WO2017101243A1 - 慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法 - Google Patents

慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法 Download PDF

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WO2017101243A1
WO2017101243A1 PCT/CN2016/080421 CN2016080421W WO2017101243A1 WO 2017101243 A1 WO2017101243 A1 WO 2017101243A1 CN 2016080421 W CN2016080421 W CN 2016080421W WO 2017101243 A1 WO2017101243 A1 WO 2017101243A1
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casp9
gene
dual
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sequence
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杨世成
毛侃琅
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深圳精准医疗科技有限公司
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    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the field of biotechnology, in particular to a lentiviral expression vector, a preparation method and application thereof, and a preparation method of the recombinant lentivirus.
  • Gene expression is the process by which a cell converts its genetic information stored in a DNA sequence into a biologically active protein molecule during its life. Through genetic engineering, the expression of foreign genes in biological cells and even organisms can be realized, thereby achieving the purpose of artificially regulating cells or physical objects.
  • the introduction of foreign genes in eukaryotic cells, especially mammalian cells, is mainly accomplished by physical, chemical or biological methods.
  • Physical methods mainly include DNA microinjection, electroporation and metal particle bombardment; chemical methods mainly include liposome-mediated and receptor-mediated methods; biological methods are mainly implemented by various viruses, such as Adenovirus, adeno-associated virus, retrovirus, lentivirus, etc.
  • Physical methods and chemical methods can load a large number of foreign gene fragments, and some methods can be used to insert foreign genes into the genome of cells or organisms, but in general, the efficiency of exogenous gene expression is achieved by these two methods.
  • lentiviruses are favored by the scientific community for their advantages of being able to infect non-dividing cells and accommodating exogenous gene fragments.
  • lentivirus applications are very extensive, both in the laboratory and in the industry. As the field of application continues to expand, so does the demand for lentiviruses. At present, in scientific research and practical application, the need to introduce two genes at one time and achieve high expression through lentivirus is also increasing. For example, the top ten scientific breakthroughs selected by Science magazine in 2013 - cancer immunotherapy , including TCR-T And other technologies. Because TCR-T technology can express specific receptors to target specific cells such as tumor cells, it has received extensive attention and research, and has changed from the initial basic immunological research to the current clinical application.
  • TCR-T technology requires the simultaneous introduction of DNA sequences corresponding to the two subunits of the T cell receptor (TCR) into T cells, the need for rapid introduction of the two genes is urgent, and the existing lentiviruses are urgent.
  • the carrier is not yet well suited to this need.
  • the load capacity of the virus foreign gene is generally about 8000 bp to 10000 bp, and the size of the starting element is generally about 500 bp to 700 bp. It can be seen that the ratio of a single promoter element to the lentiviral load capacity is close to 10%, so the introduction of a single promoter element means that the foreign gene sequence introduced by the lentivirus means a reduction in the capacity of the foreign gene to be introduced. Therefore, lentiviral double gene expression using both methods is not particularly desirable. There are no lentiviral vectors in the prior art that can solve the above problems.
  • a lentiviral expression vector is pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE vector, and the pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE vector is pRRLSIN.cPPT.MSCV/GFP.
  • the GFP sequence of the WPRE vector was replaced with a double gene expression cassette Dual-Casp9 with a suicide switch;
  • the double-gene expression cassette Dual-Casp9 with a suicide switch includes the following structures arranged in order from the 5' to the 3' end: a first multiple cloning site - a first furin cleavage site - a V5 tag - a first Spacer a first 2A peptide-Casp9-second multiple cloning site, wherein "-" represents a linkage;
  • the Casp9 includes the following structures arranged in order from the 5' to the 3' end: iCasp9 - second furin cleavage site - second Spacer - second 2A peptide.
  • sequence of the iCasp9 is set forth in SEQ ID No. 1.
  • sequence of the double gene expression cassette Dual-Casp9 with a suicide switch is set forth in SEQ ID No. 2.
  • sequence of the first multiple cloning site is as set forth in SEQ ID No. 3;
  • the sequence of the second multiple cloning site is shown in SEQ ID No. 4.
  • the sequence of the first furin cleavage site and the second furin cleavage site are both shown in SEQ ID No. 5;
  • V5 tag The sequence of the V5 tag is as shown in SEQ ID No. 6;
  • the sequence of the first Spacer and the second Spacer is as shown in SEQ ID No. 7.
  • the sequence of the first 2A peptide and the second 2A peptide is selected from the group consisting of the sequence shown in SEQ ID No. 8, the sequence shown in SEQ ID No. 9, the sequence shown in SEQ ID No. 10, and the SEQ ID No. In one of the sequences shown in .11, the sequences of the first 2A peptide and the second 2A peptide are different from each other.
  • the first multiple cloning site is inserted with a first gene of interest, and the second multiple cloning site is inserted with a second gene of interest;
  • the first gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452619
  • the second gene of interest is mouse TCR ⁇
  • the NCBI number of the mouse TCR ⁇ gene is DQ452620.
  • a method for preparing the above lentiviral expression vector comprising the steps of:
  • the double gene expression cassette Dual-Casp9 with suicide switch was designed, and the double gene expression cassette Dual-Casp9 with suicide switch was entrusted to obtain E. coli bacterial solution containing pUC-Dual-Casp9 plasmid, the pUC-
  • the Dual-Casp9 plasmid contains the double gene expression cassette Dual-Casp9 with a suicide switch
  • the double gene expression cassette Dual-Casp9 with a suicide switch includes the following structures arranged in order from the 5' to the 3' end: a multiple cloning site - a first furin cleavage site - V5 tag - a first Spacer - first 2A peptide - Casp9 - a second multiple cloning site, wherein "-" represents a linkage, and the Casp9 comprises from 5 'The following structures are arranged in order from the 3' end: iCasp9 - second furin cleavage site -
  • the Escherichia coli liquid containing the pUC-Dual-Casp9 plasmid is mixed with the selective LB liquid medium, and cultured at 300 rpm for 12 to 16 hours to an OD 600 of 0.6 to 0.8 at 37 ° C in a constant temperature shaker, and the obtained bacterial solution is centrifuged. Thereafter, the first precipitate is retained, and the pUC-Dual-Casp9 plasmid is extracted after cleavage of the first precipitate;
  • the pUC-Dual-Casp9 plasmid was treated with Asc I endonuclease and Sal I endonuclease at 37 ° C, and fully recovered and recovered to obtain a fragment of the double gene expression cassette Dual-Casp9 with a suicide switch;
  • the pRRLSIN.cPPT.MSCV/GFP.WPRE vector was treated with Asc I endonuclease and Sal I endonuclease at 37 ° C, and the GFP sequence of the pRRLSIN.cPPT.MSCV/GFP.WPRE vector was excised and fully recovered. , obtaining a linearized pRRLSIN.cPPT.MSCV/GFP.WPRE vector;
  • the fragment of the double gene expression cassette Dual-Casp9 with a suicide switch was mixed with the linearized pRRLSIN.cPPT.MSCV/GFP.WPRE vector at a molar ratio of 3 to 10:1, and then T4 DNA ligase was added, 4 Ligation overnight at °C to obtain a ligation product containing the ligation plasmid;
  • the ligation product containing the ligation plasmid was transformed into competent Top10 Escherichia coli and uniformly coated on a selective LB plate, inverted culture at 37 ° C for 12 h to 16 h, and positive colonies were picked and placed in the selective LB liquid medium.
  • the culture was carried out at 300 rpm for 12 h to 16 h to an OD 600 of 0.6 to 0.8, and the obtained bacterial liquid was centrifuged to retain a second precipitate, and the second precipitate was lysed and the ligated plasmid was extracted.
  • the ligation plasmid is the lentiviral expression vector.
  • the first gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452619
  • the second gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452620.
  • a lentiviral expression kit comprising the lentiviral expression vector described above.
  • a method for preparing a recombinant lentivirus comprising the following steps:
  • the lentiviral expression vector carrying the gene of interest, pMDLg/pRRE vector, pRSV-Rev vector and pMD-G vector were mixed at a molar ratio of 2:1:1:1, and then transfected into 293FT cells, after transfection. The cells were replaced with complete medium for 4h-6h. After 48h, the culture solution was collected. After centrifugation, the supernatant was retained and the supernatant was filtered with a 0.45 ⁇ m filter to retain the filtrate. The filtrate was a solution of recombinant lentivirus.
  • the method further comprises the step of detecting the titer of the solution of the recombinant lentivirus after obtaining the solution of the recombinant lentivirus, specifically:
  • the dilution ratio of the solution of the recombinant lentivirus was 1 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 , and the recombination was slow with the medium.
  • the solution of the virus was serially diluted, and then 100 ⁇ L of the gradient-diluted solution of the recombinant lentivirus was separately mixed with the cell culture medium in a 100 ⁇ L multiwell plate in different wells of the multiwell plate, and the medium was aspirated 24 h after the start of transfection.
