CN108203706B

CN108203706B - Helper adenovirus for improving packaging efficiency of high-capacity adenovirus

Info

Publication number: CN108203706B
Application number: CN201711239774.XA
Authority: CN
Inventors: 夏海滨; 单琳琳; 朱贺; 张伟锋
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2021-03-26
Anticipated expiration: 2037-11-30
Also published as: CN108203706A

Abstract

The invention discloses a helper adenovirus for improving the packaging efficiency of high-capacity adenovirus. Based on the type 5 adenovirus genome, the DNA sequence and 27893bp between 193bp and 5196bp from the 5' end of the type 5 adenovirus genome are removed. DNA sequence to 30990bp; insert loxp sequence, adenovirus packaging signal Ψ, Cre gene expression box, loxp sequence between the 192nd base and the 5197th base from the 5' end of the adenovirus type 5 genome in turn; Between the 27892nd base and the 30991st base from the 5' end of the adenovirus type 5 genome, the pIX gene expression cassette and the IVa2 gene expression cassette were inserted. By adding the Cre gene, under the action of the CRE enzyme of the packaging cells, while removing the helper virus packaging signal Ψ, the expression of the Cre gene carried by the helper virus itself is induced, the amount of CRE enzyme is increased, and the packaging signal Ψ is removed more thoroughly. . At the same time, the genome structure of the helper virus is adjusted to avoid the recombination of the helper virus and the virus genome in the packaging cell and the production of the wild-type virus.

Description

Helper adenovirus for improving packaging efficiency of high-capacity adenovirus

Technical Field

The invention belongs to the technical field of biology, relates to a helper adenovirus, and particularly relates to a helper adenovirus for improving the packaging efficiency of high-capacity adenovirus.

Background

Currently, in gene therapy, the most effective means for introducing foreign genes into target cells is via viral vectors. Commonly used viral vectors include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and the like. Adeno-associated virus vectors have the advantages of low immunogenicity, no random chromosomal insertions, etc., but they are too small in capacity and are limited in use. Since lentiviral vectors are randomly inserted into chromosomes, they are prone to cause cancer, and their safety is questioned. The adenovirus vector has the advantages of high capacity, high titer, no chromosome integration and the like, but has high immunogenicity and is easy to initiate stronger immune response in vivo treatment. The third generation of adenoviral vectors that have been developed today remove most of the adenovirus genome, leaving only its elements for replication and packaging, ITR and Ψ, and thus greatly reduce its immunogenicity. Is considered to be a virus vector with great application prospect.

Because most of the viral genomic sequence is removed, packaging of third generation adenoviruses requires helper viruses to provide the various capsid proteins required for packaging, while avoiding contamination by helper viruses. In the currently used method for removing helper virus, it is better to add loxp sequences on both sides of the packaging signal Ψ, and in the process of packaging helper high-capacity adenovirus, the packaging signal Ψ is removed after the loxp sequence is acted by CRE enzyme expressed in the packaging cell, so that the helper virus genome cannot be packaged. However, because the CRE expression level is insufficient in these methods and the helper virus and a part of the viral genome sequence contained in the packaging cell may be recombined to form a wild-type virus during the packaging process, the helper virus cannot be well removed from the packaging virus, and a certain amount of contamination still exists. There is currently no better way to further reduce helper virus contamination.

Disclosure of Invention

In view of the technical problem of contamination of helper adenoviruses as described above, it is an object of the present invention to provide a novel helper adenovirus that is capable of more effectively removing the packaging signal and avoiding the production of wild-type viruses.

In order to realize the task, the invention adopts the following technical solution:

a helper adenovirus for improving the packaging efficiency of high-capacity adenovirus is characterized in that based on a type 5 adenovirus genome, a DNA sequence between 193bp and 5196bp and a DNA sequence between 27893bp and 30990bp which are away from a 5' end of the type 5 adenovirus genome are removed; sequentially inserting a loxp sequence, an adenovirus packaging signal psi, a Cre gene expression frame and a loxp sequence between 192 th base and 5197 th base of the 5' -end distance of the type 5 adenovirus genome, wherein the directions of the two inserted loxp sequences are kept consistent; the pIX gene expression cassette and the IVa2 gene expression cassette are inserted between 27892 th base and 30991 th base from the 5' end of the type 5 adenovirus genome.

