CN120138052A

CN120138052A - A dual regulation system of Cap and Rep proteins for producing high-quality recombinant adeno-associated virus and its construction method

Info

Publication number: CN120138052A
Application number: CN202510392573.1A
Authority: CN
Inventors: 聂简琪; 任和; 宋紫川; 白仲虎
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2025-03-31
Filing date: 2025-03-31
Publication date: 2025-06-13

Abstract

The invention discloses a Cap and Rep protein dual regulation system for producing high-quality recombinant adeno-associated virus and a construction method thereof. The dual regulation system comprises a Rep2-off protein expression system and a Cap-on protein expression system, wherein the Cap-on protein expression system realizes the delayed expression of Cap protein by adding mifepristone which is a small molecular compound, and the Rep-off protein expression system shuts off the later expression of Rep protein by adding tetracycline. The invention improves the virus titer and the real heart rate by constructing a dual regulation and control system for delaying the expression of the capsid protein Cap and closing the later expression of the Rep protein, thereby realizing the production of high-quality recombinant adeno-associated virus.

Description

Cap and Rep protein dual regulation system for producing high-quality recombinant adeno-associated virus and construction method thereof

Technical Field

The invention relates to the technical field of gene and cell therapy drug carriers, in particular to a Cap and Rep protein dual regulation system for producing high-quality recombinant adeno-associated virus and a construction method thereof.

Background

Heavy adeno-associated virus (rAAV) plays an important role in the field of gene therapy due to its high safety, broad tissue tropism and durable gene expression characteristics. As a non-pathogenic virus, rAAV can achieve efficient transduction of a variety of tissues. For example, AAV8 and AAV9 perform excellently in liver and heart tissue, whereas AAV10 has a higher infection efficiency in the central nervous system. In addition, rAAV vectors have been successfully used in the treatment of various diseases, such as Zolgensma for the treatment of spinal muscular atrophy and Luxturna for the treatment of hereditary eye diseases, due to their ability to express therapeutic genes for a long period and not integrate into host chromosomes.

Currently, three plasmids co-transfect HEK293 cells in the most common commercial rAAV production format. The method produces rAAV vectors containing therapeutic genes by co-transfecting HEK293 cells with a plasmid containing the target gene (p-GOI), a helper plasmid (pHelper, providing adenovirus helper functions) and a packaging plasmid (pRepCap, containing AAV Cap capsid protein and Rep protein). Although this method is relatively simple to operate and suitable for small-scale laboratory preparation, it has drawbacks including low production efficiency, high empty virus ratio, low purity, and the like. Therefore, optimizing the production mode of rAAV to more efficiently prepare high quality viral vectors has become a key issue to be addressed currently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a Cap and Rep protein dual regulation system for producing high-quality recombinant adeno-associated virus and a construction method thereof. The invention constructs a dual regulation system for delaying the expression of capsid protein Cap and closing the later expression of Rep protein, wherein the Cap-on inducible expression system realizes the delayed expression of the Cap protein by adding mifepristone which is a small molecular compound, and the Rep-off inducible expression system closes the later expression of the Rep protein by adding tetracycline. The double regulation system constructed by the invention improves the virus titer and the real heart rate, thereby realizing the production of high-quality recombinant adeno-associated virus.

The technical scheme of the invention is as follows:

the double regulation and control system for the Cap protein and the Rep protein comprises a Rep2-off protein expression system and a Cap-on protein expression system, wherein the Rep2-off protein expression system expresses the Rep2 protein, the expression of the Rep protein is closed by adding tetracycline, the Cap-on system expresses the Cap protein, and the expression of the Cap protein is started by adding mifepristone.

Further, the construction method of the Rep2-off protein expression system comprises the following steps:

(1) Taking a PCW57.1-MAT2A vector as a template, and carrying out enzyme digestion by utilizing restriction enzymes NheI and SalI to obtain a linearized PCW57.1-MAT2A vector;

(2) Amplifying by using pRep2Cap5 plasmid as a template through Rep2-F and Rep2-R to obtain a Rep2 fragment;

(3) Carrying out homologous recombination on the linearized PCW57.1-MAT2A vector obtained in the step (1) and the Rep2 fragment obtained in the step (2) through Gibson assembly to obtain a Rep2-off vector;

the Rep2-off vector is a Rep2-off protein expression system.