  • the medium in each well of the multiwell plate was aspirated, and the cells were digested by adding 500 ⁇ L of trypsin-EDTA solution, and reacted at 37 ° C for 1 minute, followed by adding the medium to terminate the digestion reaction and purging the cells.
  • the cells in each well are collected by centrifugation, the total RNA of each well is extracted, and then the total cDNA of each well is obtained by reverse transcription;
  • the total cDNA of the obtained cells per well was subjected to real-time quantitative PCR to obtain the Ct value of each well cell, and the experimental group with the smallest difference from the control group Cc value but exceeding 2 was selected, and the dilution factor was obtained, and the calculation was performed according to the following formula.
  • Virus titer The total cDNA of the obtained cells per well was subjected to real-time quantitative PCR to obtain the Ct value of each well cell, and the experimental group with the smallest difference from the control group Cc value but exceeding 2 was selected, and the dilution factor was obtained, and the calculation was performed according to the following formula.
  • Virus titer The total cDNA of the obtained cells per well was subjected to real-time quantitative PCR to obtain the Ct value of each well cell, and the experimental group with the smallest difference from the control group Cc value but exceeding 2 was selected, and the dilution factor was obtained, and the calculation was performed according to the following formula.
  • Virus titer The total
  • T R ⁇ 20 ⁇ 10 3 , wherein T is a lentivirus titer, T is in units of TU/mL, and R is a dilution factor.
  • This lentiviral expression vector utilizes the 2A peptide to achieve the expression of a double foreign gene in mammalian cells.
  • the 2A peptide has a "self-shearing" property.
  • the 2A peptide changes the activity of the ribosome and promotes Hydrolysis of the ester chain between the 2A peptide residue Gly and tRNA Gly , releasing the upstream polypeptide from the transcription complex while initiating translation of the downstream polypeptide, and achieving double gene expression at the translational level.
  • the 2A peptide can almost completely cut. Therefore, this lentiviral expression vector can theoretically ensure the equal expression of the double foreign gene in mammalian cells, compared to the traditional lentiviral vector for double gene expression, the lentiviral expression vector Gene expression is better.
  • Figure 1 is a schematic view showing the structure of a lentiviral expression vector of an embodiment
  • Figure 2 is a flow chart showing the preparation method of the lentiviral expression vector shown in Figure 1;
  • Figure 3 is a graph showing the results of flow cytometry in Example 6 for detecting the expression of TCR on the surface of CD4+ T lymphocytes;
  • Figure 4 is a graph showing the results of flow cytometry in Example 6 for detecting the expression of TCR on the surface of CD8+ T lymphocytes;
  • Fig. 5 is a graph showing the results of detecting apoptosis induced by flow cytometry in Example 7.
  • lentiviral expression vector The lentiviral expression vector, the preparation method and application thereof, and the preparation method of the recombinant lentivirus are further described in detail below with reference to the accompanying drawings and specific examples.
  • the pharmaceuticals and reagents not described in the present invention are commercial products in the field (purchased from Bioengineering (Shanghai) Co., Ltd., Bao Bioengineering (Dalian) Co., Ltd., QIAGEN, Germany, Life Technologies, USA, GIBCO, etc.
  • the unexplained operation adopts the conventional methods in the art, and the specific steps can be found in: "Molecular Cloning: A Laboratory Manual” (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
  • the lentiviral expression vector of one embodiment as shown in Figure 1 is the pRRLSIN.cPPT.MSCV-Dual.WPRE vector.
  • the pRRLSIN.cPPT.MSCV-Dual.WPRE vector was obtained by replacing the GFP sequence of the pRRLSIN.cPPT.MSCV/GFP.WPRE vector with the double gene expression cassette Dual-Casp9 with a suicide switch.
  • the pRRLSIN.cPPT.MSCV/GFP.WPRE vector is from a commercial plasmid purchased from addgene.
  • the pGRLSIN.cPPT.PGK/GFP.WPRE was modified, and the PGK promoter of the plasmid pRRLSIN.cPPT.PGK/GFP.WPRE was replaced with the MSCV promoter.
  • the replacement of the GFP sequence has the following advantages: 1.
  • the replacement GFP can accommodate a larger foreign gene (the GFP gene itself has a sequence of more than 700 bp); 2.
  • the GFP is located just after the MSCV promoter, and replacing it can improve the foreign source. The level of expression of the gene.
  • the GFP sequence of the pRRLSIN.cPPT.MSCV/GFP.WPRE vector can be excised with Asc I endonuclease and Sal I endonuclease, and then subjected to the same treatment of the double gene expression cassette with suicide switch using T4 DNA ligase Dual The fragment of Casp9 was ligated with the pRRLSIN.cPPT.MSCV/GFP.WPRE vector excluding the GFP sequence to construct the above-mentioned pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE vector.
  • the dual-gene expression cassette Dual-Casp9 with a suicide switch includes the following structures arranged in order from the 5' to the 3' end: the first multiple cloning site - the first furin cleavage site - the V5 tag - the first Spacer - A 2A peptide-Casp9-second multiple cloning site, wherein "-" represents a linkage.
  • the suicide switch Casp9 can achieve artificially induced apoptosis, and Casp9 includes the following structures arranged in order from the 5' to the 3' end: iCasp9 - second furin cleavage site - second Spacer - second 2A peptide.
  • iCasp9 includes a FK506 binding protein 12 (FKBP12) with a F36V variant and a modified Caspase-9 protein.
  • FKBP12 FK506 binding protein 12
  • the wild-type FKBP12 has a low affinity with the compound AP1903 or AP20187, and the FKBP12 with the F36V mutation can bind well to the two compounds, thereby realizing the dimerization of the iCasp-9 element, thereby activating the Caspase-9 therein. The process of initiating apoptosis.
  • sequence of iCasp9 is as shown in SEQ ID No. 1.
  • This lentiviral expression vector utilizes the 2A peptide to achieve the expression of a double foreign gene in mammalian cells.
  • the 2A peptide has a "self-shearing" property.
  • the 2A peptide changes the activity of the ribosome and promotes Hydrolysis of the ester chain between the 2A peptide residue Gly and tRNA Gly , releasing the upstream polypeptide from the transcription complex while initiating translation of the downstream polypeptide, and achieving double gene expression at the translational level.
  • the 2A peptide can almost completely cut. Therefore, this lentiviral expression vector can theoretically ensure the equal expression of the double foreign gene in mammalian cells, compared to the traditional lentiviral vector for double gene expression, the lentiviral expression vector Gene expression is better.
  • the sequence of the double gene expression cassette Dual-Casp9 with a suicide switch is shown in SEQ ID No. 2.
  • the first multiple cloning site includes the following structures arranged in order from the 5' to the 3' end: an Asc I cleavage site, a BstBI cleavage site, a Bam HI cleavage site, and an Age I cleavage site.
  • the second multiple cloning site includes the following structures arranged in sequence from the 5' to the 3' end: Xho I cleavage site, Spe I cleavage site, Xma I cleavage site, and Sal I cleavage site.
  • the sequence of the first multiple cloning site is as shown in SEQ ID No. 3.
  • sequence of the second multiple cloning site is as shown in SEQ ID No. 4.
  • the sequence of the first furin cleavage site and the second furin cleavage site is as shown in SEQ ID No. 5.
  • sequence of the V5 tag is as shown in SEQ ID No. 6.
  • sequence of the first Spacer and the second Spacer is as shown in SEQ ID No. 7.
  • the sequences of the first 2A peptide and the second 2A peptide are selected from the group consisting of the sequence shown in SEQ ID No. 8, the sequence shown in SEQ ID No. 9, the sequence shown in SEQ ID No. 10, and the SEQ ID No. In one of the sequences shown by 11, the sequences of the first 2A peptide and the second 2A peptide are different from each other.
  • sequences of the first 2A peptide and the second 2A peptide are different from each other, and homologous recombination can be avoided.
  • the first cloning site is inserted with the first gene of interest, and the second cloning site is inserted with the second gene of interest.
  • the first gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452619
  • the second gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452620.
  • the present invention also provides a lentiviral expression kit comprising the lentiviral expression vector described above.
  • the lentiviral expression kit is convenient and efficient, can achieve high-efficiency expression of double foreign genes of mammalian cells, and can accommodate more gene fragments, and can be used as a powerful tool.
  • Cellular immunotherapy is an emerging anti-tumor therapy with remarkable curative effect, which makes up for the drawbacks of traditional surgery, radiotherapy and chemotherapy. It has been recognized as the most promising in the comprehensive treatment model of tumor, and it is the only hope to completely eliminate the tumor. The means of treatment.
