WO2017107296A1 - 一种重组腺相关病毒的制备方法及重组杆状病毒 - Google Patents

一种重组腺相关病毒的制备方法及重组杆状病毒 Download PDF

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WO2017107296A1
WO2017107296A1 PCT/CN2016/073246 CN2016073246W WO2017107296A1 WO 2017107296 A1 WO2017107296 A1 WO 2017107296A1 CN 2016073246 W CN2016073246 W CN 2016073246W WO 2017107296 A1 WO2017107296 A1 WO 2017107296A1
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gene
recombinant
associated virus
raav
adeno
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吴阳
徐富强
何晓斌
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中国科学院武汉物理与数学研究所
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Definitions

  • the invention belongs to the field of bioengineering, and more particularly to a method for preparing a recombinant adeno-associated virus and a recombinant baculovirus.
  • rAAV Recombinant adeno-associated virus
  • rAAV large-scale preparation of rAAV using a baculovirus expression system
  • Two Bac system and a One Bac system depending on the packaging cell line.
  • the main process of the two baculovirus system is to integrate the Rep gene and the Cap gene of AAV into one baculovirus genome, integrate the ITR core expression element into another baculovirus genome, and then combine the two recombination rods.
  • the virus co-infects the host cells to produce rAAV (Mol Ther. 2008 May; 16(5): 924-30, Mol Ther. 2009 Nov; 17(11): 1888-96.).
  • the main process of a baculovirus system that relies on packaging cell lines is to first establish a packaging cell line that induces expression of the Rep gene and the Cap gene.
  • This packaging cell line integrates the Rep gene and the Cap gene expression element, and the Rep gene and the Cap gene are respectively
  • the hr2 enhancer sequence and the Rep protein binding sequence of AAV were added upstream of the PH promoter under the control of the baculovirus late gene expression strong promoter PH.
  • the Rep gene and the Cap gene in the packaging cell line are induced to express, thereby producing rAAV (Proc Natl Acad Sci U S A. 2009 Mar 31; 106(13): 5059 -64, Hum Gene Ther. 2014 Mar; 25(3): 212-22.).
  • the former is inefficient in co-infecting cells with two baculoviruses, unable to fully utilize the productivity of each cell, and the infection is a random process, which is easy to produce empty-shell defective rAAV particles without nucleic acid, and the preparation process conditions are optimized.
  • Complex, rAAV quality prepared in different batches is unstable.
  • the latter relies on the packaging cell line that induces the expression of Rep gene and Cap gene, and the construction is complicated.
  • the screening of excellent packaging cell lines is difficult.
  • To prepare rAAV with different serotypes it is necessary to establish a packaging cell line expressing the corresponding serotype Cap gene, which is flexible. Sex and versatility are poor and difficult to promote.
  • the present invention provides a method for preparing a recombinant adeno-associated virus for gene therapy and a recombinant baculovirus, which aims to adopt a Rep gene, a Cap gene and an ITR core expression element of AAV.
  • a method for producing a recombinant adeno-associated virus comprising the following step:
  • the preparation method, the step (1) of the recombinant baculovirus is constructed using a pFast.Bac.Dual shuttle vector.
  • the preparation method, the specific operation method of the step (1) is as follows:
  • the preparation method wherein the recombinant baculovirus constructed with a recombinant adeno-associated virus ITR core expression element and a Cap gene (or Rep gene) carries a target gene in an ITR core expression element thereof.
  • the method of preparation wherein the host packaging cell line is used to assist in the replication and assembly of a recombinant adeno-associated virus.
  • the host cell genome is integrated with an expression cassette element which induces expression of an adeno-associated virus Rep gene or an expression frame element which induces expression of an adeno-associated virus Cap gene.
  • a recombinant baculovirus having a gene associated with an adeno-associated virus ITR core expression element and a corresponding serotype Cap gene is provided on the genome.
  • the recombinant baculovirus has a sequence of the Cap gene which is a codon-optimized sequence according to the ribosome leak scanning principle.
  • a recombinant baculovirus having an adeno-associated virus ITR core expression element and a Rep gene of a corresponding serotype is genomically constructed.
  • the recombinant baculovirus has a sequence of a Rep gene whose codon is optimized according to the principle of ribosome leakage.
  • the Rep gene (or Rep gene) of AAV and the ITR core expression element are placed in a recombinant baculovirus
  • the Rep gene (or Cap gene) is integrated into the host cell genome, and the packaging packaging cell line provides packaging rAAV. Some of the auxiliary functions required.
  • the Rep gene and the Cap gene are all integrated into the host cell line, and the difficulty in constructing the packaging cell line is greatly reduced; in particular, the Cap gene and the ITR core element are placed.
  • a method in the baculovirus genome which usually supports the packaging of various serotypes of rAAV by using the Rep gene of type 2 AAV, so it is only necessary to establish a Rep gene that induces expression of type 2 AAV to satisfy different serotypes of rAAV.
  • the preparation, flexibility and versatility will also be greatly enhanced.
  • Figure 1 is a schematic diagram of the packaging schematic of rAAV (A) and the shuttle plasmid pFast.Bac.Dual (pFBD) in the baculovirus expression system (Bac to Bac) (B);
  • FIG. 2 is a schematic illustration of the structure of the components required for the integration of recombinant baculovirus and packaging cell lines required for the preparation of rAAV in the Examples.
  • 2A is a structural diagram of the recombinant shuttle plasmid pFD/Cap-(ITR-GFP) in Example 1;
  • FIG. 2A is a structural diagram of the recombinant shuttle plasmid pFD/Cap-(ITR-GFP) in Example 1;
  • FIG. 2B is a structural diagram of the recombinant shuttle plasmid pFD/Cap-(ITR-GFP) in Example 2;
  • Figure 3 is a structural diagram of the recombinant shuttle plasmid pFD/Rep-(ITR-GFP);
  • Figure 2D is a structural diagram of the recombinant shuttle plasmid pFD/Rep-(ITR-GFP) in Example 4;
  • Figure 2E is established in Example 1 and Example 2.
  • FIG. 1 The structure of the pIR-rep78-hr2-RBE-bsd-GFP plasmid used in the Sf9/Rep packaging cell line;
  • Figure 2F is the pIR-VP-hr2-RBE-bsd- used to establish the Sf9/Cap packaging cell line in Example 3 and Example 4. a structural diagram of the GFP plasmid;
  • FIG. 3 is a schematic diagram showing the process of preparing rAAV by infecting a host packaging cell line with recombinant baculovirus in the examples.
  • 3A is a schematic diagram of processes of Embodiment 1 and Embodiment 2;
  • FIG. 3B is a schematic diagram of processes of Embodiment 3 and Embodiment 4;
  • Figure 4 is a verification diagram of the horizontal transduction activity of rAAV and rAAV cells prepared by the recombinant baculovirus-infected packaging cell line of Example 1; wherein, Figure 4A is a recombinant baculovirus BEV/Cap-(ITR-GFP)-infected Sf9 cell, The selected Sf9/Rep packaging cell line and its fluorescence microscopic image after infection with recombinant BEV; FIG. 4B is a fluorescence microscopic image of rAAV infected with HEK293 and Sf9 cells prepared in Example 1; FIG. 4C is the purification of Example 1. Fluorescence microscopy image of HEA293 cells infected with rAAV;
  • Figure 5 is a fluorescence microscopic image of purified rAAV-infected HEK293 cells prepared in Example 2;
  • FIG. 6 is a verification diagram showing the horizontal transduction activity of rAAV and rAAV cells in the recombinant baculovirus-infected packaging cell line of Example 3.
  • 6A is a fluorescent fluoroscopic image of the Sf9/Cap packaging cell line and the recombinant BEV infected by the recombinant baculovirus BEV/Rep-(ITR-GFP)-infected Sf9 cells
  • FIG. 6B is an example 4 Fluorescence microscopic image of the prepared rAAV infected HEK293 and Sf9 cells
  • Figure 6C is a fluorescence microscopic image of the purified rAAV-infected HEK293 cells of Example 4;
  • Figure 7 is a fluorescence micrograph of the purified rAAV-infected HEK293 cells prepared in Example 4.