According to the invention, the adenovirus packaging signal Ψ is a packaging signal of a type 5 adenovirus, specifically a DNA sequence between the 193 rd base and the 440 th base from the 5' end of the type 5 adenovirus genome, and the direction thereof is forward or reverse.

The Cre gene expression frame consists of a promoter, a Cre gene and a terminator; the Cre gene sequence comprises an N-terminal sequence, a C-terminal sequence, an intron shearing donor sequence and an intron shearing acceptor sequence, wherein the N-terminal sequence is a DNA fragment with the length of 4 bases to 1049 bases at the 5 'end of a Cre gene coding region, the C-terminal sequence is a DNA fragment with the length of 4 bases to 1049 bases at the 3' end of the Cre gene coding region, an intron shearing donor sequence is inserted at the 3 'end of the N-terminal sequence, an intron shearing acceptor sequence is inserted at the 5' end of the C-terminal sequence, and the total length of the intron shearing donor sequence and the shearing acceptor sequence is not more than 500 bp; the intron shearing donor sequence, the Cre gene N-terminal sequence, the promoter, the terminator, the Cre gene C-terminal sequence and the intron shearing acceptor sequence are sequentially inserted into the 3 ' end of the adenovirus packaging signal psi according to the direction from 3 ' to 5 '.

The Cre gene expression frame sequence is as follows:

AAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCAT。

furthermore, the pIX gene expression frame is formed by respectively adding a promoter and a terminator at the 5 'end and the 3' end of the pIX gene coding region of the type 5 adenovirus; the IVa2 gene expression is that promoter and terminator are added to 5 'end and 3' end of IVa2 gene coding region of type 5 adenovirus respectively.

The promoter is a eukaryotic promoter, and the terminator is a eukaryotic terminator.

The promoter is any one of CMV, EF1 alpha, PGK, SV40, CAG, Tet on and Tet off, and the terminator is any one of SV40pA, BGHpA, TKpA and hGHpA.

According to the helper adenovirus for improving the packaging efficiency of the high-capacity adenovirus, the Cre gene is added into the helper adenovirus, and under the action of the CRE enzyme of a packaging cell, the expression of the Cre gene carried by the helper virus is induced while the helper virus packaging signal psi is removed, so that the quantity of the CRE enzyme is improved, and the packaging signal psi is removed more completely. Meanwhile, the genome structure of the helper virus is adjusted, so that recombination of the helper virus and the virus genome in the packaging cell is avoided, and generation of wild-type viruses is avoided. By both of these aspects, contamination by helper virus is further reduced. Thus, high-capacity adenovirus with higher purity can be obtained.

Drawings

FIG. 1 is a schematic diagram of the structure and operation of Ad helper 1.

FIG. 2 is a photograph of a lesion formed by assisted adenovirus packaging.

FIG. 3 is a graph showing the results of examination of the removal effect of the helper adenovirus packaging signal.

FIG. 4 is a graph showing the results of detection of helper adenovirus contamination.

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

Detailed Description

The applicant inserts a loxp sequence, an adenovirus packaging signal Ψ, a Cre gene expression cassette and a loxp sequence between 192 th base and 5197 th base of 5' end distance of a 5-type adenovirus genome in sequence, so that in an adenovirus packaging cell for expressing CRE enzyme, an auxiliary adenovirus can remove the self packaging signal Ψ and simultaneously induce the expression of a self Cre gene, thereby increasing the amount of CRE enzyme in the packaging cell and further completely removing the packaging signal Ψ. Meanwhile, the pIX gene expression frame and the IVa2 gene expression frame in the 5-type adenovirus genome are moved to positions between the 27892 th base and the 30991 th base away from the 5' end from the original positions, so that the possibility of generating wild-type viruses by recombination of the helper adenovirus genome and partial virus genome sequences existing in the packaging cell genome is avoided. Through the improvement, helper virus pollution caused by helper adenovirus in high-capacity adenovirus packaging assistance is further reduced.