Further, the nucleotide sequence of the Rep2-off vector is shown as SEQ ID NO.1, the nucleotide sequence of the Rep2-F vector is shown as SEQ ID NO.3, and the nucleotide sequence of the Rep2-R vector is shown as SEQ ID NO. 4.

Further, the construction method of the Cap-on protein expression system comprises the following steps:

(1) Using a pSwitch vector as a template, and amplifying by using a primer pSwitch-F and a primer pSwitch-R to obtain a GSTA module;

(2) Using Pgene/v5-His carrier as template to amplify primer Pgene-F and Pgene-R to obtain Pgene carrier module;

(3) Carrying out homologous recombination on the GSTA module obtained in the step (1) and the Pgene carrier module obtained in the step (2) through Gibson assembly to construct a pGSTA-on carrier;

(4) Carrying out enzyme digestion on pGSTA-on vector by using restriction enzymes KpnI and NotI to obtain linearized pGSTA-on plasmid;

(5) Amplifying to obtain Cap gene fragments by taking pRep2CapX plasmid as a template;

(6) Carrying out homologous recombination on the linearization pGSTA-on plasmid obtained in the step (4) and the Cap gene fragment obtained in the step (5) through Gibson assembly, and cloning the Cap gene to the downstream of the mifepristone induced promoter to obtain a Cap-on vector;

the Cap-on vector is a Cap-on protein expression system.

Further, in the step (1), the nucleotide sequence of the pSwitch-F is shown as SEQ ID NO.5, and the nucleotide sequence of the pSwitch-R is shown as SEQ ID NO. 6.

Further, in the step (2), the nucleotide sequence of Pgene-F is shown as SEQ ID NO.7, and the nucleotide sequence of Pgene-R is shown as SEQ ID NO. 8.

Further, in the step (6), the pRep2CapX plasmid comprises any one of pRep2Cap1 plasmid, pRep2Cap2 plasmid, pRep2Cap4 plasmid, pRep2Cap5 plasmid, pRep2Cap6 plasmid, pRep2Cap8 plasmid, pRep2Cap9 plasmid and pRep2Cap10 plasmid, and the Cap-on vector comprises any one of Cap1-on, cap2-on, cap4-on, cap5-on, cap6-on, cap7-on, cap8-on, cap9-on and Cap 10-on.

Use of the dual regulation system for the production of high quality recombinant adeno-associated virus.

Further, the method for producing the high-quality recombinant adeno-associated virus comprises the following steps:

Culturing the suspension HEK293 cells to a proper transfection density, then co-transfecting the HEK293 cells with a Rep2-off vector, a Cap-on vector, a pHelper plasmid and a p-GOI plasmid, adding mifepristone to induce Cap protein expression when the HEK293 cells are transfected for 12 hours, adding doxycycline to close the Rep protein expression when the HEK293 cells are transfected for 36 hours, and continuing culturing for 72 hours, and collecting the recombinant adeno-associated virus.

Further, the p-GOI plasmid refers to a plasmid carrying a target gene, wherein the plasmid has two inverted repeat sequences, and the target gene is carried between the inverted repeat sequences at two ends.

The beneficial technical effects of the invention are as follows:

The invention discloses a novel rAAV production method, which realizes the remarkable improvement of rAAV titer produced by HEK293 cells by double expression regulation and control of induced Rep protein and Cap protein, saves actual production cost and reduces the difficulty of downstream purification process.

The invention discloses a rAAV production method which can greatly improve the real heart rate of rAAV, reduce the immune response and inflammatory response brought by empty viral capsids, maintain the transduction effect of viral particles, and improve the safety of gene therapy while ensuring the treatment effect.