  • the preparation method of the recombinant adeno-associated virus provided by the invention comprises the following steps:
  • a recombinant baculovirus constructed with a recombinant adeno-associated virus ITR core expression element and a Cap gene or a Rep gene is infected with a corresponding host packaging cell line.
  • the recombinant baculovirus is used to provide an ITR core expression element and a Cap gene or a Rep gene required for the preparation of rAAV, and to induce expression of a host packaging cell line by infecting a host packaging cell line to activate a baculovirus-specific promoter PH or P10.
  • the Rep gene or the Cap gene thus aids in the replication and assembly of rAAV.
  • the recombinant baculovirus is preferably constructed using the pFast.Bac. Dual (pFBD) shuttle vector of the Bac to Bac system (Fig. 1B), as follows:
  • the ITR core expression element was cloned into the P10 promoter and PH promoter spacer sequences of the pFBD vector.
  • the corresponding recombinant baculovirus is then prepared according to the method of the Bac to Bac system.
  • the ITR core expression element is linked to the expression cassette of the Cap gene or Rep gene via a 5'-end ligation nucleic acid fragment or a 3'-end ligation nucleic acid fragment, and the ITR core expression element comprises an inverted repeat in the AAV genome at both ends Sequence (ITR) and the target gene sequence (GOI) located in the middle.
  • ITR inverted repeat in the AAV genome at both ends Sequence
  • GOI target gene sequence located in the middle.
  • the ITR core expression element selects the GFP gene expression cassette containing the CMV promoter, GFP gene and SV40ploy A component, which facilitates the verification of the technical scheme.
  • the Cap gene encodes three structural proteins VP1, VP2, and VP3 of AAV, which constitute the capsid of the virus.
  • AAV binds to the surface of host cells by binding of capsid proteins to receptors on the cell surface.
  • AAVs of different serotypes have different range and efficiency of infection when infecting different types of tissues or cells, mainly due to differences in different serotype Cap genes. Caused. Therefore, to prepare rAAV of different serotypes, the Cap gene of the corresponding serotype is used.
  • the Cap gene was codon-optimized according to the ribosome leak scanning principle, and one mRNA was transcribed by the P10 promoter or the PH promoter to achieve three capsid proteins of VP1, VP2 and VP3 to a natural ratio (1:1:10). Functional expression.
  • the Rep gene encodes four non-structural proteins Rep78, Rep68, Rep52 and Rep40 of AAV, which are mainly responsible for viral genome replication, transcriptional regulation, site-specific integration and the like.
  • Rep gene was codon-optimized according to the principle of ribosome leakage scanning, and an mRNA was transcribed by the P10 promoter or the PH promoter to realize functional expression of the Rep gene.
  • the recombinant baculovirus provided by the present invention can be prepared as follows:
  • the ITR core expression elements were obtained by gene synthesis, PCR amplification and restriction enzyme ligation.
  • step B The ITR core expression element obtained in step A and the Cap gene or Rep gene were constructed on the pFast.Bac.Dual (pFBD) shuttle vector by molecular cloning method, and the recombinant baculovirus was obtained according to the Bac to Bac system operation method. .
  • pFBD pFast.Bac.Dual
  • the host packaging cells are used to aid in the replication and assembly of rAAV.
  • the host cell when the recombinant baculovirus does not contain a Rep gene, the host cell must integrate a Rep gene expression cassette element that expresses AAV on the genome; when the recombinant baculovirus does not contain a Cap gene, the host cell
  • the Cap gene expression cassette element that induces expression of AAV must be integrated into the genome, and the plasmid carrying the corresponding gene expression element can be randomly integrated into the genome of the host cell by transfecting the host cell with the plasmid.
  • the host cell preferably Sf9 cells, which induces expression of the Rep gene expression cassette element of AAV is randomly integrated, and the specific preparation method is as follows: First, in the existing pIR-rep78-hr2-RBE plasmid (refer to Proc Natl Acad Sci U S A. 2009Mar31;106(13):5059-64) was modified based on the fusion of the green fluorescent protein (GFP) gene by FMDV self-cleaving polypeptide 2A at the C-terminus of the blasticidin (Bsd) gene. As shown in Figure 2E.
  • GFP green fluorescent protein
  • the engineered plasmid was transfected into Sf9 cells, and a Sf9/Rep packaging cell line in which a Rep gene expression element which induces expression of AAV was integrated was screened by Bsd antibiotic.
  • the cell line constitutively expresses GFP, and can be further isolated to obtain a Sf9/Rep packaging cell line with high rAAV yield by monoclonal isolation culture or flow cytometry.
  • the host cell which is a Sf9 cell, is randomly integrated with a Cap gene expression cassette element which induces expression of AAV, and the specific preparation method is as follows: First, in the existing pIR-VP-hr2-RBE plasmid (refer to Proc Natl Acad Sci U S A .2009Mar31;106(13):5059-64) was modified on the basis of FMDV at the C-terminus of the blasticidin (Bsd) gene. The cleavage polypeptide 2A fused the green fluorescent protein (GFP) gene as shown in Figure 2F.
  • GFP green fluorescent protein
  • the engineered plasmid was transfected into Sf9 cells, and the Sf9/Cap packaging cell line in which the Cap gene expression element which induces expression of AAV was integrated was screened by Bsd antibiotic.
  • the cell line constitutively expresses GFP, and can be further isolated to obtain a Sf9/Cap packaging cell line with high rAAV yield by monoclonal isolation culture or flow cytometry.
  • the corresponding host packaging cell line ( Figure 3) was infected with the recombinant baculovirus (BEV) described above.
  • the host packaging cell line infected with the recombinant baculovirus in the step (1) is expanded and cultured to produce a large amount of recombinant adeno-associated virus.
  • BEV complex baculovirus
  • MOI multiplicity of infection
  • rAAV is mainly present in cell pellets. Separating and purifying the prepared rAAV,
  • the recombinant baculovirus provided by the present invention is characterized in that an adeno-associated virus ITR core expression element and a Cap gene or a Rep gene of a corresponding serotype are constructed on the genome.
  • the sequence of the Cap gene or Rep gene is a sequence that is codon optimized according to the principle of ribosome leak scanning.
  • AAV2 adeno-associated virus
  • Example 1 Recombinant baculovirus BEV/Cap-(ITR-GFP) infection Sf9/Rep packaging cell line preparation rAAV
  • the recombinant baculovirus which is a recombinant baculovirus BEV/Cap-(ITR-GFP), constructed with the recombinant adeno-associated virus ITR core expression element and the corresponding serotype Cap gene, was prepared and amplified as follows:
  • the Cap gene sequence is as SEQ ID No. 1, SEQ ID No. 2 (the two types correspond to CapA, CapB).
  • the ITR core expression element ITR selects the ITR nucleic acid sequence of type 2 AAV, ie the sequence shown in SEQ ID No. 3, and the core expression element of ITR adopts the expression cassette containing green fluorescent protein (GFP), which is initiated by CMV. The sub-controls the expression of GFP to facilitate detection of the activity of the recombinant virus.
  • the ITR core expression element is linked to the Cap gene expression cassette or vector via a 5' ligated nucleic acid fragment or a 3' ligated nucleic acid fragment.
  • the 5'-end or 3'-end ligated nucleic acid fragment is the sequence shown as SEQ ID No. 4 (link A) or SEQ ID No. 5 (link B).
  • the main component combination scheme in the recombinant baculovirus is as follows:
  • the pFBD/Cap-(ITR-GFP) recombinant shuttle plasmid was constructed by placing the ITR core expression element (ITR-GFP) on one side of the pFBD/Cap vector by ligation of the nucleic acid fragment using conventional molecular cloning techniques.
  • the recombinant shuttle plasmid was transformed into DH10Bac bacteria containing the AcMNPV baculovirus genome, following the Bac to Bac system operating instructions.
  • the recombinant baculovirus genome (Bacmid) was obtained by Tn7 transposon mediated recombination. Positive bacteria containing recombinant Bacmid were obtained by blue-white spot screening and PCR identification.