  • CTL cytotoxic T cells
  • the method for preparing the above lentiviral expression vector shown in FIG. 2 comprises the following steps:
  • the pUC-Dual-Casp9 plasmid contains a double gene expression cassette Dual-Casp9 with a suicide switch, and the double-gene expression cassette Dual-Casp9 with a suicide switch includes the following structures arranged in order from the 5' to the 3' end: the first Cloning site - first furin cleavage site - V5 tag - first Spacer - first 2A peptide - Casp9 - second multiple cloning site, where "-" represents the linkage and Casp9 comprises from 5' to 3'
  • the following structures are arranged in sequence: iCasp9-second furin cleavage site-second Spacer-second 2A peptide
  • the selective LB liquid medium was an LB liquid medium containing 100 ⁇ g/mL ampicillin.
  • the pRRLSIN.cPPT.MSCV/GFP.WPRE vector was treated with Asc I endonuclease and Sal I endonuclease at 37 ° C, and pRRLSIN.cPPT.MSCV/GFP.WPRE was carried.
  • the GFP sequence of the body was excised and recovered after sufficient reaction to obtain a linearized pRRLSIN.cPPT.MSCV/GFP.WPRE vector.
  • the pRRLSIN.cPPT.MSCV/GFP.WPRE vector was engineered from the commercialized pRRLSIN.cPPT.PGK/GFP.WPRE purchased from addgene, and the PGK promoter was replaced with the MSCV promoter.
  • the preparation method of the lentiviral expression vector further comprises, after obtaining a fragment of the double gene expression cassette Dual-Casp9 with a suicide switch, in the first multiple position of the fragment of the double gene expression cassette Dual-Casp9 with a suicide switch.
  • the insertion of the first gene of interest into the second cloning site of the fragment of the dual gene expression cassette Dual-Casp9 with a suicide switch is inserted into the second gene of interest.
  • the first gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452619
  • the second gene of interest is a mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452620.
  • a method for preparing a recombinant lentivirus comprising the steps of:
  • a second cloning site of the fragment is inserted into the second gene of interest to obtain a lentiviral expression vector carrying the gene of interest; a lentiviral expression vector carrying the gene of interest, pMDLg/pRRE vector, pRSV-Rev vector and pMD-G
  • the vector was mixed at a molar ratio of 2:1:1:1 and transfected into 293FT cells. After transfection, the cells were replaced with complete medium at 4h-6h. After 48h, the culture medium was collected. After centrifugation, the supernatant was retained and the supernatant was used. The 0.45 ⁇ m filter was filtered, and the filtrate was retained as a solution of recombinant lentivirus.
  • the first gene of interest is the mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452619
  • the second gene of interest is the mouse TCR ⁇ gene
  • the NCBI number of the mouse TCR ⁇ gene is DQ452620.
  • the sequencing primers are: MCS I upstream primer and MCS I Downstream primers, after sequencing is completed, select the vector whose sequencing result is exactly the same as expected for the next step.
  • Dual-Casp9 with a suicide switch requires sequencing of the primers: MCS II upstream primer and MCS II Downstream primers, after sequencing is completed, select the vector whose sequencing result is exactly the same as expected for the next step.
  • the sequence of the MCS I upstream primer is shown in SEQ ID No. 12.
  • the sequence of the MCS I downstream primer is shown in SEQ ID No. 13.
  • the sequence of the MCS II upstream primer is shown in SEQ ID No. 14.
  • the sequence of the MCS II downstream primer is shown in SEQ ID No. 15.
  • the pMDLg/pRRE vector, the pRSV-Rev vector and the pMD-G vector are all conventional lentiviral assembly vectors.
  • the preparation method of the recombinant lentivirus further comprises the step of detecting the titer of the recombinant lentivirus solution after obtaining the solution of the recombinant lentivirus, specifically:
  • the dilution ratio of the recombinant lentivirus solution was 1 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 , and the solution gradient of the recombinant lentivirus was used.
  • 100 ⁇ L of the gradient diluted recombinant lentivirus solution and 100 ⁇ L of the multi-well plate were mixed and transfected in different wells of the multi-well plate.
  • the medium was aspirated and replaced with 500 ⁇ L containing 5 U.
  • the fresh medium of DNaseI was cultured at 37 ° C for 30 min to remove residual plasmid DNA which may adhere to the cell surface, and then the medium was changed to 1 mL of normal medium, and the culture was continued for 48 hours;
  • the medium in each well of the multiwell plate was aspirated, and 500 ⁇ L of trypsin-EDTA solution was added.
  • the cells were reacted at 37 ° C for 1 minute, then the medium was added to terminate the digestion reaction and the cells were purged.
  • the cells of each well were collected by centrifugation, and the total RNA of each well was extracted, followed by reverse transcription to obtain the total amount of cells per well.
  • the MCS I upstream primer and the MCS II downstream primer were respectively used as upstream and downstream primers, and were loaded according to the following table, and then subjected to real-time PCR to obtain the Ct value of the fluorescence quantitative PCR reaction of each well.
  • the experimental group with the smallest difference from the control group but exceeding 2 was selected, and the dilution factor was obtained, and the lentivirus titer was calculated according to the following formula:
  • T R ⁇ 20 ⁇ 10 3 , wherein T is a lentivirus titer, T is in units of TU/mL, and R is a dilution factor.
  • the plasmid used in the specific examples was modified with pRRLSIN.cPPT.MSCV, and GFP.WPRE and 293FT cells were commercially available, and the reagents used were all commercially available.
  • the core of the double gene expression cassette is the expression of the first 2A peptide (SEQ ID No. 8), having the first multiple cloning site (SEQ ID No. 3), the first 2A peptide (SEQ ID No. 8) and the second The multiple cloning site (SEQ ID No. 4) can be used to achieve equal expression of the double foreign gene.
  • the expression level of the foreign gene obtained by this method is not high, and 13 amino acid residues derived from the first 2A peptide are added to the end of the polypeptide sequence upstream of the first 2A peptide, which may have characteristics of the polypeptide. Have a certain impact.
  • first Spacer SEQ ID No. 7
  • a first furin cleavage site (SEQ ID No. 5) is inserted between the first multiple cloning site of the double gene expression cassette obtained in the previous step and the first Spacer, and the first 2A peptide can be upstream. After the polypeptide is cleaved by furin after expression, only 4 amino acid residues remain at the end. Unexpectedly, the expression level of foreign genes has been further improved.
  • insertion of the V5 tag (SEQ ID No. 6) between the first furin cleavage site and the first Spacer of the double gene expression cassette obtained in the previous step can again increase the expression level of the foreign gene, achieving double The most efficient isobaric expression of foreign genes.
  • Dual-Casp9 a double-gene expression cassette with a suicide switch
  • the protocol of Dual-Casp9 was commissioned by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the resulting sequence was contained in the pUC-Dual-Casp9 vector in E. coli.
  • the Escherichia coli was expanded and extracted, and the pUC-Dual-Casp9 vector was extracted, and then the purity and concentration were measured. The results are shown in the following table.
  • the pUC-Dual-Casp9 vector and the pRRLSIN.cPPT.MSCV/GFP.WPRE vector were digested with Asc I and SalI, respectively, and the small fragments of the pUC-Dual-Casp9 vector digested product and pRRLSIN were recovered after electrophoresis.
  • Example 3 Construction of a vector expressing mouse targeting melanoma-associated antigen gp100 (154-162) TCR ⁇ and TCR ⁇
  • E. coli strains Two E. coli strains were obtained by synthesis, which contained pUC57-TCR ⁇ and pUC57-TCR ⁇ vectors, respectively. These two strains of Escherichia coli were expanded and extracted, and the pUC57-TCR ⁇ and pUC57-TCR ⁇ vectors were separately extracted, and the purity and concentration were measured. The results are shown in the following table.
  • the pUC57-TCR ⁇ vector was treated with Asc I and Age I enzyme, and the pUC57-TCR ⁇ vector was treated with Xho I and Sal I enzymes. After electrophoresis, the small fragments TCR ⁇ and TCR ⁇ were recovered.
  • the pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE vector obtained in the previous step was double-digested with Asc I and Age I enzyme, and the vector was recovered after electrophoresis, and then ligated with TCR DNA by T4 DNA ligase to obtain pRRLSIN. .cPPT.MSCV-Dual-Casp9.WPRE-TCR ⁇ vector. Sequencing was performed using the MCS I upstream primer (SEQ ID No. 12) and the MCS I downstream primer (SEQ ID No. 13), and the results were completely in agreement with expectations.
  • the pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCR ⁇ vector was digested with Xho I and Sal I enzymes, and the vector was recovered after electrophoresis, and then ligated with TCR DNA by T4 DNA ligase to obtain pRRLSIN.cPPT. .MSCV-Dual-Casp9.WPRE-TCR ⁇ -TCR ⁇ vector.
  • MCS The II upstream primer (SEQ ID No. 14) and the MCSII downstream primer (SEQ ID No. 15) were sequenced and the results were exactly as expected.
