电泳后切胶回收piggybac-CBA-loxp-stop-loxp载体片段。
(2)设计无缝克隆引物gag-pol-F
(GCTGGCGCGAGCTGGTCGACAAGCTgaattcgggccgcgtt)和gag-pol-R(Taaccattataagctgcaataaacaagttaactgagagacacaaaaaattcc)，将gag-pol片段(序列为SEQ ID NO：4)从psPAX2质粒载体(addgene plasmid#12260)进行PCR扩增，琼脂糖电泳后切胶回收gag-pol片段。
(3)将胶回收试剂盒分别回收的piggybac-CBA-loxp-stop-loxp-G2P载体片段和gag-pol片段，采用CloneSmarter的无缝克隆试剂盒进行连接，10ul体系50℃反应15min。
(4)将5ul连接产物转化至大肠杆菌：连接产物转化感受态trans1-t1，轻轻混匀，冰浴20min；42℃热休克80s，立刻冰浴5min，加入无抗生素的LB培养液37℃振荡约60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(5)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h。
(6)用axygen质粒提取试剂盒提取piggybac-CBA-loxp-stop-loxp-gag-pol质粒，进行NdeI和HpaI(KspAI)双酶切鉴定后进行测序鉴定，获得piggybac-CBA-loxp-stop-loxp-gag-pol质粒。
(三)构建piggybac-CBA-loxp-stop-loxp-VSV.G质粒，其过程为：
(1)为了简便，取上述构建的piggybac-CBA-loxp-stop-loxp-G2P质粒用限制性内切酶HpaI(KspAI)和HindIII于37℃进行双酶切30min，琼脂糖电泳后切胶回收piggybac-CBA-loxp-stop-loxp载体片段。
(2)设计无缝克隆引物VSV.G-F
(GCTGGCGCGAGCTGGTCGACAAGCTgaattctgacactatgaagtgcct)和VSV.G-R(aaccattataagctgcaataaacaagttaactaaaatacagcatagcaaaac)，将VSV.G片段(序列为SEQ ID NO：5)从pMD2.G质粒载体(addgene plasmid#12259)进行PCR扩增，琼脂糖电泳后切胶回收VSV.G片段。
(3)将胶回收试剂盒分别回收的piggybac-CBA-loxp-stop-loxp载体片段和VSV.G片段，采用CloneSmarter的无缝克隆试剂盒进行连接，10ul体系50℃反应15min。
(4)将5ul连接产物转化至大肠杆菌：连接产物转化感受态trans1-t1，轻轻混匀，冰浴20min；42℃热休克80s，立刻冰浴5min，加入无抗生素的LB培养液37℃振荡约60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(5)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h
(6)用axygen质粒提取试剂盒提取piggybac-CBA-loxp-stop-loxp-VSV.G质粒，进行XbaI和HpaI(KspAI)双酶切鉴定后进行测序鉴定，获得piggybac-CBA-loxp-stop-loxp-VSV.G质粒。
(二)将上述三个质粒，转染进入悬浮293细胞中，筛选符合要求的细胞株作为生产慢病毒的稳转株
稳定细胞系筛选是将质粒通过转染进入悬浮293细胞中，筛选出整合到悬浮293细胞基因组的Cell pool，具体步骤如下：
(1)将293细胞以2x10^6/孔铺一块35mm平皿，37℃培养1h；
(2)取piggybac-CBA-loxp-stop-loxp-G2P质粒,piggybac-CBA-loxp-stop-loxp-gag-pol质粒和piggybac-CBA-loxp-stop-loxp-VSV.G质粒各2μg用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，取8μl CellfectinII转染试剂用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，稀释的转染试剂加入稀释的质粒中，轻弹混匀，室温静置15min。
(3)将转染试剂质粒混合物加入293细胞中，于37℃培养6h后换成新鲜的培养基。
(4)转染后24h，293细胞换成含有8μg/ml Puromycin的培养基，继续于37℃培养，每三天换新鲜的8μg/ml Puromycin的培养基，直至细胞不再出现死亡，存活的细胞均为抗Puromycin的细胞，至此获得诱导型产慢病毒的悬浮293细胞系。