  • Recombinant Bacmid was purified by extraction and transfected into adherently cultured Sf9 cells. Sf9 cells were gradually infected with the recombinant baculovirus produced by transfection and showed significant cytopathic effect (CPE) of BV-infected Sf9 cells.
  • CPE cytopathic effect
  • the supernatant was infected with adherently cultured Sf9 cells and cultured for 3 days. It can be seen that the uninfected Sf9 cells in the control group were in a normal state without GFP expression.
  • the recombinant baculovirus BEV/Cap- was infected. Significant CPE and significant GFP expression were observed in Sf9 cells (ITR-GFP), and the results are shown in Figure 4A.
  • the BEV prepared by transfecting Sf9 cells was infected with adherent or suspension cultured Sf9 cells.
  • the titer of the virus was determined by a method of real-time quantitative PCR, which is a reference (Proc Natl Acad Sci U S A, 2009. 106 (13): p. 5059-64).
  • the corresponding host packaging cell line the Sf9/Rep packaging cell line that induces expression of the Rep gene, was established as follows:
  • the existing pIR-rep78-hr2-RBE plasmid (Proc Natl Acad Sci U S A. 2009 Mar 31; 106 (13): 5059-64) was modified to kill rice.
  • the C-terminus of the blasticidin (Bsd) gene was fused to the green fluorescent protein (GFP) gene by FMDV self-cleaving polypeptide 2A to obtain a pIR-rep78-hr2-RBE-bsd-GFP plasmid (Fig. 2E).
  • the plasmid was transfected into Sf9 cells, and a Sf9/Rep packaging cell line in which a Rep gene expression element which induces expression of AAV was integrated was screened by Bsd antibiotic.
  • the cell line constitutively expresses GFP, and can be further isolated by monoclonal isolation or flow cytometry to obtain a Sf9/Rep packaging cell line having a higher rAAV yield, as shown in Fig. 4A.
  • CPE cytopathic effect
  • rAAV has no envelope, the activity is not affected after treatment at 60 ° C for 30 minutes; while the recombinant baculovirus (BEV) has an envelope and is inactivated after treatment at 60 ° C for 30 minutes.
  • BEV baculovirus
  • the results showed that the rAAV control group prepared by the conventional three-plasmid transfection of HEK293 cells showed no GFP fluorescence, indicating that rAAV was not infectious to Sf9 cells.
  • the supernatant of the medium after BEV infection of Sf9 cells showed strong GFP expression before inactivation, and no GFP expression after inactivation.
  • untreated GFP was expressed, and no GFP expression was observed after treatment.
  • Sf9/Rep cell pellet of about 1 ⁇ 108 cells was collected, and 10 ml of lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 2 mM MgCl 2 , pH 8.0) was added, followed by repeated freeze-thaw three times and centrifugation at 5000 rpm for 5 min. After the supernatant was collected, the nuclease Benzonase was added to the supernatant to a final concentration of 50 U/ml, and treated in a water bath at 37 ° C for 60 min. The supernatant was collected by centrifugation at 5000 rpm for 10 min after the treatment.
  • lysis buffer 50 mM Tris-Cl, 150 mM NaCl, 2 mM MgCl 2 , pH 8.0
  • the supernatant was extracted with chloroform, and the extracted supernatant was further purified by two-phase precipitation using a solution containing 13.2% of (NH4)2SO4 and 10% of PEG 8000. Reference method (J Virol Methods, 2007. 139(1) ): p. 61-70, J Virol Methods, 2012. 179(1): p.276-80.).
  • the supernatant after the two-phase precipitation was subjected to dialysis desalting treatment with a PBS solution, and concentrated by centrifugation to a final volume of 1 ml using an Amicon ultra-4 (100 KD cutoff) dialysis column, and frozen at -80 ° C after aseptic dispensing.
  • the titer of rAAV was determined by fluorescence quantitative PCR, and the titer unit was expressed by VG/ml (VG, virus genomes).
  • the rAAV yield of the purification process is shown in Table 1.
  • the experimental results showed that the yield of rAAV in a single Sf9/Rep packaging cell could reach 8.62 ⁇ 104 VG. After purification by this method, the recovery rate reached about 36.9%.
  • HEK293 cells were seeded at 1 x 104 cells/well into 96-well plates, and after 6 h, the corresponding concentration gradients of purified rAAV were infected. After 48 hours of infection, the expression of GFP was observed by fluorescence microscopy, as shown in Fig. 4C. The results show that the method of the invention is prepared The obtained rAAV has a good cell-level transduction activity.
  • Example 2 Preparation of rAAV by recombinant baculovirus BEV/Cap-(ITR-GFP) infection of Sf9/Rep packaging cell line
  • the prepared rAAV was purified.
  • the rAAV yield of the purification process is shown in Table 2.
  • the experimental results show that the yield of rAAV in a single Sf9/Rep packaging cell can reach 7.20 ⁇ 104 VG. After purification by this method, the recovery rate is about 31.3%.
  • HEK293 cells were seeded at 1 x 104 cells/well into 96-well plates, and after 6 h, the corresponding concentration gradients of purified rAAV were infected. After 48 hours of infection, the expression of GFP was observed by fluorescence microscopy, as shown in Fig. 5. The results showed that the rAAV prepared by the method of the present invention has good cell-level transduction activity.
  • Example 3 Recombinant baculovirus BEV/Rep-(ITR-GFP) infection Sf9/Cap packaging cell line preparation rAAV
  • the recombinant baculovirus which is a recombinant baculovirus BEV/Rep-(ITR-GFP), constructed with the recombinant adeno-associated virus ITR core expression element and the phase Rep gene, was prepared and amplified as follows:
  • the ITR core expression element selects the ITR nucleic acid sequence of type 2 AAV, ie the sequence shown in SEQ ID No. 3, and the core expression element of ITR adopts the expression cassette containing green fluorescent protein (GFP), which is initiated by CMV. The sub-controls the expression of GFP to facilitate detection of the activity of the recombinant virus.
  • the ITR core expression element is ligated to the Rep gene expression cassette or vector via a 5' ligated nucleic acid fragment or a 3' ligated nucleic acid fragment.
  • the 5'-end or 3'-end ligated nucleic acid fragment is the sequence shown in SEQ ID No. 4 (link A) or SEQ ID No. 5 (link B).
  • the main component combination scheme in the recombinant baculovirus is as follows:
  • ITR core expression element (ITR-GFP) was placed on the pFBD/Rep vector by ligation of nucleic acid fragments using conventional molecular cloning techniques
  • a pFBD/Rep-(ITR-GFP) recombinant shuttle plasmid was constructed.
  • the recombinant shuttle plasmid was transformed into DH10Bac bacteria containing the AcMNPV baculovirus genome, following the Bac to Bac system operating instructions.
  • the recombinant baculovirus genome (Bacmid) was obtained by Tn7 transposon mediated recombination. Positive bacteria containing recombinant Bacmid were obtained by blue-white spot screening and PCR identification.
  • Recombinant Bacmid was purified by extraction and transfected into adherently cultured Sf9 cells. Sf9 cells were gradually infected with the recombinant baculovirus produced by transfection and showed significant cytopathic effect (CPE) of BV-infected Sf9 cells.
  • CPE cytopathic effect
  • the supernatant was infected with adherently cultured Sf9 cells and cultured for 3 days. It can be seen that the uninfected Sf9 cells in the control group were in a normal state without GFP expression.
  • the recombinant baculovirus BEV/Rep- was infected. Significant CPE and significant GFP expression were observed in Sf9 cells (ITR-GFP), and the results are shown in Figure 6A.
  • the BEV prepared by transfecting Sf9 cells was infected with adherent or suspension cultured Sf9 cells.
  • the titer of the virus was determined by a method of real-time quantitative PCR, which is a reference (Proc Natl Acad Sci U S A, 2009. 106 (13): p. 5059-64).
  • the corresponding host packaging cell line the Sf9/Cap packaging cell line that induces expression of the Cap gene, was established as follows:
  • the pIR-VP-hr2-RBE plasmid (refer to Proc Natl Acad Sci U S A.2009 Mar31; 106(13): 5059-64) was modified on the rice blast fungus.