  • the 293FT cells were cultured, and the cells with good growth state were inoculated into a 10 cm culture dish, and each dish was inoculated with 5 ⁇ 10 6 cells, and the cells were cultured in DMEM-free medium for about 18 hours until the fusion degree reached 80-90%.
  • 293FT cells were cultured, the 293FT take grew well were seeded into 24-well plates, each well inoculated with 2 ⁇ 10 5 cells was added 500 ⁇ L medium, 37 °C, 5% CO 2 overnight.
  • the virus stock solution the dilution ratio of the medium was 10 0 - 10 8 , respectively, 100 ⁇ L of each of the slow-toxic gradient dilutions were prepared, and then 100 ⁇ L of the original culture medium of each well was taken, and 100 ⁇ L of the lentivirus dilution solution was added to start the transfer. dye.
  • the lentivirus-containing medium was aspirated and replaced with 500 ⁇ L of fresh medium containing 5UDNaseI, and cultured at 37 ° C for 30 min to remove residual plasmid DNA which may adhere to the cell surface. The medium was then changed to 1 mL of normal medium and incubation was continued for 48 h.
  • RNA from each well was extracted and then reverse transcribed into cDNA.
  • the MCSI upstream primer and the MCSII downstream primer are the upstream and downstream primers respectively, and the samples are loaded according to the following table:
  • Downstream primer (10 ⁇ M) 0.4 ⁇ L 0.2 ⁇ M ROX dye (50 ⁇ ) 0.4 ⁇ L 1 ⁇ cDNA template 2.0 ⁇ L
  • Premixed Taq enzyme (2 ⁇ ) 10 ⁇ L 1 ⁇ dH 2 O 6.8 ⁇ L Total amount 20.0 ⁇ L
  • Pre-denaturation 95 ° C for 30 seconds, cycle 1 time;
  • T R ⁇ 20 ⁇ 10 3 , wherein T is a lentivirus titer, T is in units of TU/mL, and R is a dilution factor.
  • the lentivirus titer of this package was greater than 10 7 TU/mL, indicating that the lentivirus packaging was successful.
  • IL-2 60 IU/mL
  • protamine sulfate 10 ⁇ g/mL
  • human Jurkat T cells with anti-CD3 antibody (1 ⁇ g/mL) and anti-CD28 antibody (1 ⁇ g/mL).
  • anti-CD3 antibody 1 ⁇ g/mL
  • anti-CD28 antibody 1 ⁇ g/mL
  • they were implanted into a 24-well plate at 2 mL per well. After centrifugation of the 24-well plate at 1000 °C for 90 min at 32 ° C, it was placed at 37 ° C, 5% CO 2 for 24 h, then the medium was changed to fresh complete medium, and then placed at 37 ° C, 5% CO 2 Cultivate for 48 hours.
  • the TCR ⁇ and the captured gp100 tetramer were then detected by flow cytometry, and the results are shown in Figures 3 and 4.
  • the expression level of TCR on the cell surface of the experimental group Jurkat T lymphocytes treated with lentivirus was significantly higher than that of the untreated control group, and the gp100 was captured. The proportion also increased significantly, indicating that the pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE vector can be well used for the efficient expression of double foreign genes in mammalian cells.
  • Control cells not treated with lentivirus and cells treated with lentivirus were set without the addition of dimerization inducer (CID) (AP20187; ARIAD Pharmaceuticals, Cambridge, MA) and CID to a final concentration of 50 nmol/ L groups were placed at 37 ° C, 5% CO 2 for 24 h. After 24 h, the cells were stained with Annexin V-PE and 7-AAD for flow cytometry, and the apoptosis and necrosis of the cells were observed. The results are shown in Fig. 5.
  • CID dimerization inducer

Abstract

提供了一种用于双基因表达的慢病毒表达载体及其制备方法和应用。该慢病毒表达载体为pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列被替换为带有自杀开关的双基因表达盒子Dual-Casp9得到。

Description

慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法 技术领域
本发明涉及生物技术领域，特别是涉及一种慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法。
背景技术
基因表达是细胞在生命过程中，把储存在DNA序列中的遗传信息经过转录和翻译，转变成具有生物活性的蛋白质分子的过程。通过基因工程可以实现外源基因在生物细胞甚至生物体内的表达，从而达到人为调控细胞或实物体性状的目的。
真核细胞，尤其是哺乳动物细胞的外源基因的导入，主要通过物理方法、化学方法或生物学方法完成。物理方法主要包括DNA显微注射法、电穿孔法和金属颗粒轰击法等方法；化学方法主要包括脂质体介导和受体介导等方法；生物学方法主要是通过各种病毒实现，如腺病毒、腺相关病毒、逆转录病毒、慢病毒等。物理方法和化学方法可以负载大量外源基因片段，并且有部分方法可以实现将外源基因插入到细胞或生物体的基因组中，但总体来说，通过这两类方法实现外源基因表达的效率不高，而且大多数时候无法很好地实现外源基因插入细胞或生物体基因组，进而无法实现外源基因的稳定永久表达。生物学方法中的腺相关病毒、慢病毒等方法则可以很好地实现这一目标，它们可以将外源基因整合到细胞或生物体的基因组中，从而实现外源基因的稳定永久表达。其中，慢病毒以其具有可以感染非分裂期细胞、容纳外源性基因片段更大等优点受到科学界的青睐。
目前，不管是在实验室还是在产业界，慢病毒的应用都非常广泛。随着应用领域的不断扩大，对慢病毒的要求也在不断提高。当前，在科研和实际运用中，通过慢病毒一次导入两个基因并实现其高效表达的需求也越来越大，例如2013年《Science》杂志评选出的十大科学突破之首—癌症免疫疗法，包括TCR-T 等技术。由于TCR-T技术能够表达特异性受体靶向识别特异性的细胞如肿瘤细胞，受到广泛的关注和研究，并从初期的基础免疫研究转变为现在的临床应用。由于TCR-T技术需要将T细胞受体(TCR)两个亚基对应的DNA序列同时导入到T细胞中，因此其对能实现双基因快速导入技术的需求非常迫切，而现有的慢病毒载体尚不能很好地满足这一需求。