Drawings

FIG. 1 is a diagram showing a comparison of the technical routes of the Cap and Rep protein dual regulation system and the three plasmid transfection system of the present invention for producing rAAV virus.

FIG. 2 is a graph of rAAV viral titers produced by example 2 and comparative example 1 of the present invention.

FIG. 3 is a real heart rate plot of rAAV produced in comparative example 1 of the present invention.

FIG. 4 is a real heart rate plot of rAAV produced in example 2 of the present invention.

FIG. 5 is a graph comparing the transduction effect of rAAV produced in example 2 of the present invention and comparative example 1.

FIG. 6 is a graph comparing titers of Cap and Rep protein Dual regulatory systems and three plasmid transfection systems of the present invention for production of different serotypes of rAAV virus.

FIG. 7 is a graph comparing the percent filled in of the Cap and Rep protein dual regulatory system and the three plasmid transfection system of the present invention for the production of different serotypes of rAAV virus.

FIG. 8 is a graph comparing transduction effects of the Cap and Rep protein double regulatory system and the three plasmid cotransfection system of the present invention for producing rAAV viruses of different serotypes.

FIG. 9 is a schematic representation of a Rep2-off plasmid constructed in accordance with the present invention.

FIG. 10 is a schematic diagram of a Cap5-on plasmid constructed in accordance with the present invention.

FIG. 11 is a graph showing rAAV gene replication efficiency versus Cap protein expression rate during production of recombinant adeno-associated virus according to example 1 and comparative example 1 of the present invention.

FIG. 12 is a graph showing cell density and cell viability of example 1 and comparative example 1 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the drawings and examples.

The pHelper plasmid and pRep2Cap5 plasmid of the present invention are purchased from Takara corporation, pRep2Cap1 plasmid, pRep2Cap2 plasmid, pRep2Cap4 plasmid, pRep2Cap6 plasmid, pRep2Cap8 plasmid, pRep2Cap9 plasmid and pRep2Cap10 plasmid are purchased from Sieimer's Feiche technology, pAAV2-CMV-EGFP plasmid (regarded as p-GOI) of the present invention is purchased from Sieimer's Feiche technology, PCW57.1-MAT2A vector is purchased from Orno gene, pSwitch vector and Pgene/v5-His vector are purchased from Sieimer's Feiche technology.

The nucleotide sequence of pTet-off-Rep2 plasmid constructed in the invention is shown as SEQ ID NO.1, and the nucleotide sequence of pGSTA-on-Cap5 plasmid is shown as SEQ ID NO. 2.

Rep2-off,SEQ ID NO.1:

AATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCCGCGAATCGATATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTTAGTAATGAACCGGTCCACCACCACCACCACCACTAAGGATCCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGCAATTCACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCGAAGGTAGAGGTTCTCTCCTCACTTGTGGTGATGTTGAAGAAAACCCTGGTCCAATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGGCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGATCAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCATCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAAGGATCCCGGGCCCGTCGACTGCAGAGGCCTTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTTTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGAC

Cap5-on,SEQ ID NO.2:

CCGAGCTCTTACGCGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGAGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGACTCTAGAGGGTATATAATGGATCTCGAGATATCGGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGCATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCTGTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTGTATTTTTGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACCAAGCTTGGTACATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAGAAAGAAGGCCCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTCTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGCGGCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACATCTAGCTAGAGCTTGGCGTAATTTGCATGCCTGCAGGTCGAAGCGGAGTACTGTCCTCCGAGTTTAAAAGCGGAGTACTGTCCTCCGAGGATATCAGCGGAGTACTGTCCTCCGAGTCGCGAAGCGGAGTACTGTCCTCCGAGATCGATGTCGACCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCTGTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTGTATTTTTGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACCAAGCTACCGGTCCACCATGGACTCCCAGCAGCCAGATCTGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAGAATTCCCGGGTGTCGACCAGAAAAAGTTCAATAAAGTCAGAGTTGTGAGAGCACTGGATGCTGTTGCTCTCCCACAGCCAGTGGGCGTTCCAAATGAAAGCCAAGCCCTAAGCCAGAGATTCACTTTTTCACCAGGTCAAGACATACAGTTGATTCCACCACTGATCAACCTGTTAATGAGCATTGAACCAGATGTGATCTATGCAGGACATGACAACACAAAACCTGACACCTCCAGTTCTTTGCTGACAAGTCTTAATCAACTAGGCGAGAGGCAACTTCTTTCAGTAGTCAAGTGGTCTAAATCATTGCCAGGTTTTCGAAACTTACATATTGATGACCAGATAACTCTCATTCAGTATTCTTGGATGAGCTTAATGGTGTTTGGTCTAGGATGGAGATCCTACAAACACGTCAGTGGGCAGATGCTGTATTTTGCACCTGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGTGGCAGATCCCACAGGAGTTTGTCAAGCTTCAAGTTAGCCAAGAAGAGTTCCTCTGTATGAAAGTATTGTTACTTCTTAATACAATTCCTTTGGAAGGGCTACGAAGTCAAACCCAGTTTGAGGAGATGAGGTCAAGCTACATTAGAGAGCTCATCAAGGCAATTGGTTTGAGGCAAAAAGGAGTTGTGTCGAGCTCACAGCGTTTCTATCAACTTACAAAACTTCTTGATAACTTGCATGATCTTGTCAAACAACTTCATCTGTACTGCTTGAATACATTTATCCAGTCCCGGGCACTGAGTGTTGAATTTCCAGAAATGATGTCTGAAGTTATTGCTGGGTCGACGCCCATGGAATTCCAGTACCTGCCAGATACAGACGATCGTCACCGGATTGAGGAGAAACGTAAAAGGACATATGAGACCTTCAAGAGCATCATGAAGAAGAGTCCTTTCAGCGGACCCACCGACCCCCGGCCTCCACCTCGACGCATTGCTGTGCCTTCCCGCAGCTCAGCTTCTGTCCCCAAGCCAGCACCCCAGCCCTATCCCTTTACGTCATCCCTGAGCACCATCAACTATGATGAGTTTCCCACCATGGTGTTTCCTTCTGGGCAGATCAGCCAGGCCTCGGCCTTGGCCCCGGCCCCTCCCCAAGTCCTGCCCCAGGCTCCAGCCCCTGCCCCTGCTCCAGCCATGGTATCAGCTCTGGCCCAGGCCCCAGCCCCTGTCCCAGTCCTAGCCCCAGGCCCTCCTCAGGCTGTGGCCCCACCTGCCCCCAAGCCCACCCAGGCTGGGGAAGGAACGCTGTCAGAGGCCCTGCTGCAGCTGCAGTTTGATGATGAAGACCTGGGGGCCTTGCTTGGCAACAGCACAGACCCAGCTGTGTTCACAGACCTGGCATCCGTCGACAACTCCGAGTTTCAGCAGCTGCTGAACCAGGGCATACCTGTGGCCCCCCACACAACTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGACAGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCCCCAATGGCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTTCTCAGCCCTGCTGAGTCAGATCAGCTCCTAAGGATCCTCCGGACTAGAAAAGCCGAATTCTGCAGGAATTGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCTCGAGGGGGGGCCCGAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGCGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCGTA

The primers used in the following examples of the present invention are shown in Table 1:

TABLE 1 primer list

Example 1 establishment of Dual control System for Cap and Rep proteins

(1) Construction of Rep2-off protein expression System

① Firstly, taking a PCW57.1-MAT2A vector as a template, and carrying out enzyme digestion by utilizing restriction enzymes NheI and SalI to obtain the linearized PCW57.1-MAT2A vector.

② Rep2 fragment (NCBI SEQ ID NO: AF043303.1, nucleic acid positions 336-2278) was obtained by amplification of Rep2-F and Rep2-R using pRep2Cap5 plasmid as template.