  • the C-terminus of the blasticidin (Bsd) gene was fused to the green fluorescent protein (GFP) gene by FMDV self-cleaving polypeptide 2A to obtain a pIR-VP-hr2-RBE-bsd-GFP plasmid (Fig. 2F).
  • the plasmid was transfected into Sf9 cells, and a Sf9/Cap packaging cell line in which a Cap gene expression element which induces expression of AAV was integrated was screened by Bsd antibiotic.
  • the cell line constitutively expresses GFP, and can be further isolated by monoclonal isolation or flow cytometry to obtain a Sf9/Cap packaging cell line with higher rAAV yield, as shown in Fig. 6A.
  • CPE cytopathic effect
  • active rAAV was prepared.
  • the experimental method of virus infection of HEK293 cells and Sf9 cells confirmed that the system prepared rAAV, and the experimental results are shown in Fig. 6B.
  • the specific implementation process and results were analyzed as follows: After the cell pellet was repeatedly freeze-thawed and lysed 3 times, the cell lysis supernatant was collected by centrifugation at 5000 rpm for 5 min.
  • rAAV has no envelope, the activity is not affected after treatment at 60 ° C for 30 minutes; while the recombinant baculovirus (BEV) has an envelope and is inactivated after treatment at 60 ° C for 30 minutes.
  • BEV baculovirus
  • the results showed that the rAAV control group prepared by the conventional three-plasmid transfection of HEK293 cells showed no GFP fluorescence, indicating that rAAV was not infectious to Sf9 cells.
  • the supernatant of the medium after BEV infection of Sf9 cells showed strong GFP expression before inactivation, and no GFP expression after inactivation.
  • untreated GFP was expressed, and no GFP expression was observed after treatment.
  • Sf9/Cap cell pellet of about 1 ⁇ 108 cells was collected, and 10 ml of lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 2 mM MgCl 2 , pH 8.0) was added, followed by repeated freeze-thaw three times and centrifugation at 5000 rpm for 5 min. After the supernatant was collected, the nuclease Benzonase was added to the supernatant to a final concentration of 50 U/ml, and treated in a water bath at 37 ° C for 60 min. The supernatant was collected by centrifugation at 5000 rpm for 10 min after the treatment.
  • lysis buffer 50 mM Tris-Cl, 150 mM NaCl, 2 mM MgCl 2 , pH 8.0
  • the supernatant was extracted with chloroform, and the extracted supernatant was further purified by two-phase precipitation using a solution containing 13.2% of (NH4)2SO4 and 10% of PEG 8000. Reference method (J Virol Methods, 2007. 139(1) ): p. 61-70. and J Virol Methods, 2012. 179(1): p.276-80.).
  • the supernatant after the two-phase precipitation was subjected to dialysis desalting treatment with a PBS solution, and concentrated by centrifugation to a final volume of 1 ml using an Amicon ultra-4 (100 KD cutoff) dialysis column, and frozen at -80 ° C after aseptic dispensing.
  • the titer of rAAV was determined by fluorescence quantitative PCR, and the titer unit was expressed by VG/ml (VG, virus genomes).
  • the rAAV yield of the purification process is shown in Table 3.
  • the experimental results show that the yield of rAAV in a single Sf9/Cap cell can reach 6.84 ⁇ 104 VG. After purification by this method, the recovery rate is about 28.8%.
  • HEK293 cells were seeded at 1 x 104 cells/well into 96-well plates, and after 6 h, the corresponding concentration gradients of purified rAAV were infected. After 48 hours of infection, the expression of GFP was observed by fluorescence microscopy, as shown in Fig. 6C. The results showed that the rAAV prepared by the method of the present invention has good cell-level transduction activity.
  • Example 4 Recombinant baculovirus BEV/Rep-(ITR-GFP) infection Sf9/Cap packaging cell line preparation rAAV
  • the prepared rAAV was purified.