当前的慢病毒技术主要通过两种方式实现双基因的导入：在慢病毒载体的基因表达区构建双启动子或一个启动子+一个内部核糖体进入位点序列(IRES)，通过两个启动元件在转录水平实现两个外源基因的表达。这两种方式均可以实现双基因的表达，但存在以下两个主要的缺点：1、位于载体上游和下游基因的表达水平不一致。由于不同启动元件效率的不同以及两者之间可能存在的相互作用，从而导致上下游基因的表达水平差异很大(如10倍的差异)，甚至出现其中一个基因不表达的情况；2、慢病毒对外源基因的负载容量一般在8000bp～10000bp左右，而启动元件的大小一般在500bp～700bp左右。可以看到，单个启动元件占慢病毒负载容量的比例接近10％，因此多引入一个启动元件就意味着通过慢病毒导入的外源基因序列，就意味着可用于导入外源基因容量的减少。因此使用这两种方式实现的慢病毒双基因表达均不是特别理想。现有技术中尚无可解决以上问题的慢病毒载体。
发明内容
基于此，有必要提供一种双基因表达效果较好的慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法。
一种慢病毒表达载体，所述慢病毒表达载体为pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体，所述pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体为pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列被替换为带有自杀开关的双基因表达盒子Dual-Casp9得到；
所述带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接；
所述Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽。
在一个实施例中，所述iCasp9的序列如SEQ ID No.1所示。
在一个实施例中，所述带有自杀开关的双基因表达盒子Dual-Casp9的序列如SEQ ID No.2所示。
在一个实施例中，所述第一多克隆位点的序列如SEQ ID No.3所示；
所述第二多克隆位点的序列如SEQ ID No.4所示；
所述第一弗林蛋白酶切割位点和所述第二弗林蛋白酶切割位点的序列均如SEQ ID No.5所示；
所述V5标签的序列如SEQ ID No.6所示；
所述第一Spacer和所述第二Spacer的序列如SEQ ID No.7所示；
所述第一2A肽和所述第二2A肽的序列选自SEQ ID No.8所示的序列、SEQ ID No.9所示的序列、SEQ ID No.10所示的序列和SEQ ID No.11所示的序列中的一种，所述第一2A肽和所述第二2A肽的序列彼此不同。
在一个实施例中，所述第一多克隆位点插入有第一目的基因，所述第二多克隆位点插入有第二目的基因；
所述第一目的基因为小鼠TCRα基因，所述小鼠TCRα基因的NCBI编号为DQ452619，所述第二目的基因为小鼠TCRβ，所述小鼠TCRβ基因的NCBI编号为DQ452620。
一种上述的慢病毒表达载体的制备方法，包括如下步骤：
设计带有自杀开关的双基因表达盒子Dual-Casp9，并委托合成所述带有自杀开关的双基因表达盒子Dual-Casp9，得到含有pUC-Dual-Casp9质粒的大肠杆菌菌液，所述pUC-Dual-Casp9质粒中含有所述带有自杀开关的双基因表达盒子Dual-Casp9，所述带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接，所述Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽；
将所述含有pUC-Dual-Casp9质粒的大肠杆菌菌液跟选择性LB液体培养基混合，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第一沉淀，将所述第一沉淀裂解后提取所述pUC-Dual-Casp9质粒；
在37℃下用Asc I内切酶和Sal I内切酶处理pUC-Dual-Casp9质粒，充分反应后回收，得到所述带有自杀开关的双基因表达盒子Dual-Casp9的片段；
在37℃下用Asc I内切酶和Sal I内切酶处理pRRLSIN.cPPT.MSCV/GFP.WPRE载体，将所述pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列切除，充分反应后回收，得到线性化的pRRLSIN.cPPT.MSCV/GFP.WPRE载体；
按照摩尔比为3～10：1将所述带有自杀开关的双基因表达盒子Dual-Casp9的片段与所述线性化pRRLSIN.cPPT.MSCV/GFP.WPRE载体混合，再加入T4DNA连接酶，4℃下连接过夜，得到含有连接质粒的连接产物；以及
将所述含有连接质粒的连接产物转化感受态Top10大肠杆菌，并均匀涂布到选择性的LB平板上，37℃倒置培养12h～16h，挑取阳性菌落置于所述选择性LB液体培养基中，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第二沉淀，将所述第二沉淀裂解后提取所述连接质粒，所述连接质粒即为所述慢病毒表达载体。
在一个实施例中，还包括在得到所述带有自杀开关的双基因表达盒子Dual-Casp9的片段之后，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第一多克隆位点插入第一目的基因，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第二多克隆位点插入第二目的基因的操作；
所述第一目的基因为小鼠TCRα基因，所述小鼠TCRα基因的NCBI编号为DQ452619，所述第二目的基因为小鼠TCRβ基因，所述小鼠TCRβ基因的NCBI编号为DQ452620。
一种慢病毒表达试剂盒，包括上述的慢病毒表达载体。
一种重组慢病毒的制备方法，包括如下步骤：
提供上述的慢病毒表达载体，并在所述带有自杀开关的双基因表达盒子 Dual-Casp9的片段的所述第一多克隆位点插入第一目的基因，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第二多克隆位点插入第二目的基因，得到携带有目的基因的慢病毒表达载体；
将所述携带有目的基因的慢病毒表达载体、pMDLg/pRRE载体、pRSV-Rev载体和pMD-G载体按照摩尔比为2：1：1：1混合后转染到293FT细胞中，转染后4h～6h更换为完全培养基培养，48h后收集培养液，离心后保留上清并将所述上清用0.45μm过滤头过滤，保留滤液，所述滤液即为重组慢病毒的溶液。
在一个实施例中，还包括在得到所述重组慢病毒的溶液后，对所述重组慢病毒的溶液的滴度进行检测的步骤，具体为：
第一天，将293FT细胞接种到多孔板中，每个孔接种2×105个细胞，每个孔加入500μL培养基，37℃、5％CO2培养过夜；
第二天，按所述重组慢病毒的溶液的稀释比例为1、101、102、103、104、105、106、107和108，用培养基将所述重组慢病毒的溶液梯度稀释，接着分别将100μL梯度稀释的所述重组慢病毒的溶液与100μL多孔板中的细胞培养液在多孔板的不同孔中混合转染，转染开始后24h，吸去培养基并换成500μL含5U DNaseI的新鲜培养基，37℃下培养30min以去除可能附着于细胞表面的残余质粒DNA，然后将培养基换成1mL正常培养基，继续培养48h；
第四天，吸去所述多孔板的每个孔中的培养基，加入500μL胰酶-EDTA溶液消化细胞，在37℃反应1分钟，接着加入培养基终止消化反应并将细胞吹洗下，离心收集每个孔的细胞，抽提每孔细胞的总RNA，接着逆转录得到每孔细胞的总cDNA；以及
分别对得到的所述每孔细胞的总cDNA进行荧光定量PCR，得到每孔细胞的Ct值，选择与对照组Ct值差异最小但超过2的实验组，得到其稀释倍数，按照以下公式计算慢病毒滴度：
T＝R×20×103，其中，T为慢病毒滴度，T的单位为TU/mL，R为稀释倍数。
这种慢病毒表达载体利用2A肽实现双外源基因在哺乳动物细胞内的表达，2A肽具有“自我剪切”的特性，在蛋白质的翻译过程中，2A肽改变了核糖体的活性，促进2A肽残基Gly与tRNAGly之间酯链的水解，从转录复合物上释放 上游多肽的同时又起始下游多肽的翻译，在翻译水平上实现的双基因表达。据研究，2A肽能几乎实现完全剪切。因此，这种慢病毒表达载体可以从理论上保证了双外源基因在哺乳动物细胞内的等量表达，相对于传统的用于双基因表达的慢病毒载体，这种慢病毒表达载体的双基因表达效果较好。
附图说明
图1为一实施方式的慢病毒表达载体的结构示意图；
图2为如图1所示的慢病毒表达载体的制备方法的流程图；
图3为实施例6中流式细胞术检测TCR在CD4+ T淋巴细胞表面表达情况的结果；
图4为实施例6中流式细胞术检测TCR在CD8+ T淋巴细胞表面表达情况的结果；
图5为实施例7中流式细胞术检测人工诱导细胞凋亡的结果示意图。
具体实施方式
下面主要结合附图及具体实施例对慢病毒表达载体及其制备方法和应用、重组慢病毒的制备方法作进一步详细的说明。
本发明中未说明的药品、试剂均为本领域商用产品(购自生工生物工程(上海)股份有限公司、宝生物工程(大连)有限公司、德国QIAGEN公司、美国Life Technologies公司、美国GIBCO公司等)，未说明的操作均采用本领域常规方法，具体步骤可参见：《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor)。
如图1所示的一实施方式的慢病毒表达载体，为pRRLSIN.cPPT.MSCV-Dual.WPRE载体。pRRLSIN.cPPT.MSCV-Dual.WPRE载体为pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列被替换为带有自杀开关的双基因表达盒子Dual-Casp9得到。