在本实施例中，没有对所述三个质粒转染成功逐一进行筛选，但抗Puromycin的细胞中必然包含已稳定整合了上述三个质粒的悬浮293细胞 株，因此筛选出的抗Puromycin的细胞可用作为产慢病毒的悬浮细胞(参见下文中的验证过程)。
在其他实施例和实际上生产中，可根据实际情况对三个质粒进行逐一转染和逐一筛选确认，以筛选出稳定整合了所述三个质粒的悬浮293细胞株。
二、构建表达Cre重组酶的杆状病毒
事实上，任何可感染293细胞且能够表达Cre重组酶的病毒都可以作为感染病毒。在实际上生产中考虑到成本和工艺性，选择复制型的杆状病毒作为表达Cre重组酶的载体。表达Cre重组酶的杆状病毒的构建过程包括pFast-Bac-Cre质粒的构建和重组获得表达Cre重组酶的杆状病毒。过程如下：
(一)构建pFast-Bac-Cre质粒
将合成的Cre基因表达序列(序列为SEQ ID NO：6)无缝克隆至pFast Bac1质粒(invitrogen，Cat.No.10359-016)中，得到pFast-Bac-Cre质粒；该克隆过程包括如下步骤：
(1)将pFast Bac1质粒用限制性内切酶SnaBI和HpaI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pFast Bac1载体片段，回收后的pFast Bac1载体片段用碱性磷酸酶CIAP于50℃进行去磷处理60min，用65℃进行灭活处理15min。
(2)将碱性磷酸酶处理的pFast Bac1载体片段和含有Cre表达盒基因片段，采用无缝克隆重组酶连接，50℃反应10min，得到无缝连接重组产物。
(3)取无缝连接重组产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(4)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pFast-Bac-Cre质粒，进行SnaBI和HpaI双酶切鉴定后进行测序鉴定，至此带有Cre表达盒的杆状病毒质粒构 建成功。
(二)重组获得表达Cre重组酶的杆状病毒
用pFast-Bac-Cre质粒转化DH10bac感受态细胞，获取Bacmid-Cre，随后用Bacmid转染Sf9细胞，转染预定时间后，收集上清，获得Baculovirus-Cre。具体过程包括如下步骤：
(1)将pFast-Bac-Cre质粒转化至DH10Bac感受态细胞：取pFast-Bac-Cre质粒转化感受态DH10Bac，轻轻混匀，冰浴30min；42℃热休克45s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有卡那霉素、庆大霉素、四环素、X-gal和IPTG的LB琼脂平板上，37℃倒置培养28h。
(2)挑取白色单克隆接种于含有卡那霉素、庆大霉素和四环素的LB液体培养液中，37℃振荡16h，用酚氯仿抽提法提取Bacmid-Cre，获取Bacmid-Cre的用PCR方法进行鉴定。
(3)按照2×10 6个细胞/孔，铺一块6孔板，用20μL Bacmid-Cre转染Sf9细胞，转染后8h换液，转染后96h收集上清，获得携带Cre表达盒的杆状病毒。
三、杆状病毒感染悬浮293细胞产慢病毒的诱导型系统的验证
验证包括两部分，一部分是杆状病毒感染悬浮293细胞后，细胞中慢病毒包装蛋白的表达验证；另一部分是杆状病毒感染悬浮293细胞后，上清液的层析纯化产生的慢病毒感染靶细胞后目的基因表达的验证。
(一)、利用携带Cre重组酶表达盒的杆状病毒感染稳定整合慢病毒包装系统的悬浮293细胞，Western Blot检测感染前后慢病毒包装组分的表达。结果如图2所示，感染前由于loxp-stop-loxp的存在gag-pol和VSV.G均无表达。感染后明显表达。过程包括如下步骤：
(1)按照2×10 6个细胞/孔，铺一块6孔板，Baculovirus-Cre按照MOI1000感染悬浮293细胞，感染后72h收集细胞裂解获得裂解液。
(2)配制10％SDS-PAGE胶，将裂解液用BCA法进行蛋白浓度测定，向6％SDS-PAGE胶中加入相同蛋白量的细胞裂解液，用80V的电压跑至 压缩胶与分离胶交界处，用120V电压跑至胶的底部。
(3)用400mA恒定电流将含有蛋白的SDS-PAGE胶转至NC膜上。
(4)将转有蛋白的NC膜用5％脱脂牛奶在室温孵育1h，用TBST清洗膜3次，每次5min。
(5)用gag-pol和VSV.G蛋白的一抗于4℃孵育过夜。
(6)第二天用TBST清洗膜3次，每次10min，用含有HRP的二抗室温孵育1h，用TBST清洗膜3次，每次5min。
(7)用ECL法进行显影，获得感染前后gag-pol和VSV.G的表达水平比较结果。结果如图2所示，在感染后可以清楚看到gag-pol和VSV.G的表达。