  • the rAAV yield of the purification process is shown in Table 4. The experimental results showed that the yield of rAAV in a single Sf9/Cap packaging cell could reach 8.16 ⁇ 104 VG. After purification by this method, the recovery rate reached about 34.7%.
  • HEK293 cells were seeded at 1 x 104 cells/well into 96-well plates, and after 6 h, the corresponding concentration gradients of purified rAAV were infected. After 48 hours of infection, the expression of GFP was observed by fluorescence microscopy, as shown in Fig. 7. The results showed that the rAAV prepared by the method of the present invention has good cell-level transduction activity.

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Abstract

提供了一种重组腺相关病毒的制备方法和重组杆状病毒，所述制备方法包括以下步骤：(1)将构建有重组腺相关病毒ITR核心表达元件和Cap基因或Rep基因的重组型杆状病毒感染相应的宿主包装细胞系；(2)将步骤(1)中感染了重组杆状病毒的宿主包装细胞系扩大培养，使产生重组腺相关病毒；(3)将步骤(2)中获得的重组腺相关病毒分离并纯化。

Description

一种重组腺相关病毒的制备方法及重组杆状病毒 [技术领域]
本发明属于生物工程领域，更具体地，涉及一种重组腺相关病毒的制备方法及重组杆状病毒。
[背景技术]
重组腺相关病毒(rAAV)具有安全性高、免疫原性低、宿主范围广、能介导外源基因在动物体内长期稳定表达等特点，是基因治疗领域最具应用前景的载体之一。rAAV在神经科学研究以及疾病的基因治疗等领域具有重要作用和巨大需求。有研究数据表明，一次大型灵长类动物试验或临床基因治疗试验就需要约1015VG(VG，virus genomes)的rAAV，是一般体外细胞试验或小鼠试验用量的成百上千倍(Hum Gene Ther.2002Nov 1；13(16):1935-43.)。
目前利用杆状病毒表达系统大规模制备rAAV的方法主要有以下两种：两杆状病毒系统(Two Bac system)和依赖包装细胞系的一杆状病毒系统(One Bac system)。两杆状病毒系统的主要流程是，将AAV的Rep基因和Cap基因整合在一个杆状病毒基因组中，将ITR核心表达元件整合到另外一个杆状病毒基因组中，然后将上述两种重组杆状病毒共同感染宿主细胞，产生出rAAV(Mol Ther.2008May；16(5):924-30，Mol Ther.2009Nov；17(11):1888-96.)。依赖包装细胞系的一杆状病毒系统的主要流程是，先建立诱导表达Rep基因和Cap基因的包装细胞系，这种包装细胞系整合了Rep基因和Cap基因表达元件，Rep基因和Cap基因分别置于杆状病毒晚期基因表达强启动子PH调控下，在PH启动子的上游加入了hr2增强子序列和AAV的Rep蛋白结合序列。在感染整合了ITR核心表达元件的重组杆状病毒后，包装细胞系中的Rep基因和Cap基因被诱导表达，进而产生rAAV(Proc Natl Acad Sci U S A.2009Mar 31；106(13):5059-64，Hum Gene Ther.2014Mar；25(3):212-22.)。
然而，前者由于两种杆状病毒共同感染细胞效率较低，无法充分利用每个细胞的产能，而且感染是个随机的过程，容易产生不含核酸的空壳缺陷rAAV颗粒，制备工艺条件优化起来比较复杂，不同批次制备的rAAV质量不稳定。后者由于依赖诱导表达Rep基因和Cap基因的包装细胞系，构建复杂，优良包装细胞系的筛选难度大，制备不同血清型的rAAV，就需要建立表达相应血清型Cap基因的包装细胞系，灵活性和通用性较差，难以推广。
[发明内容]
针对现有技术的以上缺陷或改进需求，本发明提供了一种基因治疗用重组腺相关病毒的制备方法及重组杆状病毒，其目的在于通过将AAV的Rep基因、Cap基因和ITR核心表达元件进行优化整合，构建到杆状病毒基因组中或者整合到宿主包装细胞基因组中，实现利用一种重组杆状病毒感染宿主包装细胞系制备rAAV，由此解决现有的大规模制备rAAV方法存在的构建复杂、灵活性差、通用性低的技术问题。
为实现上述目的，按照本发明的一个方面，提供了一种重组腺相关病毒的制备方法，包括以下 步骤：
(1)将构建有重组腺相关病毒ITR核心表达元件和Cap基因或Rep基因的重组型杆状病毒感染相应的宿主包装细胞系；
(2)将步骤(1)中感染了重组杆状病毒的宿主包装细胞系扩大培养，使产生重组腺相关病毒；
(3)将步骤(2)中获得的重组腺相关病毒分离并纯化。
优选地，所述制备方法，其步骤(1)利用pFast.Bac.Dual穿梭载体构建所述重组型杆状病毒。
优选地，所述制备方法，其所述步骤(1)具体操作方法如下：
a.将ITR核心表达元件克隆到pFast.Bac.Dual穿梭载体的P10启动子和PH启动子间隔序列中；
b.将Cap基因或Rep基因克隆到pFast.Bac.Dual穿梭载体的P10启动子或PH启动子下游的多克隆位点中，得到相应的穿梭质粒。
优选地，所述制备方法，其所述构建有重组腺相关病毒ITR核心表达元件和Cap基因(或Rep基因)的重组型杆状病毒其ITR核心表达元件中携带有目标基因。
优选地，所述制备方法，其所述宿主包装细胞系用于辅助重组腺相关病毒的复制和组装。
优选地，所述制备方法，其所述宿主细胞基因组上相应整合有诱导表达腺相关病毒Rep基因的表达框元件或诱导表达腺相关病毒Cap基因的表达框元件。
按照本发明的另一方面，提供了一种重组杆状病毒，其基因组上构建有腺相关病毒ITR核心表达元件和相应血清型的Cap基因。
优选地，所述重组杆状病毒，其Cap基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列。
按照本发明的另一方面，提供了一种重组杆状病毒，其基因组上构建有腺相关病毒ITR核心表达元件和相应血清型的Rep基因。
优选地，所述重组杆状病毒，其Rep基因其序列为依据核糖体泄露原理进行密码子优化的序列。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能取得以下有益效果：
由于将AAV的Cap基因(或Rep基因)与ITR核心表达元件放在一个重组杆状病毒中，相应的将Rep基因(或Cap基因)整合到宿主细胞基因组中，由宿主包装细胞系提供包装rAAV所需的部分辅助功能。相比现有的依赖包装细胞系的一杆状病毒系统将Rep基因和Cap基因全部都整合到宿主细胞系中，构建包装细胞系的难度大大降低；尤其是将Cap基因与ITR核心元件置于一个杆状病毒基因组中的方法，由于通常采用2型AAV的Rep基因就可以辅助各种血清型rAAV的包装，所以只需要建立一种诱导表达2型AAV的Rep基因就可以满足不同血清型rAAV的制备，灵活性和通用性也将大大增强。
[附图说明]
图1是rAAV的包装原理图(A)和杆状病毒表达系统(Bac to Bac)中穿梭质粒pFast.Bac.Dual(pFBD)的结构示意图(B)；
图2是实施例中整合了制备rAAV所需的重组杆状病毒和包装细胞系所需组件的结构示意图。 其中，图2A是实例1中重组穿梭质粒pFD/Cap-(ITR-GFP)的结构图；图2B是实例2中重组穿梭质粒pFD/Cap-(ITR-GFP)的结构图；图2C是实例3中重组穿梭质粒pFD/Rep-(ITR-GFP)的结构图；图2D是实例4中重组穿梭质粒pFD/Rep-(ITR-GFP)的结构图；图2E是实例1和实例2中建立Sf9/Rep包装细胞系所用pIR-rep78-hr2-RBE-bsd-GFP质粒的结构图；图2F是实例3和实例4中建立Sf9/Cap包装细胞系所用pIR-VP-hr2-RBE-bsd-GFP质粒的结构图；
图3是实施例中利用重组杆状病毒感染宿主包装细胞系制备rAAV的过程示意图。其中，图3A是实施例1和实施例2的过程示意图；图3B是实施例3和实施例4的过程示意图；
图4是实施例1中重组杆状病毒感染包装细胞系制备rAAV以及rAAV细胞水平转导活性的验证图；其中，图4A是重组杆状病毒BEV/Cap-(ITR-GFP)感染Sf9细胞，筛选出的Sf9/Rep包装细胞系及其被重组BEV感染后的荧光显微成像图；图4B是实例1制备的rAAV感染HEK293和Sf9细胞后的荧光显微成像图；图4C是实例1纯化的rAAV感染HEK293细胞的荧光显微成像图；
图5是实施例2中制备纯化的rAAV感染HEK293细胞的荧光显微成像图；