The following are examples given by the inventors, and it should be noted that these examples are merely preferred embodiments of the present invention, and the present invention is not limited to these examples.

Example 1:

taking the helper adenovirus Ad helper1 with self-removed packaging signal and controllable pIX gene expression as an example, the helper adenovirus genome structure is as follows:

based on the adenovirus type 5 genome, the adenovirus type 5 genome sequence NCBI is numbered AC _ 000008. Removing a DNA sequence between 193bp and 5196bp and a DNA sequence between 27893bp and 30990bp away from the 5' end in the adenovirus 5 genome; sequentially inserting a loxp sequence, an adenovirus packaging signal psi, a Cre gene expression frame and a loxp sequence between 192 th base and 5197 th base of the 5' -end distance of the type 5 adenovirus genome, wherein the directions of the two inserted loxp sequences are kept consistent; a pIX gene expression cassette and an IVa2 gene expression cassette are inserted between 27892 th base and 30991 th base from the 5' end of the adenovirus type 5 genome.

The adenovirus packaging signal Ψ is a forward placement, specifically a DNA sequence between the 193 rd and 440 th bases from the 5' end of the adenovirus type 5 genome.

The Cre gene N-terminal sequence is a DNA fragment with 453 bases at the 5 'end of the Cre gene coding region, and the Cre gene C-terminal sequence is a DNA fragment with 600 bases at the 3' end of the Cre gene coding region.

The DNA sequence inserted between the 192 nd base and the 5197 th base from the 5' end of the adenovirus type 5 genome is as follows:

ATAACTTCGTATAATGTATGCTATACGAAGTTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGTTAT。

the pIX gene expression cassette and IVa2 gene expression cassette inserted between 27892 th base and 30991 th base from 5' end of type 5 adenovirus genome are as follows:

GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGGCTGCAGGTCGAAAGGCCCGGAGATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTCCATCTGCACGAGACTAGCTAGTAGTGAGACGTGCTACTCCCATTTGTCACGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATGTGGAATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAT。

the structure and action principle of the virus genome are shown in figure 1.

The process for constructing helper adenovirus Ad helper1 is as follows:

(1) construction of helper adenovirus E3 region shuttle vector carrying pIX and IVa2 expression cassettes

Primers were synthesized in Huada Gene, having the following sequences:

P1EPXN for：AATTTTAATTAATTCTCGAGAATCGATAAGCGGCCGC；

P2EPXN reverse：AGCTGCGGCCGCTTATCGATTCTCGAGAATTAATTAA；

the above two primers were annealed to form an EPXN linker (EcoRI M-PacI-XhoI-ClaI-NotI-HindIIIM), and the EPXN linker was ligated with EcoRI-and HindIII-digested pUC19 vector to obtain pUC 19/EPXN.

Primers were synthesized by Huada Gene, having the following sequences:

P3E3up PacI for：ATTAATTAAatcgacccgcgagcttagaaacag；

P4E3up XhoI reverse：ACTCGAGtttcaggcgcagttgctctgcctc；

P5E3down ClaI for：AATCGATgtttcctcctgttcctgtccatc；

P6E3down NotI reverse：AGCGGCCGCtgagctgcccggggagtttatt；

P3/P4 and P5/P6 are respectively used as primers and a 5-type adenovirus genome site template, and E3up and E3 down are obtained by PCR amplification. E3up and E3 down were digested with PacI/XhoI and ClaI/NotI, respectively, and fragments were recovered, followed by ligation of pUC19/EPXN vectors digested with the same enzymes, to obtain phepperE 3 shunt.