③ And (3) carrying out homologous recombination on the linearized PCW57.1-MAT2A vector obtained in the step (1) and the Rep2 fragment obtained in the step (2) through Gibson assembly to construct a Rep2-off vector (the nucleotide sequence is shown as SEQ ID NO.1, and the plasmid map is shown as FIG. 9). In the Rep2-off expression vector, rep2 is placed at the downstream of a Tet promoter, and transcription termination of the Rep2 gene is realized through addition of doxycycline, so that expression of the Rep2 is turned off.

(2) Construction of Cap-on protein expression System

The mifedone-induced Cap-on protein expression system was constructed using GENESWITCH system (sameir feier technologies, cat No. K106001).

① Firstly, using pSwitch vector as template, using primer pSwitch-F and pSwitch-R to make amplification so as to obtain GSTA module (nucleic acid sequence position is 25-3002 bp)

② Using Pgene/v5-His vector as template and primers Pgene-F and Pgene-R, pgene vector module (nucleic acid sequence position: 2815-2476 bp) was obtained

③ And (3) carrying out homologous recombination on the GSTA module obtained in the step (1) and the Pgene vector module obtained in the step (2) through Gibson assembly to construct a pGSTA-on vector.

④ The pGSTA-on vector was digested with restriction enzymes KpnI and NotI to give linearized pGSTA-on plasmid.

⑤ PRep2CapX (X represents different serotypes) of different serotypes is used as a template, and corresponding Cap gene fragments are obtained through amplification by the corresponding primers in the table 1, and AAV Cap template information of the different serotypes is as follows:

AAV1 Cap (NCBI sequence No. AF063497.1, nucleic acid positions 2223-4433),

AAV2 Cap (NCBI sequence No. AF043303.1, nucleic acid positions 1853-4506),

AAV4 Cap (NCBI sequence No. NC001829.1, nucleic acid positions 2260-4464),

AAV5 Cap (NCBI sequence No. AF085716.1, nucleic acid positions 2210-4382),

AAV6 Cap (NCBI sequence No. AF028704.1, nucleic acid positions 2208-4418),

AAV7 Cap (NCBI sequence No. NC006260.1, nucleic acid positions 2214-4435),

AAV8 Cap (NCBI sequence No. AF513852.1, nucleic acid positions 2121-4337),

AAV9 Cap (NCBI sequence number AY530579.1, nucleic acid positions 1-2211),

AAV10 Cap (NCBI sequence number AY243015.1, nucleic acid positions 1-2217).

⑥ Carrying out homologous recombination on pGSTA-on fragments obtained in the step (4) and various serotypes of Cap gene fragments obtained in the step (5) through Gibson assembly, wherein the Cap gene is cloned to the downstream of a mifepristone-induced promoter to obtain a Cap-on vector which comprises Cap1-on, cap2-on, cap4-on and Cap5-on (the nucleotide sequence is shown as SEQ ID NO.2, the plasmid map is shown as figure 10), cap6-on, cap7-on, cap8-on, cap9-on and Cap10-on.

Example 2

A method for producing high-quality rAAV5 virus by utilizing Cap and Rep protein dual regulation and control system refers to the technical route of figure 1, and specifically comprises the following steps:

Suspension HEK293 cells were cultured at a density of 1.3X10 ⁶ cells/mL, plasmids were manipulated according to the transfection system in Table 1, HEK293 cells were co-transfected in 125mL baffled shake flasks using the Rep2-off plasmid (SEQ ID NO. 1), cap5-on plasmid (SEQ ID NO. 2), pHelper plasmid and pAAV2-CMV-EGFP plasmid constructed in example 1, mifepristone was added at a final concentration of 10. Mu.M to induce Cap protein expression when transfected for 12h, dox (doxycycline) was added at a final concentration of 2. Mu.g/mL to shut off Rep protein expression when transfected for 36h, and after further culture for 72h, recombinant adeno-associated virus was initially formed and collected for subsequent detection.