图6是实施例3中重组杆状病毒感染包装细胞系制备rAAV以及rAAV细胞水平转导活性的验证图。其中，图6A是重组杆状病毒BEV/Rep-(ITR-GFP)感染Sf9细胞，筛选出的Sf9/Cap包装细胞系及其被重组BEV感染后的荧光显微成像图；图6B是实例4制备的rAAV感染HEK293和Sf9细胞后的荧光显微成像图；图6C是实例4纯化的rAAV感染HEK293细胞的荧光显微成像图；
图7是实施例4中制备纯化的rAAV感染HEK293细胞的荧光显微成像图。
[具体实施方式]
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的重组腺相关病毒的制备方法，包括以下步骤：
(1)将构建有重组腺相关病毒ITR核心表达元件和Cap基因或Rep基因的重组型杆状病毒感染相应的宿主包装细胞系。
所述重组杆状病毒用于提供制备rAAV所需的ITR核心表达元件和Cap基因或Rep基因，并通过感染宿主包装细胞系激活杆状病毒特异性启动子PH或P10，诱导宿主包装细胞系表达Rep基因或Cap基因从而辅助rAAV的复制和组装。
所述重组杆状病毒优选利用Bac to Bac系统的pFast.Bac.Dual(pFBD)穿梭载体(如图1B)构建，具体方法如下：
a.将ITR核心表达元件克隆到pFBD载体的P10启动子和PH启动子间隔序列中。
b.将Cap基因或Rep基因克隆到pFBD载体的P10启动子或PH启动子下游的多克隆位点中，得到相应的穿梭质粒。
c.然后按照Bac to Bac系统的方法制备相应的重组杆状病毒。
所述ITR核心表达元件通过5’端连接核酸片段或3’端连接核酸片段与所述Cap基因或Rep基因的表达框相连，ITR核心表达元件包括位于两端的AAV基因组中的一段末端反向重复序列(ITR)以及位于中间的目标基因序列(GOI)。ITR核心表达元件在实例中选用含CMV启动子、GFP基因、SV40ploy A组件的GFP基因表达框，便于技术方案的验证。
所述Cap基因编码AAV的三种结构蛋白VP1、VP2、VP3，构成病毒的衣壳。AAV通过衣壳蛋白与细胞表面的受体结合吸附到宿主细胞表面，不同血清型的AAV在感染不同类型组织或细胞时存在感染的范围和效率的差异，主要是由于不同血清型Cap基因的差异造成的。因此，制备不同血清型的rAAV，就要使用相应血清型的Cap基因。所述Cap基因依据核糖体泄露扫描原理进行了密码子优化，通过P10启动子或PH启动子转录出一条mRNA，实现VP1、VP2、VP3三种衣壳蛋白以接近天然比例(1:1:10)的功能性表达。
所述Rep基因编码AAV的四种非结构蛋白Rep78、Rep68、Rep52和Rep40，主要负责病毒基因组的复制，转录调控，位点特异性整合等。目前，通常使用2型AAV的Rep基因制备不同血清型的rAAV。所述Rep基因依据核糖体泄露扫描原理进行了密码子优化，通过P10启动子或PH启动子转录出一条mRNA，实现Rep基因的功能性表达。
本发明提供的重组杆状病毒，可按照如下方法制备：
A、采用基因人工合成的方法获得密码子优化的Cap基因、Rep基因；
采用基因人工合成、PCR扩增和酶切连接的方法获得ITR核心表达元件。
B、通过分子克隆的方法将步骤A中获得的ITR核心表达元件以及Cap基因或Rep基因构建到pFast.Bac.Dual(pFBD)穿梭载体上，根据Bac to Bac系统操作方法，获得重组杆状病毒。
所述宿主包装细胞，优选为Sf9细胞，用于辅助rAAV的复制和组装。优选地，所述重组杆状病毒不含有Rep基因时，所述宿主细胞其基因组上须整合诱导表达AAV的Rep基因表达框元件；所述重组杆状病毒不含有Cap基因时，所述宿主细胞其基因组上须整合诱导表达AAV的Cap基因表达框元件，可通过将携带有相应基因表达元件的质粒转染宿主细胞后随机整合到宿主细胞的基因组中。
所述随机整合了诱导表达AAV的Rep基因表达框元件的宿主细胞，优选Sf9细胞，具体制备方法如下:首先，在现有pIR-rep78-hr2-RBE质粒(参考Proc Natl Acad Sci U S A.2009Mar31；106(13):5059-64)的基础上进行了改造，在杀稻瘟菌素(blasticidin,Bsd)基因的C端通过FMDV自剪切多肽2A融合了绿色荧光蛋白(GFP)基因，如图2E所示。然后，将改造后的质粒转染Sf9细胞，通过Bsd抗生素筛选得到整合了诱导表达AAV的Rep基因表达元件的Sf9/Rep包装细胞系。该细胞系组成性表达GFP，可通过单克隆分离培养或流式细胞仪分选，进一步得到rAAV产量较高的Sf9/Rep包装细胞系。
所述随机整合了诱导表达AAV的Cap基因表达框元件的宿主细胞，优选为Sf9细胞，具体制备方法如下:首先，在现有pIR-VP-hr2-RBE质粒(参考Proc Natl Acad Sci U S A.2009Mar31；106(13):5059-64)的基础上进行了改造，在杀稻瘟菌素(blasticidin,Bsd)基因的C端通过FMDV自 剪切多肽2A融合了绿色荧光蛋白(GFP)基因，如图2F所示。然后，将改造后的质粒转染Sf9细胞，通过Bsd抗生素筛选得到整合了诱导表达AAV的Cap基因表达元件的Sf9/Cap包装细胞系。该细胞系组成性表达GFP，可通过单克隆分离培养或流式细胞仪分选，进一步得到rAAV产量较高的Sf9/Cap包装细胞系。
由于基因整合是随机的，宿主细胞转染质粒后用抗生素筛选，只有基因整合拷贝数比较高的细胞抗性基因表达量高，报告基因GFP表达量也高，在被杆状病毒感染后诱导表达的辅助基因量也高。抗生素筛选后的细胞基因组上随机整合的诱导表达Rep基因或Cap基因的表达框至少有一个拷贝，也可能有多拷贝的。但宿主包装细胞系的优劣(rAAV产量高低)与拷贝数没有绝对的关系，表达量高须处于合适的范围之内，所以包装细胞系的优劣需要通过最终感染重组杆状病毒后制备rAAV的产量来做最终评价指标。
利用上述重组杆状病毒(BEV)感染相应的宿主包装细胞系(如图3所示)。
(2)将步骤(1)中感染了重组杆状病毒的宿主包装细胞系扩大培养，使产生大量重组腺相关病毒。
具体可按照如下步骤操作：摇瓶培养所述宿主包装细胞至细胞密度达到3×106cells/ml，以感染复数(MOI＝5)感染重组杆状病毒(BEV)，27℃、120rpm/min摇床培养。感染3天后，将细胞悬液3000rpm离心5min，收集培养液上清和细胞沉淀。
(3)将步骤(2)中获得的重组腺相关病毒分离并纯化。
rAAV主要存在于细胞沉淀中。对制备出的rAAV进行分离纯化操作，
就能够得到进一步应用所需的rAAV。具体方法步骤可参考文献(J Virol Methods,2007.139(1):p.61-70，J Virol Methods,2012.179(1):p.276-80.)。
本发明提供的重组杆状病毒，其特征在于，其基因组上构建有腺相关病毒ITR核心表达元件和相应血清型的Cap基因或Rep基因。所述Cap基因或Rep基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列。
下面仅以二型腺相关病毒(AAV2)为例，具体说明如下：
实施例1：重组杆状病毒BEV/Cap-(ITR-GFP)感染Sf9/Rep包装细胞系制备rAAV
(1)将构建有重组腺相关病毒ITR核心表达元件和相应血清型的Cap基因的重组杆状病毒感染相应的宿主包装细胞系
所述构建有重组腺相关病毒ITR核心表达元件和相应血清型的Cap基因的重组杆状病毒，即重组杆状病毒BEV/Cap-(ITR-GFP)，按照如下方法制备并扩增：
为了将制备rAAV所需的ITR核心表达元件以及Cap基因置于一个重组杆状病毒中。我们利用了杆状病毒表达系统(Bac to Bac)中的pFast.Bac.Dual(pFBD)穿梭载体(如图1B)。实例中基于2型AAV的Cap基因依据核糖体泄露扫描原理进行了密码子优化，并将Cap基因置于P10启动子的调控之下(如组合方案1，如图2A)或PH启动子(如组合方案2，如图2B)的调控之下，实现VP1、VP2、VP3三种衣壳蛋白以接近天然比例(1:1:10)的功能性表达。Cap基因序列为如SEQ ID No.1、 SEQ ID No.2(2种对应为CapA、CapB)所示的序列。实例中ITR核心表达元件，ITR选用2型AAV的ITR核酸序列，即如SEQ ID No.3所示的序列，ITR的核心表达元件采用了含有绿色荧光蛋白(GFP)的表达框，由CMV启动子控制GFP的表达，便于检测重组病毒的活性。ITR核心表达元件通过5’端连接核酸片段或3’端连接核酸片段与Cap基因表达框或载体相连。5’端或3’端连接核酸片段为如SEQ ID No.4(linkA)或SEQ ID No.5(linkB)所示的序列。
在本实例中，重组杆状病毒中主要组件组合方案如下：
CapA-LinkA-(ITR-GFP)-linkB
利用常规分子克隆技术将ITR核心表达元件(ITR-GFP)通过连接核酸片段置于pFBD/Cap载体的一侧，构建pFBD/Cap-(ITR-GFP)重组穿梭质粒。