pRRLSIN.cPPT.MSCV/GFP.WPRE载体由购自addgene的商业化的质粒 pRRLSIN.cPPT.PGK/GFP.WPRE改造而来，将质粒pRRLSIN.cPPT.PGK/GFP.WPRE的PGK启动子换成了MSCV启动子即可。
GFP序列被替换具有如下优点：1、替换GFP可以容纳更大的外源基因(GFP基因本身也有超过700bp的序列)；2、GFP所在位置正好处于MSCV启动子之后，将其替换可以提高外源基因的表达水平。
pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列可以采用Asc I内切酶和Sal I内切酶切除，然后用T4 DNA连接酶将经过相同处理的带有自杀开关的双基因表达盒子Dual-Casp9的片段和切除了GFP序列的pRRLSIN.cPPT.MSCV/GFP.WPRE载体连接，构建得到上述pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体。
带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接。
自杀开关Casp9可以实现人工诱导的细胞凋亡，Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽。
iCasp9包括一个带有F36V变异的FK506结合蛋白12(FKBP12)和经修饰的Caspase-9蛋白。野生型的FKBP12与化合物AP1903或AP20187的亲和力很低，而带F36V突变的FKBP12则可以很好地与这两种化合物结合，实现iCasp-9元件的二聚化，进而活化其中的Caspase-9，起始细胞凋亡的过程。
优选的，iCasp9的序列如SEQ ID No.1所示。
这种慢病毒表达载体利用2A肽实现双外源基因在哺乳动物细胞内的表达，2A肽具有“自我剪切”的特性，在蛋白质的翻译过程中，2A肽改变了核糖体的活性，促进2A肽残基Gly与tRNAGly之间酯链的水解，从转录复合物上释放上游多肽的同时又起始下游多肽的翻译，在翻译水平上实现的双基因表达。据研究，2A肽能几乎实现完全剪切。因此，这种慢病毒表达载体可以从理论上保证了双外源基因在哺乳动物细胞内的等量表达，相对于传统的用于双基因表达的慢病毒载体，这种慢病毒表达载体的双基因表达效果较好。
优选的，带有自杀开关的双基因表达盒子Dual-Casp9的序列如SEQ ID No.2所示。
第一多克隆位点包括从5’到3’端依次排列的如下结构：Asc I酶切位点、BstBI酶切位点、Bam HI酶切位点和Age I酶切位点。
第二多克隆位点包括从5’到3’端依次排列的如下结构：Xho I酶切位点、Spe I酶切位点、Xma I酶切位点和Sal I酶切位点。
优选的，第一多克隆位点的序列如SEQ ID No.3所示。
优选的，第二多克隆位点的序列如SEQ ID No.4所示。
优选的，第一弗林蛋白酶切割位点和第二弗林蛋白酶切割位点的序列如SEQ ID No.5所示。
优选的，V5标签的序列如SEQ ID No.6所示。
优选的，第一Spacer和第二Spacer的序列如SEQ ID No.7所示。
优选的，第一2A肽和第二2A肽的序列选自SEQ ID No.8所示的序列、SEQ ID No.9所示的序列、SEQ ID No.10所示的序列和SEQ ID No.11所示的序列中的一种，第一2A肽和第二2A肽的序列彼此不同。
第一2A肽和第二2A肽的序列彼此不同，可以避免被同源重组掉。
在实际应用中，第一多克隆位点插入有第一目的基因，第二多克隆位点插入有第二目的基因。
优选的，第一目的基因为小鼠TCRα基因，小鼠TCRα基因的NCBI编号为DQ452619，第二目的基因为小鼠TCRβ基因，小鼠TCRβ基因的NCBI编号为DQ452620。
本发明还提供了一种慢病毒表达试剂盒，包括上述的慢病毒表达载体。
与现有技术相比，这种慢病毒表达试剂盒具有使用方便、高效，可很好地实现哺乳动物细胞双外源基因的高效表达，而且可以容纳更多的基因片段，可作为有力的工具应用于科研和产业界，如肿瘤的细胞免疫治疗。细胞免疫治疗是一种新兴的、具有显著疗效的抗肿瘤疗法，弥补了传统的手术、放化疗的弊端，已被公认为肿瘤综合治疗模式中最有发展前途，也是目前唯一有希望完全消灭肿瘤的治疗手段。细胞毒性T细胞(CTL)的免疫功能在抗肿瘤免疫中起 决定作用，肿瘤抗原激活CTL及CTL杀伤肿瘤细胞的前提是CTL对肿瘤抗原的有效识别，为了增强CTL对肿瘤细胞的免疫应答，多种治疗策略正处于临床试验阶段，譬如T细胞移植、肿瘤抗原或者DC细胞的免疫。目前常用的肿瘤特异性CTL包括TIL、DC诱导的CTL及基因修饰的T细胞(TCR-T和CAR-T)，这些技术都具有特异性的杀伤表达该抗原的肿瘤细胞的功能，是未来细胞免疫治疗的重要发展方向。
如图2所示的上述的慢病毒表达载体的制备方法，包括如下步骤：
S10、设计带有自杀开关的双基因表达盒子Dual-Casp9，并委托合成带有自杀开关的双基因表达盒子Dual-Casp9，得到含有pUC-Dual-Casp9质粒的大肠杆菌菌液。
带有自杀开关的双基因表达盒子Dual-Casp9的合成委托商业公司完成，直接获得含有pUC-Dual-Casp9质粒的大肠杆菌菌液。
pUC-Dual-Casp9质粒中含有带有自杀开关的双基因表达盒子Dual-Casp9，带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接，Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽
S20、将S10得到的含有pUC-Dual-Casp9质粒的大肠杆菌菌液跟选择性LB液体培养基混合，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第一沉淀，将第一沉淀裂解后提取pUC-Dual-Casp9质粒。
选择性LB液体培养基为含有100μg/mL氨苄青霉素的LB液体培养基。
S30、在37℃下用Asc I内切酶和Sal I内切酶处理S20得到的pUC-Dual-Casp9质粒，充分反应后回收，得到带有自杀开关的双基因表达盒子Dual-Casp9的片段。
S40、在37℃下用Asc I内切酶和Sal I内切酶处理pRRLSIN.cPPT.MSCV/GFP.WPRE载体，将pRRLSIN.cPPT.MSCV/GFP.WPRE载 体的GFP序列切除，充分反应后回收，得到线性化的pRRLSIN.cPPT.MSCV/GFP.WPRE载体。
pRRLSIN.cPPT.MSCV/GFP.WPRE载体由购自addgene的商业化的pRRLSIN.cPPT.PGK/GFP.WPRE改造而来，将PGK启动子换成了MSCV启动子即可。
S50、按照摩尔比为3：1将S30得到的带有自杀开关的双基因表达盒子Dual-Casp9的片段与S40得到的线性化pRRLSIN.cPPT.MSCV/GFP.WPRE载体混合，再加入T4 DNA连接酶，4℃下连接过夜，得到含有连接质粒的连接产物。
S60、将S50得到的含有连接质粒的连接产物转化感受态Top10大肠杆菌，并均匀涂布到选择性的LB平板上，37℃倒置培养12h～16h，挑取阳性菌落置于选择性LB液体培养基中，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第二沉淀，将第二沉淀裂解后提取连接质粒，连接质粒即为慢病毒表达载体。
这种慢病毒表达载体的制备方法还包括在得到带有自杀开关的双基因表达盒子Dual-Casp9的片段之后，在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第一多克隆位点插入第一目的基因，在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第二多克隆位点插入第二目的基因的操作。
优选的，第一目的基因为小鼠TCRα基因，小鼠TCRα基因的NCBI编号为DQ452619，第二目的基因为小鼠TCRβ基因，小鼠TCRβ基因的NCBI编号为DQ452620。
一种重组慢病毒的制备方法，其特征在于，包括如下步骤：
提供上述的慢病毒表达载体，并在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第一多克隆位点插入第一目的基因，在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第二多克隆位点插入第二目的基因，得到携带有目的基因的慢病毒表达载体；将携带有目的基因的慢病毒表达载体、pMDLg/pRRE载体、pRSV-Rev载体和pMD-G载体按照摩尔比为2：1：1：1混合后转染到293FT细胞中，转染后4h～6h更换为完全培养基培养，48h后收集培养液，离心后保留上清并将上清用0.45μm过滤头过滤，保留滤液即为重组慢病毒的溶液。
第一目的基因为小鼠TCRα基因，小鼠TCRα基因的NCBI编号为DQ452619，第二目的基因为小鼠TCRβ基因，小鼠TCRβ基因的NCBI编号为DQ452620。
在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第一多克隆位点插入第一目的基因后需要对插入的第一目的基因进行测序，测序引物为：MCS I上游引物和MCS I下游引物，测序完成后选取测序结果与预期完全相符的载体供下一步使用。
在带有自杀开关的双基因表达盒子Dual-Casp9的片段的第二多克隆位点插入第二目的基因后需要对插入的第二目的基因进行测序，测序引物为：MCS II上游引物和MCS II下游引物，测序完成后选取测序结果与预期完全相符的载体供下一步使用。
MCS I上游引物的序列如SEQ ID No.12所示。
MCS I下游引物的序列如SEQ ID No.13所示。
MCS II上游引物的序列如SEQ ID No.14所示。
MCS II下游引物的序列如SEQ ID No.15所示。
pMDLg/pRRE载体、pRSV-Rev载体和pMD-G载体均为常规的慢病毒组装载体。
这种重组慢病毒的制备方法还包括在得到重组慢病毒的溶液后，对重组慢病毒的溶液的滴度进行检测的步骤，具体为：