TABLE 1 high quality production of plasmids and PEI usage for rAAV Virus transfection System

Name of the name	Dosage of
		pHelper	7.5μg
pAAV2-CMV-EGFP	7.5μg
		Rep2-off	7.5μg
Cap5-on	7.5μg
		PEI	29.3μg

Comparative example 1

A method for producing recombinant adeno-associated virus by three-plasmid cotransfection takes AAV5 as an example, and refers to the technical route of figure 1, and specifically comprises the following steps:

Suspension HEK293 cells were cultured at a density of 1.3X10 ⁶ cells/mL, plasmids were manipulated according to the transfection system in Table 2, HEK293 cells were co-transfected with pHelper plasmid, packaging plasmid pRep2Cap5 and pAAV2-CMV-EGFP plasmid in 125mL baffled shake flasks, and after further culture for 72h, recombinant adeno-associated virus was initially formed and collected for subsequent detection.

TABLE 2 plasmid and PEI usage for production of rAAV Virus transfection System by three plasmid cotransfection

Test case

(1) RAAV viral titers

① The crude cell products of example 1 and comparative example 1 were diluted in TE buffer (containing 1% PF 68), 1U TURBO DNase (MERCK SCIENTIFIC) was added, treated at 37℃for 30min, then heated at 75℃for 10min to inactivate DNase, an equal volume of 1% SDS was added, and heated at 95℃to release viral genome, to obtain a sample to be tested, and the sample to be tested was subjected to real-time fluorescent quantitative PCR (qPCR) (the reaction procedure is shown in Table 3) to obtain PCR detection results of the corresponding sample. ② The pAAV2-CMV-EGFP plasmid is diluted into standard substances with different concentrations according to a proportion, a standard curve is drawn, and the PCR detection result of the sample is substituted into the standard curve to calculate the virus titer, and the result is shown in figure 2.

TABLE 3qPCR reaction procedure

As can be seen from the results of FIG. 2, the dual regulatory system of Cap protein and Rep protein produced rAAV5 virus at 2.5 times the rAAV5 titer of the three plasmid co-transfection method. Therefore, the method for producing the high-quality recombinant adeno-associated virus by utilizing the Cap and Rep protein dual regulation system can obviously improve the titer of the rAAV5 virus.

(2) Real heart rate of rAAV virus

Detection method cell cultures of inventive example 2 and comparative example 1 were collected by centrifugation, resuspended in lysis buffer composed of 20mM Tris (pH 8.0), 2mM MgCl ₂, 200mM NaCl, released rAAV5 vector by 3 freeze thawing, added 200U/mL Benzonase DNase (Millipore Sigma) and homogenized at 37℃for 1h. After centrifugation to remove cell debris, the rAAV virus was purified by affinity chromatography. The purified rAAV viral particles were buffer exchanged in 10mM Tris-propane (BTP, pH 9.0) and then analyzed for rAAV real heart rate using an anion exchange monolith (CIMac ^TM ANALYTICAL MONOLITHS 0.1.1 mL, sartorius) in a high performance liquid chromatography system. Due to differences in isoelectric points, hollow rAAV viral particles were separated before solid particles, forming two distinct peaks, and solid rate analysis was performed according to peak area, with the results shown in fig. 3 and 4, respectively. From the results shown in fig. 3 and fig. 4, the rAAV5 virus produced by the Cap and Rep protein dual regulation system has a higher solid peak area, so that the method can greatly improve the duty ratio of the solid rAAV5 virus.

(3) Transduction efficiency

Detection method rAAV5 transduction efficiency was measured in adherent HEK293 cells. Specifically, 6-well tissue culture plates were inoculated at a density of 3X 10 ⁵ cells/well into DMEM buffer containing 10% FBS and cultured for 24 hours prior to transduction. The rAAV crude products produced in example 1 and example 2 at the same titer were then added to 6-well plates according to qPCR assay results. After 72 hours of incubation, the transduction efficiency of the AAV vector was observed using a fluorescence microscope and the number of cells expressing fluorescence was counted, and the results are shown in FIG. 5. From the results of fig. 5, the transduction efficiency of the rAAV virus produced by the dual regulation system of the present invention is not different from that of the rAAV virus obtained by the three-plasmid transfection method, which indicates that the two have the same transduction effect, i.e. the method of the present invention has no influence on the living activity of the rAAV virus.