参照Bac to Bac系统操作说明，将该重组穿梭质粒转化含有AcMNPV杆状病毒基因组的DH10Bac菌。通过Tn7转座子元件介导的重组得到重组杆状病毒基因组(Bacmid)。通过蓝白斑筛选和PCR鉴定获得含有重组Bacmid的阳性菌。抽提纯化重组Bacmid，将其转染贴壁培养的Sf9细胞。Sf9细胞逐渐被转染后产生的重组杆状病毒完全感染并出现明显的BEV感染Sf9细胞的细胞病变(CPE)现象。将细胞培养液3000rpm离心5min，上清中就含有产生的重组杆状病毒。
将上清液感染贴壁培养的Sf9细胞后继续培养3天，可以看到对照组未感染病毒的Sf9细胞状态正常，没有GFP的表达；而在实验组感染了重组杆状病毒BEV/Cap-(ITR-GFP)的Sf9细胞都出现了明显的CPE现象和明显的GFP表达，结果如图4A所示。将转染Sf9细胞后制备的BEV感染贴壁或悬浮培养的Sf9细胞，感染3天后细胞出现明显CPE，将细胞培养液3000rpm离心5min，收上清即可获得大量BEV。病毒的滴度用荧光定量PCR的方法进行测定，参考文献(Proc Natl Acad Sci U S A,2009.106(13):p.5059-64)中的方法。
所述相应的宿主包装细胞系，即诱导表达Rep基因的Sf9/Rep包装细胞系，按照如下方法建立：
为了便于包装细胞系的筛选，在现有pIR-rep78-hr2-RBE质粒(Proc Natl Acad Sci U S A.2009Mar 31；106(13):5059-64)的基础上进行了改造，在杀稻瘟菌素(blasticidin,Bsd)基因的C端通过FMDV自剪切多肽2A融合了绿色荧光蛋白(GFP)基因，得到pIR-rep78-hr2-RBE-bsd-GFP质粒(如图2E)。然后，将该质粒转染Sf9细胞，通过Bsd抗生素筛选得到整合了诱导表达AAV的Rep基因表达元件的Sf9/Rep包装细胞系。该细胞系组成性表达GFP，可通过单克隆分离培养或流式细胞仪分选，进一步得到rAAV产量较高的Sf9/Rep包装细胞系，如图4A所示。
(2)利用BEV/Cap-(ITR-GFP)感染Sf9/Rep细胞系制备rAAV并验证其感染活性
将制备的BEV/Cap-(ITR-GFP)以感染复数(MOI＝5)感染培养的Sf9/Rep细胞系。感染72小时后，将细胞培养液3000rpm离心5min，分别收集培养上清和细胞沉淀。BEV产生后，主要通过分泌释放到培养基上清中，在Sf9/Rep细胞中也有部分未释放的BEV。rAAV产生后，主要存在于Sf9/Rep细胞核中，由于Sf9/Rep细胞感染后发生细胞病变(CPE)，部分细胞会裂解，rAAV也会有部分释放到上清中，如图4A所示。而因此上清和细胞沉淀中会同时存在BEV和rAAV。
为了验证用重组杆状病毒BEV/Cap-(ITR-GFP)感染Sf9/Rep细胞制备出了具有活性的rAAV。通 过病毒感染HEK293细胞和Sf9细胞的实验方法证实该系统制备出了rAAV，实验结果如图4B。具体实施过程和结果分析如下：将细胞沉淀反复冻融3次裂解后，5000rpm离心5min收集细胞裂解上清液。因为rAAV无包膜，经60℃、30分钟处理，活性不受影响；而重组杆状病毒(BEV)有包膜，经60℃、30分钟处理，失去活性。含有病毒的上清液感染HEK293细胞2天后，利用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，在处理前后都有GFP表达，表明rAAV在处理前后都有感染活性。BEV感染Sf9细胞后的培养基上清组，未处理的有较强GFP表达，处理后仅有无GFP表达，表明BEV可以感染HEK293细胞，灭活处理后BEV不感染。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后GFP强度有降低但仍有较强荧光，表明灭活处理后BEV不感染，但上清中大量的rAAV仍能够感染。用与感染HEK293细胞组相同的样品感染Sf9细胞2天后，结果显示：利用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，没有GFP荧光，表明rAAV对Sf9细胞没有感染性。BEV感染Sf9细胞后的培养基上清组，灭活前有较强GFP表达，灭活后的没有GFP表达。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后的没有GFP表达。
(3)将步骤(2)中获得的重组腺相关病毒分离并纯化。
rAAV的纯化、滴度测定与细胞水平转导活性验证：
将重组BEV感染后收集得到约为1×108cells的Sf9/Rep细胞沉淀，加入10ml裂解缓冲液(50mM Tris-Cl，150mM NaCl，2mM MgCl2，pH 8.0)，后反复冻融3次，5000rpm离心5min后收集上清，在上清中加入核酸酶Benzonase至终浓度50U/ml，37℃水浴处理60min。处理后5000rpm离心10min收上清。上清用氯仿抽提，抽提后的上清再用含13.2％的(NH4)2SO4和10％的PEG8000的溶液进行两相沉淀的方法进一步纯化，方法参考文献(J Virol Methods,2007.139(1):p.61-70，J Virol Methods,2012.179(1):p.276-80.)。两相沉淀后的上清，用PBS溶液进行透析脱盐处理，用Amicon ultra-4(100KD cutoff)透析柱离心浓缩至终体积1ml，无菌分装后-80℃冻存。rAAV的滴度测定采用荧光定量PCR的方法，滴度单位用VG/ml(VG，virus genomes)表示。
纯化过程的rAAV得率见表1。实验结果表明，单个Sf9/Rep包装细胞rAAV的产量可达8.62×104VG，经过本方法的纯化后，回收率达到36.9％左右。
表1rAAV纯化过程的得率分析
Figure PCTCN2016073246-appb-000001
将HEK293细胞以1×104cells/well接种到96孔板中，6h后感染相应浓度梯度含量的纯化的rAAV。感染48h后，用荧光显微镜观察GFP的表达情况，如图4C。结果表明，本发明的方法制备 得到的rAAV具有较好的细胞水平转导活性。
实施例2：重组杆状病毒BEV/Cap-(ITR-GFP)感染Sf9/Rep包装细胞系制备rAAV
本实施例与实施例1相似，区别仅在于所述重组杆状病毒，其主要组件组合方案如下：
LinkA-(ITR-GFP)-linkB-CapB
将本实例中的重组杆状病毒BEV/Cap-(ITR-GFP)感染Sf9/Rep包装细胞系后，将制备得到的rAAV进行纯化。纯化过程的rAAV得率见表2。实验结果表明，单个Sf9/Rep包装细胞rAAV的产量可达7.20×104VG，经过本方法的纯化后，回收率达到31.3％左右。
表2rAAV纯化过程的得率分析
Figure PCTCN2016073246-appb-000002
将HEK293细胞以1×104cells/well接种到96孔板中，6h后感染相应浓度梯度含量的纯化的rAAV。感染48h后，用荧光显微镜观察GFP的表达情况，如图5。结果表明，本发明的方法制备得到的rAAV具有较好的细胞水平转导活性。
实施例3：重组杆状病毒BEV/Rep-(ITR-GFP)感染Sf9/Cap包装细胞系制备rAAV
(1)将构建有重组腺相关病毒ITR核心表达元件和Rep基因的重组杆状病毒感染相应的宿主包装细胞系
所述构建有重组腺相关病毒ITR核心表达元件和相Rep基因的重组杆状病毒，即重组杆状病毒BEV/Rep-(ITR-GFP)，按照如下方法制备并扩增：
为了将制备rAAV所需的ITR核心表达元件以及Rep基因置于一个重组杆状病毒中。我们利用了杆状病毒表达系统(Bac to Bac)中的pFast.Bac.Dual(pFBD)穿梭载体(如图1B)。实例中基于2型AAV的Rep基因依据核糖体泄露扫描原理进行了密码子优化，并将Rep基因置于P10启动子的调控之下(如图2C)或PH启动子(如图2D)的调控之下，实现Rep基因的功能性表达。Rep基因序列为如SEQ ID No.6、SEQ ID No.7(2种对应为RepA、RepB)所示的序列。实例中ITR核心表达元件，ITR选用2型AAV的ITR核酸序列，即如SEQ ID No.3所示的序列，ITR的核心表达元件采用了含有绿色荧光蛋白(GFP)的表达框，由CMV启动子控制GFP的表达，便于检测重组病毒的活性。ITR核心表达元件通过5’端连接核酸片段或3’端连接核酸片段与Rep基因表达框或载体相连。5’端或3’端连接核酸片段为如SEQ ID No.4(linkA)或SEQ ID No.5(linkB)所示的序列。
在本实例中，重组杆状病毒中主要组件组合方案如下：
RepA-LinkA-(ITR-GFP)-linkB
利用常规分子克隆技术将ITR核心表达元件(ITR-GFP)通过连接核酸片段置于pFBD/Rep载体的 一侧，构建pFBD/Rep-(ITR-GFP)重组穿梭质粒。
参照Bac to Bac系统操作说明，将该重组穿梭质粒转化含有AcMNPV杆状病毒基因组的DH10Bac菌。通过Tn7转座子元件介导的重组得到重组杆状病毒基因组(Bacmid)。通过蓝白斑筛选和PCR鉴定获得含有重组Bacmid的阳性菌。抽提纯化重组Bacmid，将其转染贴壁培养的Sf9细胞。Sf9细胞逐渐被转染后产生的重组杆状病毒完全感染并出现明显的BEV感染Sf9细胞的细胞病变(CPE)现象。将细胞培养液3000rpm离心5min，上清中就含有产生的重组杆状病毒。