第一天，将293FT细胞接种到多孔板中，每个孔接种2×105个细胞，每个孔加入500μL培养基，37℃、5％CO2培养过夜；
第二天，按重组慢病毒的溶液的稀释比例为1、101、102、103、104、105、106、107和108，用培养基将重组慢病毒的溶液梯度稀释，接着分别将100μL梯度稀释的重组慢病毒的溶液与100μL多孔板中的细胞培养液在多孔板的不同孔中混合转染，转染开始后24h，吸去培养基并换成500μL含5U DNaseI的新鲜培养基，37℃下培养30min以去除可能附着于细胞表面的残余质粒DNA，然后将培养基换成1mL正常培养基，继续培养48h；
第四天，吸去多孔板的每个孔中的培养基，加入500μL胰酶-EDTA溶液消 化细胞，在37℃反应1分钟，接着加入培养基终止消化反应并将细胞吹洗下，离心收集每个孔的细胞，抽提每孔细胞的总RNA，接着逆转录得到每孔细胞的总cDNA；以及
以上述的MCS I上游引物和MCS II下游引物分别为上下游引物，按照下表加样，然后进行荧光定量PCR，获取各孔细胞荧光定量PCR反应的Ct值。
试剂 使用量(μL) 终浓度
上游引物(10μM) 0.4μL 0.2μM
下游引物(10μM) 0.4μL 0.2μM
ROX染料(50×) 0.4μL 1×
cDNA模板 2.0μL  
预混合Taq酶(2×) 10μL 1×
dH2O 6.8μL  
总量 20.0μL  
根据得到的每孔细胞的Ct值，选择与对照组Ct值差异最小但超过2的实验组，得到其稀释倍数，按照以下公式计算慢病毒滴度：
T＝R×20×103，其中，T为慢病毒滴度，T的单位为TU/mL，R为稀释倍数。
一般认为，只要慢病毒滴度达到107TU/mL以上，即认为成功获得所需慢病毒液。
具体实施例。具体实施例中使用的质粒为pRRLSIN.cPPT.MSCV改造得到，GFP.WPRE、293FT细胞为市售商品，所使用的试剂均为市售商品。
实施例1 双基因表达盒的设计
双基因表达盒的核心是表达第一2A肽(SEQ ID No.8)，在有第一多克隆位点(SEQ ID No.3)、第一2A肽(SEQ ID No.8)和第二多克隆位点(SEQ ID No.4)存在的情况下即可用于实现双外源基因的等量表达。但通过该方式得到的外源基因表达量不高，且在第一2A肽上游的多肽序列末端会被加上13个源自第一2A肽的氨基酸残基，这可能会对该多肽的特性产生一定的影响。
进一步地，在上一步的双基因表达盒的第一多克隆位点和第一2A肽之间插入第一Spacer(SEQ ID No.7)能有效提高外源基因的表达水平。
进一步地，在上一步得到的双基因表达盒的第一多克隆位点和第一Spacer之间插入第一弗林蛋白酶切位点(SEQ ID No.5)，可以使第一2A肽上游的多肽在表达后再经弗林蛋白酶的切割，其末端仅会残留4个氨基酸残基。出乎意料的，外源基因的表达水平又有了进一步的提高。
最优地，在上一步得到的双基因表达盒的第一弗林蛋白酶切位点和第一Spacer之间插入V5标签(SEQ ID No.6)能再次提升外源基因的表达水平，实现双外源基因的最高效等量表达。
如图1所示，最终得到的双基因表达盒的序列如SEQ ID No.2所示。
实施例2 pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体的构建。
委托上海生工生物工程技术服务有限公司对带有自杀开关的双基因表达盒子Dual-Casp9的序列进行合成，得到的序列包含在大肠杆菌体内的pUC-Dual-Casp9载体中。对该大肠杆菌进行扩大培养，并提取其中的pUC-Dual-Casp9载体，然后测定其纯度和浓度，结果如下表所示。
pUC-Dual-Casp9质粒的纯度和浓度
重组载体 A260/A280 浓度(ng/μL)
pUC-Dual-Casp9 1.98 704.0
用Asc I和SalI分别对pUC-Dual-Casp9载体和pRRLSIN.cPPT.MSCV/GFP.WPRE载体进行双酶切，电泳后分别回收重pUC-Dual-Casp9载体酶切产物中的小片段以及pRRLSIN.cPPT.MSCV/GFP.WPRE载体酶切产物的大片段。按pUC-Dual-Casp9载酶切产物的小段：线性化的pRRLSIN.cPPT.MSCV/GFP.WPRE载体的摩尔比为3:1混匀后，并加入T4 DNA连接酶，置于4℃连接过夜。
将连接产物转化感受态Top10大肠杆菌，并均匀涂布到含100μg/mL氨苄青霉素的LB平板上。37℃倒置培养12-16h。挑取多个阳性菌落，分别置于5mL LB液体培养基(含100μg/mL氨苄青霉素)中，于恒温空气摇床上37℃，300rpm 培养12-16h至OD600＝0.6-0.8，将得到的菌液置于离心机中，10000rpm离心1min，弃上清，获得所需菌体，并进行测序，测序验证后提取其中的质粒。测序结果与预期完全相符，说明成功构建pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体。
实施例3 表达小鼠靶向黑色素瘤相关抗原gp100(154-162)TCRα和TCRβ的载体构建
根据NCBI中提供的基因序列(TCRα和TCRβ，编号分别为DQ452619和DQ452620)，并在TCRα基因的上下游分别加入Asc I和Age I酶切位点，并去除其终止密码子；TCRβ基因的上下游分别加入Xho I和Sal I酶切位点。委托上海生工生物工程技术服务有限公司进行合成。
合成获得两株大肠杆菌，分别包含pUC57-TCRα和pUC57-TCRβ载体。对这两株大肠杆菌进行扩大培养，分别提取其中的pUC57-TCRα和pUC57-TCRβ载体，然后测定其纯度和浓度，结果如下表所示。
pUC57-TCRα和pUC57-TCRβ载体的纯度和浓度
重组载体 A260/A280 浓度(ng/μL)
pUC57-TCRα 1.86 813.7
pUC57-TCRβ 1.97 759.4
用Asc I与Age I酶对pUC57-TCRα载体进行处理，Xho I和Sal I酶对pUC57-TCRβ载体进行处理，电泳后分别回收获得两者的小片段TCRα和TCRβ。对上一步获得的pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体用Asc I与Age I酶进行双酶切处理，电泳后回收载体，然后用T4 DNA连接酶将其与TCRα进行连接，获得pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCRα载体。用MCS I上游引物(SEQ ID No.12)和MCS I下游引物(SEQ ID No.13)进行测序，结果与预期完全相符。
用Xho I和Sal I酶对pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCRα载体进行双酶切处理，电泳后回收载体，然后用T4 DNA连接酶将其与TCRα进行连接，获得pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCRα-TCRβ载体。用MCS Ⅱ上游引物(SEQ ID No.14)和MCSⅡ下游引物(SEQ ID No.15)进行测序，结果与预期完全相符。至此，用于表达小鼠靶向黑色素瘤相关抗原gp100(154-162)TCRα和TCRβ的pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCRα-TCRβ载体已成功构建完成。
实施例4 慢病毒包装
培养293FT细胞，取生长状态良好的细胞接种到10cm培养皿中，每个皿接种5×106个细胞，加入DMEM无双抗培养基培养细胞约18h后至融合度达到80-90％后，取pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE-TCRα-TCRβ载体10μg、pMDLg/pRRE、pRSV-Rev和pMD-G载体各5μg，用Lipofectamine 2000转染至293FT细胞中，转染后4-6h更换成DMEM完全培养基。48h后收集含病毒的上清培养基，6000g离心10min后，取上清液再用0.45μm过滤头进行过滤，获得病毒液。
实施例5 慢病毒滴度的检测
培养293FT细胞，取生长状态良好的293FT细胞接种到24孔板中，每个孔接种2×105个细胞，加入500μL培养基，37℃，5％CO2培养过夜。第二天，按病毒原液：培养基的稀释比例为100-108分别制备病慢毒梯度稀释液各100μL，然后吸取各孔原培养基各100μL，再加入慢病毒稀释液各100μL开始转染。转染开始后24h，吸出含慢病毒的培养基，换成500μL含5UDNaseI的新鲜培养基，37℃下培养30min以去除可能附着于细胞表面的残余质粒DNA。然后将培养基换成1mL正常培养基，继续培养48h。
小心吸走每个孔的全部培养基，加入500μL胰酶-EDTA溶液消化细胞，在37℃反应1分钟。接着加入培养基终止消化反应并将细胞吹洗下，离心收集每个孔的细胞。抽提每孔细胞的总RNA，接着逆转录为cDNA。以MCSI上游引物和MCSII下游引物分别为上下游引物，按照下表加样：
试剂 使用量(μL) 终浓度
上游引物(10μM) 0.4μL 0.2μM
下游引物(10μM) 0.4μL 0.2μM
ROX染料(50×) 0.4μL 1×
cDNA模板 2.0μL  
预混合Taq酶(2×) 10μL 1×
dH2O 6.8μL  
总量 20.0μL  
然后进行荧光定量PCR，获取各孔细胞荧光定量PCR反应的Ct值。反应条件如下：
预变性：95℃30秒，循环1次；
PCR反应：95℃5秒，58℃30秒，循环40次；
解离：95℃15秒。
一般认为，与对照组细胞相比，Ct值差异达到2以上即可认为存在显著差异，因此找出与对照组Ct值差异最小但超过2的实验组，得到其稀释倍数，按照以下公式计算慢病毒滴度：
T＝R×20×103，其中，T为慢病毒滴度，T的单位为TU/mL，R为稀释倍数。
经计算，本次包装的慢病毒滴度大于107TU/mL，表明此次慢病毒的包装是成功的。
实施例6 细胞转染、筛选与基因表达情况检测