(4) Other serotype testing

The production of rAAV of different serotypes was carried out by the method of example 2, following the substitution of the Cap5-on plasmid of example 2 of the present invention with Cap1-on, cap2-on, cap4-on, cap6-on, cap7-on, cap8-on, cap9-on, cap10-on plasmids, and the purification and analysis of the virus.

The pRep2Cap5 plasmid in comparative example 1 of the present invention was replaced with pRep2Cap1, pRep2Cap2, pRep2Cap4, pRep2Cap6, pRep2Cap8, pRep2Cap9, pRep2Cap10 plasmid, and then production of rAAV of different serotypes was performed by the method in comparative example 1, and the viruses were purified and analyzed.

The purified rAAV viruses of different serotypes were tested for virus titer, real heart rate and transduction efficiency according to the methods described above, and the results are shown in FIGS. 6-8, respectively. From the results of fig. 6-8, it can be seen that compared with the commercial three-plasmid production, the rAAV viruses of different serotypes produced by the dual regulation system of the invention have improved yield and solidity, and the transduction effect is not different, i.e. the Cap and Rep protein dual regulation system constructed by the invention is also suitable for the production of rAAV viruses of other serotypes. Therefore, the technical scheme of the invention can improve the yield and the true heart rate of all rAAV serotypes without affecting the virus transduction effect.

(5) RAAV viral gene replication efficiency and Cap protein expression Rate testing

The gene replication efficiency and Cap protein expression rate of rAAV viruses in the production of recombinant adeno-associated viruses of example 2 and comparative example 1 of the present invention were examined.

The detection method comprises ① Cap protein expression rate, collecting transfected cell samples of 12h, 24h, 36h, 48h and 60h, extracting RNA by using an RNA extraction kit, and then performing cDNA synthesis by using a reverse transcription kit. qPCR test was performed using specific primers for Cap protein to quantify the transcription level of Cap, ② extracting rAAV genome in the sample using genome extraction kit, qCPR detection using specific primers for rAAV, and analysis of gene replication efficiency, and the results are shown in FIG. 11. As can be seen from the results of FIG. 11, when the rAAV is produced by adopting the three-plasmid transfection system, the Cap protein expression is saturated within 12 hours after transfection and is far greater than the genome replication efficiency, and the double regulation system of the patent ensures that the Cap protein is continuously expressed within 36 hours after transfection by delaying the expression time of the Cap protein, so that the Cap protein is coordinated with the viral genome replication rate, thereby improving rAAV production and the real heart rate of the rAAV virus.

(6) Cell density and cell viability test

Samples of HEK293 cells 72h post transfection of example 2 and example 3 were collected and tested for cell density and cell viability using a Countess 2 automatic cytometer (Siemens technologies Co.) and the results are shown in FIG. 12. As can be seen from the results of FIG. 12, compared with the conventional three-plasmid transfection system, the rAAV produced by the Cap and Rep protein dual regulation system disclosed by the invention can remarkably improve the living cell density and the cell viability of host HEK293 cells, which is probably because the regulation system of the invention can prevent excessive Rep protein from causing toxicity to host HEK293 cells by closing the expression of Rep genes, thereby further improving the rAAV yield.

The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above examples. It is to be understood that other modifications and variations which may be directly derived or contemplated by those skilled in the art without departing from the spirit and concepts of the present invention are deemed to be included within the scope of the present invention.

Claims

1. A dual regulation system of Cap and Rep proteins, characterized in that the dual regulation system comprises a Rep2-off protein expression system and a Cap-on protein expression system; the Rep2-off protein expression system expresses Rep2 protein and shuts down the expression of Rep protein by adding tetracycline; the Cap-on system expresses Cap protein and starts the expression of Cap protein by adding mifepristone.