将上清液感染贴壁培养的Sf9细胞后继续培养3天，可以看到对照组未感染病毒的Sf9细胞状态正常，没有GFP的表达；而在实验组感染了重组杆状病毒BEV/Rep-(ITR-GFP)的Sf9细胞都出现了明显的CPE现象和明显的GFP表达，结果如图6A所示。将转染Sf9细胞后制备的BEV感染贴壁或悬浮培养的Sf9细胞，感染3天后细胞出现明显CPE，将细胞培养液3000rpm离心5min，收上清即可获得大量BEV。病毒的滴度用荧光定量PCR的方法进行测定，参考文献(Proc Natl Acad Sci U S A,2009.106(13):p.5059-64)中的方法。
所述相应的宿主包装细胞系，即诱导表达Cap基因的Sf9/Cap包装细胞系，按照如下方法建立：
为了便于包装细胞系的筛选，在pIR-VP-hr2-RBE质粒(参考Proc Natl Acad Sci U S A.2009Mar31；106(13):5059-64)的基础上进行了改造，在杀稻瘟菌素(blasticidin,Bsd)基因的C端通过FMDV自剪切多肽2A融合了绿色荧光蛋白(GFP)基因，得到pIR-VP-hr2-RBE-bsd-GFP质粒(如图2F)。然后，将该质粒转染Sf9细胞，通过Bsd抗生素筛选得到整合了诱导表达AAV的Cap基因表达元件的Sf9/Cap包装细胞系。该细胞系组成性表达GFP，可通过单克隆分离培养或流式细胞仪分选，进一步得到rAAV产量较高的Sf9/Cap包装细胞系，如图6A所示。
(2)利用BEV/Rep-(ITR-GFP)感染Sf9/Cap细胞系制备rAAV并验证其感染活性
将制备的BEV/Rep-(ITR-GFP)以感染复数(MOI＝5)感染悬浮培养的获得的Sf9/Cap细胞系。感染72小时后，将细胞培养液3000rpm离心5min，分别收集培养上清和细胞沉淀。BEV产生后，主要通过分泌释放到培养基上清中，在Sf9/Cap细胞中也有部分未释放的BEV。rAAV产生后，主要存在于Sf9/Cap细胞核中，由于Sf9/Cap细胞感染后发生细胞病变(CPE)，部分细胞会裂解，rAAV也会有部分释放到上清中，如图6A所示。而因此上清和细胞沉淀中会同时存在BEV和rAAV。
为了验证用重组杆状病毒BEV/Rep-(ITR-GFP)感染Sf9/Cap细胞制备出了具有活性的rAAV。通过病毒感染HEK293细胞和Sf9细胞的实验方法证实该系统制备出了rAAV，实验结果如图6B。具体实施过程和结果分析如下：将细胞沉淀反复冻融3次裂解后，5000rpm离心5min收集细胞裂解上清液。因为rAAV无包膜，经60℃、30分钟处理，活性不受影响；而重组杆状病毒(BEV)有包膜，经60℃、30分钟处理，失去活性。含有病毒的上清液感染HEK293细胞2天后，利用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，在处理前后都有GFP表达，表明rAAV在处理前后都有感染活性。BEV感染Sf9细胞后的培养基上清组，未处理的有较强GFP表达，处理后仅有无GFP表达，表明BEV可以感染HEK293细胞，灭活处理后BEV不感染。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后GFP强度有降低但仍有较强荧光，表明灭活处理后BEV 不感染，但上清中大量的rAAV仍能够感染。用与感染HEK293细胞组相同的样品感染Sf9细胞2天后，结果显示：利用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，没有GFP荧光，表明rAAV对Sf9细胞没有感染性。BEV感染Sf9细胞后的培养基上清组，灭活前有较强GFP表达，灭活后的没有GFP表达。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后的没有GFP表达。
(3)将步骤(2)中获得的重组腺相关病毒分离并纯化。
rAAV的纯化、滴度测定与细胞水平转导活性验证
将重组BEV感染后收集得到约为1×108cells的Sf9/Cap细胞沉淀，加入10ml裂解缓冲液(50mM Tris-Cl，150mM NaCl，2mM MgCl2，pH 8.0)，后反复冻融3次，5000rpm离心5min后收集上清，在上清中加入核酸酶Benzonase至终浓度50U/ml，37℃水浴处理60min。处理后5000rpm离心10min收上清。上清用氯仿抽提，抽提后的上清再用含13.2％的(NH4)2SO4和10％的PEG8000的溶液进行两相沉淀的方法进一步纯化，方法参考文献(J Virol Methods,2007.139(1):p.61-70.和J Virol Methods,2012.179(1):p.276-80.)。两相沉淀后的上清，用PBS溶液进行透析脱盐处理，用Amicon ultra-4(100KD cutoff)透析柱离心浓缩至终体积1ml，无菌分装后-80℃冻存。rAAV的滴度测定采用荧光定量PCR的方法，滴度单位用VG/ml(VG，virus genomes)表示。
纯化过程的rAAV得率见表3。实验结果表明，单个Sf9/Cap细胞rAAV的产量可达6.84×104VG，经过本方法的纯化后，回收率达到28.8％左右。
表3rAAV纯化过程的得率分析
Figure PCTCN2016073246-appb-000003
将HEK293细胞以1×104cells/well接种到96孔板中，6h后感染相应浓度梯度含量的纯化的rAAV。感染48h后，用荧光显微镜观察GFP的表达情况，如图6C。结果表明，本发明的方法制备得到的rAAV具有较好的细胞水平转导活性。
实施例4：重组杆状病毒BEV/Rep-(ITR-GFP)感染Sf9/Cap包装细胞系制备rAAV
本实施例与实施例3相似，区别仅在于所述重组杆状病毒，其主要组件组合方案如下：
LinkA-(ITR-GFP)-linkB-RepB
将本实例中的重组杆状病毒BEV/Rep-(ITR-GFP)感染Sf9/Cap包装细胞系后，将制备得到的rAAV进行纯化。纯化过程的rAAV得率见表4。实验结果表明，单个Sf9/Cap包装细胞rAAV的产量可达8.16×104VG，经过本方法的纯化后，回收率达到34.7％左右。
表4rAAV纯化过程的得率分析
Figure PCTCN2016073246-appb-000004
将HEK293细胞以1×104cells/well接种到96孔板中，6h后感染相应浓度梯度含量的纯化的rAAV。感染48h后，用荧光显微镜观察GFP的表达情况，如图7。结果表明，本发明的方法制备得到的rAAV具有较好的细胞水平转导活性。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种重组腺相关病毒的制备方法，其特征在于，包括以下步骤：
    (1)将构建有重组腺相关病毒ITR核心表达元件和Cap基因或Rep基因的重组型杆状病毒感染相应的宿主包装细胞系；
    (2)将步骤(1)中感染了重组杆状病毒的宿主包装细胞系扩大培养，使产生重组腺相关病毒；
    (3)将步骤(2)中获得的重组腺相关病毒分离并纯化。
  2. 如权利要求1所述的制备方法，其特征在于，其步骤(1)利用pFast.Bac.Dual穿梭载体构建所述重组型杆状病毒。
  3. 如权利要求2所述的制备方法，其特征在于，所述步骤(1)具体操作方法如下：
    a.将ITR核心表达元件克隆到pFast.Bac.Dual穿梭载体的P10启动子和PH启动子间隔序列中；
    b.将Cap基因或Rep基因克隆到pFast.Bac.Dual穿梭载体的P10启动子或PH启动子下游的多克隆位点中，得到相应的穿梭质粒。
  4. 如权利要求1所述的制备方法，其特征在于，所述构建有重组腺相关病毒ITR核心表达元件和Cap基因或Rep基因的重组型杆状病毒其ITR核心表达元件中携带有目标基因。
  5. 如权利要求1所述的制备方法，其特征在于，所述宿主包装细胞系用于辅助重组腺相关病毒的复制和组装。
  6. 如权利要求5所述的制备方法，其特征在于，所述宿主细胞基因组上相应整合有诱导表达腺相关病毒Rep基因的表达框元件或诱导表达腺相关病毒Cap基因的表达框元件。
  7. 一种重组杆状病毒，其特征在于，其基因组上构建有腺相关病毒ITR核心表达元件和相应血清型的Cap基因。
  8. 如权利要求7所述的重组杆状病毒，其特征在于，所述Cap基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列。
  9. 一种重组杆状病毒，其特征在于，其基因组上构建有腺相关病毒ITR核心表达元件和相应血清型的Rep基因。
  10. 如权利要求9所述的重组杆状病毒，其特征在于，所述Rep基因其序列为依据核糖体泄露原理进行密码子优化的序列。
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