在慢病毒液中加入IL-2(60IU/mL)和硫酸鱼精蛋白(10μg/mL)，然后与经抗CD3抗体(1μg/mL)和抗CD28抗体(1μg/mL)的人Jurkat T细胞(2.5×105～5×105个)混合均匀后，植入24孔板中，每孔2mL。在32℃下以1000g的速度对24孔板离心90min后，置于37℃，5％CO2培养24h，然后将培养基换成新鲜的完全培养基，再置于37℃，5％CO2培养48h。接着利用流式细胞术对TCRβ和捕获的gp100四聚体进行检测，其结果如图3和图4所示。
有图3和图4可以看出，经慢病毒处理后的实验组Jurkat T淋巴细胞，其TCR在细胞表面的表达水平与未经处理的对照组均有显著提高，捕获gp100的 比例也有显著升高，说明pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体能很好地用于哺乳动物细胞双外源基因的高效表达。
实施例7 人工诱导细胞凋亡
取未经慢病毒处理的对照组细胞和经慢病毒处理的实验组细胞，分别设置不加二聚化诱导剂(CID)(AP20187；ARIAD Pharmaceuticals,Cambridge,MA)和加入CID至终浓度50nmol/L两组，置于37℃，5％CO2下培养24h。24h后，使用Annexin V-PE和7-AAD染色后进行流式细胞术检测，观察细胞的凋亡和坏死情况，其结果如图5所示。
由图5可以看出，未经CID处理的对照组和实验组细胞以及经CID处理的对照组细胞的凋亡和坏死比例无显著差异，而经CID处理的实验组细胞的凋亡和坏死比例则高达93％，显著高于其他组的细胞，说明借助CID和iCasp-9能人工诱导细胞凋亡，进而说明借助pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体可以实现细胞的人工诱导凋亡。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Figure PCTCN2016080421-appb-000001
Figure PCTCN2016080421-appb-000002
Figure PCTCN2016080421-appb-000003
Figure PCTCN2016080421-appb-000004
Figure PCTCN2016080421-appb-000005
Figure PCTCN2016080421-appb-000006

Claims (10)

  1. 一种慢病毒表达载体，其特征在于，所述慢病毒表达载体为pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体，所述pRRLSIN.cPPT.MSCV-Dual-Casp9.WPRE载体为pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列被替换为带有自杀开关的双基因表达盒子Dual-Casp9得到；
    所述带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接；
    所述Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽。
  2. 根据权利要求1所述的慢病毒表达载体，其特征在于，所述iCasp9的序列如SEQ ID No.1所示。
  3. 根据权利要求1所述的慢病毒表达载体，其特征在于，所述带有自杀开关的双基因表达盒子Dual-Casp9的序列如SEQ ID No.2所示。
  4. 根据权利要求1所述的慢病毒表达载体，其特征在于，所述第一多克隆位点的序列如SEQ ID No.3所示；
    所述第二多克隆位点的序列如SEQ ID No.4所示；
    所述第一弗林蛋白酶切割位点和所述第二弗林蛋白酶切割位点的序列均如SEQ ID No.5所示；
    所述V5标签的序列如SEQ ID No.6所示；
    所述第一Spacer和所述第二Spacer的序列如SEQ ID No.7所示；
    所述第一2A肽和所述第二2A肽的序列选自SEQ ID No.8所示的序列、SEQ ID No.9所示的序列、SEQ ID No.10所示的序列和SEQ ID No.11所示的序列中的一种，所述第一2A肽和所述第二2A肽的序列彼此不同。
  5. 根据权利要求1所述的慢病毒表达载体，其特征在于，所述第一多克隆位点插入有第一目的基因，所述第二多克隆位点插入有第二目的基因；
    所述第一目的基因为小鼠TCRα基因，所述小鼠TCRα基因的NCBI编号为DQ452619，所述第二目的基因为小鼠TCRβ，所述小鼠TCRβ基因的NCBI 编号为DQ452620。
  6. 一种如权利要求1～5中任一项所述的慢病毒表达载体的制备方法，其特征在于，包括如下步骤：
    设计带有自杀开关的双基因表达盒子Dual-Casp9，并委托合成所述带有自杀开关的双基因表达盒子Dual-Casp9，得到含有pUC-Dual-Casp9质粒的大肠杆菌菌液，所述pUC-Dual-Casp9质粒中含有所述带有自杀开关的双基因表达盒子Dual-Casp9，所述带有自杀开关的双基因表达盒子Dual-Casp9包括从5’到3’端依次排列的如下结构：第一多克隆位点-第一弗林蛋白酶切割位点-V5标签-第一Spacer-第一2A肽-Casp9-第二多克隆位点，其中，“-”代表连接，所述Casp9包括从5’到3’端依次排列的如下结构：iCasp9-第二弗林蛋白酶切割位点-第二Spacer-第二2A肽；
    将所述含有pUC-Dual-Casp9质粒的大肠杆菌菌液跟选择性LB液体培养基混合，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第一沉淀，将所述第一沉淀裂解后提取所述pUC-Dual-Casp9质粒；
    在37℃下用Asc I内切酶和Sal I内切酶处理pUC-Dual-Casp9质粒，充分反应后回收，得到所述带有自杀开关的双基因表达盒子Dual-Casp9的片段；
    在37℃下用Asc I内切酶和Sal I内切酶处理pRRLSIN.cPPT.MSCV/GFP.WPRE载体，将所述pRRLSIN.cPPT.MSCV/GFP.WPRE载体的GFP序列切除，充分反应后回收，得到线性化的pRRLSIN.cPPT.MSCV/GFP.WPRE载体；
    按照摩尔比为3～10：1将所述带有自杀开关的双基因表达盒子Dual-Casp9的片段与所述线性化pRRLSIN.cPPT.MSCV/GFP.WPRE载体混合，再加入T4DNA连接酶，4℃下连接过夜，得到含有连接质粒的连接产物；以及
    将所述含有连接质粒的连接产物转化感受态Top10大肠杆菌，并均匀涂布到选择性的LB平板上，37℃倒置培养12h～16h，挑取阳性菌落置于所述选择性LB液体培养基中，在37℃恒温摇床中300rpm培养12h～16h至OD600为0.6～0.8，将得到的菌液离心后保留第二沉淀，将所述第二沉淀裂解后提取所述连接质粒， 所述连接质粒即为所述慢病毒表达载体。
  7. 根据权利要求6所述的慢病毒表达载体，其特征在于，还包括在得到所述带有自杀开关的双基因表达盒子Dual-Casp9的片段之后，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第一多克隆位点插入第一目的基因，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第二多克隆位点插入第二目的基因的操作；
    所述第一目的基因为小鼠TCRα基因，所述小鼠TCRα基因的NCBI编号为DQ452619，所述第二目的基因为小鼠TCRβ基因，所述小鼠TCRβ基因的NCBI编号为DQ452620。
  8. 一种慢病毒表达试剂盒，其特征在于，包括如权利要求1～5中任一项所述的慢病毒表达载体。
  9. 一种重组慢病毒的制备方法，其特征在于，包括如下步骤：
    提供如权利要求1～5中任一项所述的慢病毒表达载体，并在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第一多克隆位点插入第一目的基因，在所述带有自杀开关的双基因表达盒子Dual-Casp9的片段的所述第二多克隆位点插入第二目的基因，得到携带有目的基因的慢病毒表达载体；
    将所述携带有目的基因的慢病毒表达载体、pMDLg/pRRE载体、pRSV-Rev载体和pMD-G载体按照摩尔比为2：1：1：1混合后转染到293FT细胞中，转染后4h～6h更换为完全培养基培养，48h后收集培养液，离心后保留上清并将所述上清用0.45μm过滤头过滤，保留滤液，所述滤液即为重组慢病毒的溶液。
  10. 根据权利要求9所述的重组慢病毒的制备方法，其特征在于，还包括在得到所述重组慢病毒的溶液后，对所述重组慢病毒的溶液的滴度进行检测的步骤，具体为：
    第一天，将293FT细胞接种到多孔板中，每个孔接种2×105个细胞，每个孔加入500μL培养基，37℃、5％CO2培养过夜；
    第二天，按所述重组慢病毒的溶液的稀释比例为1、101、102、103、104、105、106、107和108，用培养基将所述重组慢病毒的溶液梯度稀释，接着分别将100μL梯度稀释的所述重组慢病毒的溶液与100μL多孔板中的细胞培养液在多 孔板的不同孔中混合转染，转染开始后24h，吸去培养基并换成500μL含5UDNaseI的新鲜培养基，37℃下培养30min以去除可能附着于细胞表面的残余质粒DNA，然后将培养基换成1mL正常培养基，继续培养48h；
    第四天，吸去所述多孔板的每个孔中的培养基，加入500μL胰酶-EDTA溶液消化细胞，在37℃反应1分钟，接着加入培养基终止消化反应并将细胞吹洗下，离心收集每个孔的细胞，抽提每孔细胞的总RNA，接着逆转录得到每孔细胞的总cDNA；以及
    分别对得到的所述每孔细胞的总cDNA进行荧光定量PCR，得到每孔细胞的Ct值，选择与对照组Ct值差异最小但超过2的实验组，得到其稀释倍数，按照以下公式计算慢病毒滴度：
    T＝R×20×103，其中，T为慢病毒滴度，T的单位为TU/mL，R为稀释倍数。
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