说明利用携带Cre重组酶表达盒的杆状病毒感染稳定整合慢病毒包装系统的悬浮293细胞，可以激活慢病毒包装蛋白gag-pol和VSV.G序列进行表达。
(二)、利用携带Cre重组酶表达盒的杆状病毒感染稳定整合慢病毒包装系统的悬浮293细胞，收集感染前后的悬浮293细胞的培养基上清，纯化重组慢病毒，感染靶细胞(293T细胞)。过程包括如下步骤：
(1)将整合gag-pol,VSV.G和GFP的悬浮293细胞系扩增至细胞密度达到1×10 7个/ml。
(2)用已经测好基因组滴度的Cre重组酶杆状病毒，以MOI1感染细胞系，感染后72h后收集细胞培养基上清。
(3)纯化采用GE的AKTA avant层析系统，采用DEAE层析，切向流过滤和core700层析纯化工艺，纯化得表达GFP的慢病毒。
(4)以MOI1为0.1感染靶细胞，进行流式分析和荧光显微镜观察。
用流式细胞仪检测GFP的阳性率，感染前无阳性细胞，感染后阳性率为46％。用荧光显微镜观察，结果如图3所示，Cre重组酶感染悬浮293细胞后，从上清收集、层析纯化的慢病毒感染靶细胞后，出现荧光，说明慢病毒中携带的目的基因G2P通过感染靶细胞，在靶细胞中得到表达。由此证明，本发明方法制备的重组慢病毒具有感染靶细胞并在靶细胞中表达 目的基因的能力。
上述实施例是以目的基因为G2P为例所进行的说明。在实际生产应用制备慢病毒基因药物时，根据治疗需求可以携带不同的目的基因，以达到特定的治疗目的。因而，在上述实施例的基础上，目的基因质粒可以用通式piggybac-CBA-loxp-stop-loxp-Target Gene来表示，其中，Target Gene可以是各种目的基因，例如Target Gene是COL7A1基因(序列为SEQ ID NO：7)，可以由此生产出治疗营养不良型大疱性表皮松解症的慢基因药物。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2021093905-appb-000001
Figure PCTCN2021093905-appb-000002
Figure PCTCN2021093905-appb-000003
Figure PCTCN2021093905-appb-000004
Figure PCTCN2021093905-appb-000005
Figure PCTCN2021093905-appb-000006
Figure PCTCN2021093905-appb-000007
Figure PCTCN2021093905-appb-000008
Figure PCTCN2021093905-appb-000009
Figure PCTCN2021093905-appb-000010
Figure PCTCN2021093905-appb-000011
Figure PCTCN2021093905-appb-000012
Figure PCTCN2021093905-appb-000013
Figure PCTCN2021093905-appb-000014
Figure PCTCN2021093905-appb-000015

Claims (10)

  1. 一种基于Cre重组酶诱导的大规模慢病毒基因药物制备系统，其特征在于，其包括：悬浮293稳转细胞株和表达Cre基因的病毒；
    所述悬浮293稳转细胞株的基因组上整合了慢病毒包装蛋白表达序列和目的基因序列；且所述慢病毒包装蛋白表达序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列；loxP-Stop-loxP序列包含两个方向相同的loxP位点以及连接在两个loxP位点之间的Stop序列；
    所述表达Cre基因的病毒为可感染悬浮293细胞且能够表达Cre蛋白的病毒；
    所述表达Cre基因的病毒用于感染所述悬浮293稳转细胞株，激活慢病毒包装蛋白表达序列进行表达，以包装产生慢病毒。
  2. 根据权利要求1所述的大规模慢病毒基因药物制备系统，其特征在于，所述悬浮293稳转细胞株中，所述目的基因序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列。
  3. 一种基于Cre重组酶诱导的大规模慢病毒基因药物制备方法，其特征在于，其包括：
    S1:构建用于产慢病毒的悬浮稳转细胞株；该稳转细胞株的基因组上稳定整合了慢病毒包装蛋白表达序列和目的基因序列，且所述慢病毒包装蛋白表达序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列；loxP-Stop-loxP序列包含两个方向相同的loxP位点以及连接在两个loxP位点之间的Stop序列；