The following primers were synthesized by Huada Gene Inc.:

CMV XhoI for：ACTCGAGGTTACATAACTTACGGTAAATGGC；

CMV pIX overlap reverse：

ttccatcaaacgagttggtgctcatATCTGACGGTTCACTAAACGAGCTCT；

pIX for：atgagcaccaactcgtttgatggaa；

IVa2reverse：atggaaaccagagggcgaagaccggcagcgct；

EF1αClaI for：AATCGATAAGGATCTGCGATCGCTCCGGTGCCCG；

EF1αIVa2overlap reverse：

AGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGTGGCGTCTAGCGTAGGCGC。

the method comprises the steps of taking a 5-type adenovirus genome, a CMV promoter and an EF1 alpha promoter as templates, obtaining a CMV-pIX-IVa2-EF1 alpha fragment by an overlapping PCR method, carrying out enzyme digestion by using XhoI and ClaI, recovering the CMV-pIX-IVa2-EF1 alpha fragment, and connecting the fragment with phepperE 3shut treated by the same enzyme digestion to obtain phepperE 3/CMV-pIX-IVa2-EF1 alpha.

(2) Construction of shuttle vector carrying ITR-loxp- Ψ -cre-loxp element in E1 region of adenovirus

The following primers were synthesized by Huada Gene Inc.:

helperE1down ClaII for：AATCGATctacggcatctcgatccagcata；

helperE1down NotI reverse：AGCGGCCGCgggccaggtgaatatcaaatc。

PCR amplification is carried out by taking type 5 adenovirus genome as a template to obtain a helperi 1down fragment, the fragment is cut by ClaI and NotI and recovered into a helperi E1down fragment, and the fragment is connected with a pUC19/EPXN vector which is cut by the same enzyme to obtain a shuttle vector pheperE 1 shut of the E1 region of the helper adenovirus.

The following DNA fragment ITR-loxp- Ψ -cre-loxp was synthesized by Huada Gene corporation and the sequence was as follows:

TTAATTAAcatcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgATAACTTCGTATAATGTATGCTATACGAAGTTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGTTATCTCGAG。

carrying out enzyme digestion on ITR-loxp- Ψ -cre-loxp by using PacI and XhoI, and connecting the ITR-loxp- Ψ -cre-loxp with a likewise enzyme-digested pheperE 1 shuttl vector to obtain a shuttle vector pheperE 1/ITR-loxp- Ψ -cre-loxp of an adenovirus E1 region carrying an ITR-loxp- Ψ -cre-loxp element.

(3) Construction of helper adenovirus Ad helper1

The method comprises the steps of carrying out enzyme digestion treatment on phelper 3/CMV-pIX-IVa2-EF1 alpha by PacI, carrying out enzyme digestion treatment on an adenovirus skeleton vector pAd5 backbone (the obtaining method of the adenovirus is disclosed in the patent carrying a zinc finger nuclease expression element and donor DNA in the construction method and application thereof) with an E3 region introduced into a SwaI site by SwaI, and co-transforming the treated plasmids into escherichia coli BJ5183 for homologous recombination to obtain pAd5/CMV-pIX-IVa2-EF1 alpha.

pAd5/CMV-pIX-IVa2-EF1 alpha and phelper E1/ITR-loxp- Ψ -cre-loxp were digested with PacI, and then co-transfected into HEK293 cells, and lesion formation was observed after 7-8 days, and the lesion formed by packaging of the helper adenovirus is shown in FIG. 2, which illustrates the successful packaging of the helper adenovirus Ad helper 1. After repeated expansion to 10 150mm cell culture dishes, the virus was collected and used in Ccscl₂Virus was purified by density gradient centrifugation.

The Ad helper1 of 8MOI is used for infecting the adenovirus packaging cell HEK293-CRE which stably expresses CRE enzyme, meanwhile, the traditional helper adenovirus Ad helper of 8MOI is used for infecting another disc HEK293-CRE, the cell is collected after 48 hours, and the removal effect of a packaging signal is detected through PCR (polymerase chain reaction), the result is shown in figure 3, the smaller electrophoresis band in the experimental result is a PCR product after the packaging signal is removed, the larger electrophoresis band is a PCR product when the packaging signal is not removed, and the packaging signal removal of the Ad helper1 is more complete.

HEK293-cre cells were co-infected with 4MOI Ad helper1 and 4MOI Ad eGFP (normal adenovirus carrying eGFP gene), while another disc HEK293-cre cells were co-infected with 4MOI Ad helper and 4MOI Ad eGFP, after 48 hours the virus was collected to infect U87 cells, after 24 hours the U87 cells were collected to extract genomic DNA, and helper virus contamination was detected by Realtime PCR, as shown in FIG. 4, in which Ad helper1 was found to be less contaminated than Ad helper 1.