2. The dual regulation system according to claim 1, characterized in that the method for constructing the Rep2-off protein expression system comprises the following steps:

(1) Using PCW57.1-MAT2A vector as a template, the linearized PCW57.1-MAT2A vector was obtained by digestion with restriction endonucleases NheI and SalI;

(2) Using the pRep2Cap5 plasmid as a template, the Rep2 fragment was obtained by amplifying Rep2-F and Rep2-R;

(3) homologously recombining the linearized PCW57.1-MAT2A vector obtained in step (1) with the Rep2 fragment obtained in step (2) by Gibson assembly to obtain a Rep2-off vector;

The Rep2-off vector is a Rep2-off protein expression system.

3. The dual regulatory system according to claim 2, characterized in that the nucleotide sequence of the Rep2-off vector is shown as SEQ ID NO.1; the nucleotide sequence of the Rep2-F is shown as SEQ ID NO.3; and the nucleotide sequence of the Rep2-R is shown as SEQ ID NO.4.

4. The dual regulation system according to claim 1, characterized in that the method for constructing the Cap-on protein expression system comprises the following steps:

(1) Using the pSwitch vector as a template, primers pSwitch-F and pSwitch-R were used to amplify the GSTA module;

(2) Using the Pgene/v5-His vector as a template, primers Pgene-F and Pgene-R were used to amplify the Pgene vector module;

(3) Through Gibson assembly, the GSTA module obtained in step (1) and the Pgene vector module obtained in step (2) are homologously recombined to construct a pGSTA-on vector;

(4) The pGSTA-on vector was digested with restriction endonucleases KpnI and NotI to obtain a linearized pGSTA-on plasmid;

(5) Using the pRep2CapX plasmid as a template, amplify the Cap gene fragment;

(6) performing homologous recombination of the linearized pGSTA-on plasmid obtained in step (4) and the Cap gene fragment obtained in step (5) by Gibson assembly, cloning the Cap gene downstream of the mifepristone-inducible promoter, and obtaining a Cap-on vector;

The Cap-on vector is a Cap-on protein expression system.

5. The dual regulation system according to claim 4, characterized in that, in step (1), the nucleotide sequence of pSwitch-F is as shown in SEQ ID NO.5; the nucleotide sequence of pSwitch-R is as shown in SEQ ID NO.6.

6. The dual regulation system according to claim 4, characterized in that, in step (2), the nucleotide sequence of Pgene-F is as shown in SEQ ID NO.7; the nucleotide sequence of Pgene-R is as shown in SEQ ID NO.8.

7. The dual regulation system according to claim 4 is characterized in that in step (6), the pRep2CapX plasmid includes any one of pRep2Cap1 plasmid, pRep2Cap2 plasmid, pRep2Cap4 plasmid, pRep2Cap5 plasmid, pRep2Cap6 plasmid, pRep2Cap8 plasmid, pRep2Cap9 plasmid, and pRep2Cap10 plasmid; the Cap-on vector includes any one of Cap1-on, Cap2-on, Cap4-on, Cap5-on, Cap6-on, Cap7-on, Cap8-on, Cap9-on, and Cap10-on.

8. An application of the dual regulatory system according to claim 1, characterized in that the dual regulatory system is used to produce high-quality recombinant adeno-associated virus.

9. The use according to claim 8, characterized in that the method for producing high-quality recombinant adeno-associated virus is as follows:

The suspended HEK293 cells were cultured to a density suitable for transfection, and then the HEK293 cells were co-transfected with the Rep2-off vector, Cap-on vector, pHelper plasmid and p-GOI plasmid. Mifepristone was added 12 hours after transfection to induce the expression of Cap protein. Doxycycline was added 36 hours after transfection to shut down the expression of Rep protein. After continued culture for 72 hours, the recombinant adeno-associated virus was collected.

10. The use according to claim 9, characterized in that the p-GOI plasmid refers to a plasmid carrying a target gene, the plasmid has two inverted repeat sequences, and the target gene is carried between the inverted repeat sequences at both ends.