    S2:构建表达Cre重组酶的病毒；
    S3:大规模扩增、培养所述稳转细胞株；
    在需要生产慢病毒颗粒时，利用该表达Cre基因的病毒感染所述悬浮稳转细胞株，利用该病毒表达的Cre重组酶切除loxP-Stop-loxP序列的Stop序列，以诱导慢病毒包装蛋白表达序列得以表达，从而包装表达目的基因的重组慢病毒颗粒，该重组慢病毒颗粒即为病毒基因药物。
  4. 根据权利要求3所述的制备方法，其特征在于，步骤S1中，在 构建稳转细胞株时，在目的基因序列与驱动其表达的启动子之间插入loxP-Stop-loxP序列以构建目的基因质粒，再将该质粒整合到该宿主细胞基因组上。
  5. 根据权利要求3所述的制备方法，其特征在于，步骤S1中，构建悬浮293稳转细胞株的步骤包括：S11、构建如下三种质粒：
    gag-pol质粒：piggybac-CBA-loxp-stop-loxp-gag-pol、
    VSV.G质粒：piggybac-CBA-loxp-stop-loxp-VSV.G和
    目的质粒：piggybac-CBA-loxp-stop-loxp-Target Gene质粒；
    S12、构建悬浮293稳转细胞株：将获得的上述三种质粒，通过转染进入悬浮239细胞，并使用含有抗生素的培养基进行筛选，获得抗性细胞，该抗性细胞中即包含用于产慢病毒的239悬浮稳转细胞。
  6. 根据权利要求5所述的制备方法，其特征在于，步骤S1中，所述Target Gene为密码子优化的COL7A1基因，其序列如SEQ ID NO：7所示。
  7. 根据权利要求3所述的制备方法，其特征在于，步骤S2中，所述表达Cre重组酶的病毒为杆状病毒，其构建方法为：
    先将Cre基因表达序列无缝克隆至pFast Bac1质粒中，构建构带有Cre表达盒的杆状病毒质粒pFast-Bac-Cre；然后，将该pFast-Bac-Cre质粒转化DH10bac感受态细胞，获取Bacmid-Cre，再用Bacmid转染Sf9细胞，转染预定时间后，收集上清，获得表达Cre重组酶的杆状病毒Baculovirus-Cre。
  8. 根据权利要求7所述的制备方法，其特征在于，S2中，pFast-Bac-Cre质粒的构建方法为：
    (1)将pFast Bac1质粒用限制性内切酶SnaBI和HpaI于37℃±0.5进行双酶切50-70min，琼脂糖电泳后切胶回收pFast Bac1载体片段，将回收的pFast Bac1载体片段用碱性磷酸酶CIAP于50℃±2进行去磷处理50-70min，用65℃±2进行灭活处理10-25min；
    (2)将碱性磷酸酶处理的pFast Bac1载体片段和含有Cre表达盒基因片段采用无缝克隆重组酶连接，50℃±2反应8-15min，得到无缝连接重组产物；
    (3)取无缝连接重组产物转化感受态DH5a，混匀，冰浴、热休克，立刻再次冰浴，使用无抗生素的培养液振荡培养后，将菌液均匀涂布至含有氨苄青霉素的平板培养基上，37℃±0.5倒置培养12-18h；
    (4)挑取单克隆菌落接种于含有氨苄青霉素的液体培养液中，37℃±0.5振荡12-18h；提取pFast-Bac-Cre质粒，进行SnaBI和HpaI双酶切鉴定后进行测序鉴定，构建得到带有Cre表达盒的杆状病毒质粒。
  9. 根据权利要求7所述的制备方法，其特征在于，S2中，表达Cre重组酶的杆状病毒Baculovirus-Cre的制备方法为：
    (1)将pFast-Bac-Cre质粒转化至DH10Bac感受态细胞：取pFast-Bac-Cre质粒转化感受态DH10Bac，轻轻混匀，冰浴25-35min；42℃±0.5热休克40-50s，立刻冰浴2-5min，加入无抗生素的LB培养液37℃±0.5振荡40-80min，用无菌玻璃涂布器将菌液均匀涂布至含有卡那霉素、庆大霉素、四环素、X-gal和IPTG的LB琼脂平板上，37℃±0.5倒置培养24-32h；
    (2)挑取白色单克隆接种于含有卡那霉素、庆大霉素和四环素的液体培养液中，37℃±0.5振荡14-18h，用酚氯仿抽提法提取Bacmid-Cre，获取Bacmid-Cre的用PCR方法进行鉴定；
    (3)用孔板细胞板，按照1x10^6细胞/孔，每孔用20±1μl Bacmid-Cre转染Sf9细胞，转染后6-8h换液，转染后96h收集上清，获得携带Cre表达盒的杆状病毒。
  10. 根据权利要求5所述的制备方法，其特征在于，步骤S3包括：扩增培养悬浮293稳转株细胞系至预定密度，用Baculovirus-Cre按照MOI1感染悬浮293稳转株细胞系，感染72h后，收集细胞上清，层析、纯化，得到表达目的基因的慢病毒。
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