The method for using the helper adenovirus Ad helper1 is as follows:

the high-capacity adenovirus vector pHCAD is linearized by PacI enzyme digestion, calcium phosphate coprecipitation is carried out to transfect HEK293-cre cells in a 60mm plate, after 18 hours, 4MOI auxiliary adenovirus Ad helper1 is used to infect the HEK293-cre after pHCAD transfection, after 48-72 hours, the cells are collected, and after 3 times of repeated freeze thawing, the supernatant is collected to obtain the virus. And (3) co-infecting HEK293-cre with the virus lysate collected from 1/4 and 4MOI helper adenovirus Ad helper1, and collecting cells after 48-72 hours to obtain the virus. The virus was thus repeatedly amplified to 20 cells in a 150mm culture dish, collected and passed through Ccscl₂And (5) performing density gradient centrifugation and purifying the virus. High capacity adenovirus is obtained.

Example 2:

taking Ad helper2 as an example of an adenovirus with a self-removed packaging signal and controllable pIX gene expression, the genome structure of the adenovirus is characterized in that the N-terminal sequence of the Cre gene is a DNA fragment with 4 bases long at the 5 ' end of a coding region of the Cre gene, the C-terminal sequence of the Cre gene is a DNA fragment with 1049 bases long at the 3 ' end of the coding region of the Cre gene, the packaging signal of the adenovirus is placed in a reverse direction, and the DNA sequences inserted between the 192 th base and the 5197 th base at the 5 ' end of the adenovirus genome are as follows:

ATAACTTCGTATAATGTATGCTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAAAACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGTTAT。

the other structures are the same as those of Ad helper1 mentioned in example 1.

Example 3:

taking Ad helper3 as an example of an adenovirus with a self-removed packaging signal and controllable pIX gene expression, the genome structure of the adenovirus is characterized in that the N-terminal sequence of the Cre gene is a DNA fragment 1049 bases long at the 5 ' end of a coding region of the Cre gene, the C-terminal sequence of the Cre gene is a DNA fragment 4 bases long at the 3 ' end of the coding region of the Cre gene, the packaging signal of the adenovirus is placed in a reverse direction, and the DNA sequences inserted between the 192 th base and the 5197 th base at the 5 ' end of the adenovirus genome are as follows:

ATAACTTCGTATAGCATACATTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAAAACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACTCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAACTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAGCATACATTATACGAAGTTAT。

Nucleotide or amino acid sequence listing

<110> university of Shanxi university

<120> a helper adenovirus for improving packaging efficiency of high-capacity adenovirus

<160>

<210> 1

<211> 2467

<212> example 1 sequence inserted between 192 nd and 5197 th bases from 5' end of type 5 adenovirus genome

<213> DNA

<220>

<400>

<210> 2

<211> 2882

<212> example 1 pIX Gene expression cassette and IVa2 Gene expression cassette inserted between 27892 th and 30991 th bases from 5' end of type 5 adenovirus genome

<213> DNA

<220>

<400>

<210> 3

<211> 37

<212> primer P1 EPXN for

<213> DNA

<220>

<400>

AATTTTAATTAATTCTCGAGAATCGATAAGCGGCCGC。

<210> 4

<211> 37

<212> primer P2 EPXN reverse

<213> DNA

<220>

<400>

AGCTGCGGCCGCTTATCGATTCTCGAGAATTAATTAA。

<210> 5

<211> 33

<212> primer P3E 3up PacI for

<213> DNA

<220>

<400>

ATTAATTAAatcgacccgcgagcttagaaacag。

<210> 6

<211> 31

<212> primer P4E 3up XhoI reverse

<213> DNA

<220>

<400>

ACTCGAGtttcaggcgcagttgctctgcctc。

<210> 7

<211> 30

<212> primer P5E 3 down ClaI for

<213> DNA

<220>

<400>

AATCGATgtttcctcctgttcctgtccatc。

<210> 8

<211> 31

<212> primer P6E 3 down NotI reverse

<213> DNA

<220>

<400>

AGCGGCCGCtgagctgcccggggagtttatt。

<210> 9

<211> 31

<212> primer CMV XhoI for

<213> DNA

<220>

<400>

ACTCGAGGTTACATAACTTACGGTAAATGGC。

<210> 10

<211> 51

<212> primer CMV pIX overlap reverse

<213> DNA

<220>

<400>

ttccatcaaacgagttggtgctcatATCTGACGGTTCACTAAACGAGCTCT。

<210> 11

<211> 25

<212> primer pIX for

<213> DNA

<220>

<400>

Atgagcaccaactcgtttgatggaa。

<210> 12

<211> 32

<212> primer Iva2 reverse

<213> DNA

<220>

<400>

Atggaaaccagagggcgaagaccggcagcgct。

<210> 13

<211> 34

<212> primer EF1 alpha ClaI for

<213> DNA

<220>

<400>

AATCGATAAGGATCTGCGATCGCTCCGGTGCCCG

<210> 14

<211> 53

<212> primer EF1 alpha Iva2 overlap reverse

<213> DNA

<220>

<400>

AGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGTGGCGTCTAGCGTAGGCGC。

<210> 15

<211> 30

<212> primer hellpere 1down ClaII for

<213> DNA

<220>

<400>

AATCGATctacggcatctcgatccagcata。

<210> 16

<211> 30

<212> primer hellpere 1down NotI reverse

<213> DNA

<220>

<400>

AGCGGCCGCgggccaggtgaatatcaaatc。

<210> 17

<211> 2674

<212> ITR-loxp- Ψ -cre-loxp fragment

<213> DNA

<220>

<400>

<210> 18

<211> 2467

<212> example 2 sequence inserted between 192 nd and 5197 th bases from 5' end of adenovirus genome in order

<213> DNA

<220>

<400>

<210> 19

<211> 2467

<212> example 3 sequence inserted between 192 nd and 5197 th bases from 5' end of adenovirus genome in order

<213> DNA

<220>

<400>

Claims

1. A helper adenovirus for improving the packaging efficiency of high-capacity adenovirus is characterized in that based on a type 5 adenovirus genome, a DNA sequence between 193bp and 5196bp and a DNA sequence between 27893bp and 30990bp which are away from a 5' end of the type 5 adenovirus genome are removed; sequentially inserting a loxp sequence, an adenovirus packaging signal psi, a Cre gene expression frame and a loxp sequence between 192 th base and 5197 th base of the 5' -end distance of the type 5 adenovirus genome, wherein the directions of the two inserted loxp sequences are kept consistent; inserting a pIX gene expression frame and an IVa2 gene expression frame between 27892 th base and 30991 th base away from the 5' end of a type 5 adenovirus genome;

2. The helper adenovirus for improving the packaging efficiency of an adenovirus according to claim 1, wherein the adenoviral packaging signal Ψ is a packaging signal of a type 5 adenovirus, and specifically, a DNA sequence between 193 rd and 440 th bases from the 5' end of the type 5 adenovirus genome, in a forward or reverse direction.

3. A helper adenovirus according to claim 1, for use in increasing the packaging efficiency of an adenovirus, wherein the Cre gene expression cassette sequences are as follows:

4. the helper adenovirus for improving the packaging efficiency of an adenovirus according to claim 1, wherein the pIX gene expression cassette comprises a promoter and a terminator added to the 5 'end and the 3' end of the pIX gene coding region of the adenovirus type 5, respectively; the IVa2 gene expression is that promoter and terminator are added to 5 'end and 3' end of IVa2 gene coding region of type 5 adenovirus respectively.

5. A helper adenovirus for increasing the packaging efficiency of an adenovirus according to claim 1 or 4, wherein the promoter is a eukaryotic promoter and the terminator is a eukaryotic terminator.

6. The helper adenovirus for increasing packaging efficiency of an adenovirus according to claim 5, wherein the promoter is any one of CMV, EF1 α, PGK, SV40, CAG, Tet on, Tet off, and the terminator is any one of SV40pA, BGHpA, TKpA, hGHpA.