CN108203706A - A kind of helper adenovirus for being used to improve high capacity adenoviral packaging efficiency - Google Patents

Abstract

The invention discloses a kind of for improving the helper adenovirus of high capacity adenoviral packaging efficiency, based on 5 type adenoviral gene groups, the DNA sequence dna between the DNA sequence dna and 27893bp to 30990bp between 5 type adenoviral gene group distance, 5 ＇ ends 193bp to 5196bp is eliminated；Loxp sequences, adenoviral packaging signal Ψ, Cre gene expression frame, loxp sequences are sequentially inserted between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base；Between 5 type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base, it is inserted into pIX gene expression frames and IVa2 gene expression frames.By adding in Cre genes, under the CRE enzyme effects of incasing cells, while helper virus packaging signal Ψ is removed, the expression of the Cre genes entrained by helper virus itself is induced, improves the amount of CRE enzymes, the removal for making packaging signal Ψ is more thorough.The genome structure of helper virus is adjusted simultaneously, avoids helping virus and the virus genomic recombination in incasing cells and the generation of wild-type virus.

Description

A kind of helper adenovirus for being used to improve high capacity adenoviral packaging efficiency

Technical field

The invention belongs to biotechnologies, are related to a kind of helper adenovirus, more particularly to a kind of to be used to improve high power capacity The helper adenovirus of adenovirus packaging efficiency.

Background technology

It is to pass through viral vectors by the foreign gene importing most effective means of target cell at present in gene therapy.Often Viral vectors includes adenovirus vector, gland relevant viral vector, slow virus carrier etc..Gland relevant viral vector has immune Originality is low, not radom insertion chromosome the advantages that, but its capacity is too small, is restricted in use.Slow virus carrier due to Meeting radom insertion chromosome, easily causes cancer, safety is under suspicion.Adenovirus vector has high power capacity, high titre, not whole The advantages that being incorporated into chromosome, but its immunogenicity is high, easily causes stronger immune response in treating in vivo.Development at present The third-generation adenovirus vectors to get up remove the most gene group of adenovirus, only remain its member that is used to replicate and pack Part ITR and Ψ, thus greatly reduce its immunogenicity.It is considered as a kind of viral vectors very with application prospect.

Due to eliminating most virus genome sequence, the packaging of third-generation adenovirus needs helper virus to provide Various capsid proteins needed for packaging, while need to avoid being polluted by helper virus again.Currently used removal auxiliary disease In the method for poison, effect in the both sides of its packaging signal Ψ it is preferable to add in loxp sequences, in auxiliary high capacity adenoviral In packaging process, packaging signal Ψ is removed after loxp sequences by CRE enzyme effects expressed in incasing cells, so as to make Obtaining helper virus genome can not be packaged.But due to CRE expression quantity deficiency in these methods and in packaging process Helper virus may occur recombination with fractionated viral genome sequence included in incasing cells and form wild-type virus, because This, helper virus can not preferably remove from packaging virus, still remain certain pollution.Currently without better method Make the pollution of helper virus further reduce.

Invention content

For the polluted technical problem of above-mentioned helper adenovirus, the object of the present invention is to provide a kind of new energy Enough helper adenovirus for more efficiently removing packaging signal and avoiding generating wild-type virus.

In order to realize above-mentioned task, the present invention takes following technical solution：

A kind of helper adenovirus for being used to improve high capacity adenoviral packaging efficiency, which is characterized in that with 5 type adenovirus bases Based on group, the DNA sequence dna and 27893bp that eliminate between 5 type adenoviral gene group distance, 5 ＇ ends 193bp to 5196bp arrive DNA sequence dna between 30990bp；Between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base according to Secondary insertion loxp sequences, adenoviral packaging signal Ψ, Cre gene expression frame, loxp sequences, two loxp sequences directions of insertion It is consistent；Between 5 type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base, it is inserted into pIX bases Because of expression cassette and IVa2 gene expression frames.

According to the present invention, the adenoviral packaging signal Ψ is the packaging signal of 5 type adenovirus, specially 5 type adenopathies Virus gene group distance the 193rd, 5 ＇ ends base is to the DNA sequence dna between the 440th base, and direction is forward or backwards.

The Cre gene expression frames are made of promoter, Cre genes, terminator；Wherein, Cre gene orders include N Terminal sequence, C-terminal sequence, introne shearing donor sequences, introne shearing receptor sequence, N-terminal sequence is 5 ＇ of Cre gene coding regions The DNA fragmentation that 4 bases in end are grown to 1049 bases, C-terminal sequence are Cre gene coding regions 4,3 ＇ ends base to 1049 alkali The DNA fragmentation of base length is inserted into one section of introne shearing donor sequences at 3 ＇ ends of N-terminal sequence, one is inserted at 5 ＇ ends of C-terminal sequence Section introne shearing receptor sequence, introne shearing donor sequences and shearing receptor sequence total length are no more than 500bp；Introne Shear donor sequences, Cre gene N-terminals sequence, promoter, terminator, Cre gene Cs terminal sequence, introne shearing receptor sequence according to The secondary direction according to 3 ＇ to 5 ＇ is inserted into the 3 ＇ ends of adenoviral packaging signal Ψ.

Above-mentioned Cre gene expression frames sequence is as follows：

AAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACT TACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGA TCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGC TGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTC AGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTT TCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCA TCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGC ATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGG GGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGT GGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTA ATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGC GGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAG TTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATT GACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGG ACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAA CGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACAC CTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAA TAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCAT CAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTT CACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATT GAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACC AGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGC TGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCA GAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAG CGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAA TTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTA TTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCAT。

Further, the pIX gene expression frames are at 5 ＇ ends of the pIX gene coding regions of 5 type adenovirus and 3 ＇ ends It is separately added into promoter and terminator；The IVa2 gene expressions are at 5 ＇ ends of the IVa2 gene coding regions of 5 type adenovirus Promoter and terminator are separately added into 3 ＇ ends.

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

The promoter is any one in CMV, EF1 α, PGK, SV40, CAG, Tet on, Tet off, described Terminator is any one in SV40pA, BGHpA, TKpA, hGHpA.

Provided by the present invention for improving the helper adenovirus of high capacity adenoviral packaging efficiency, by helper adenovirus Middle addition Cre genes, under the CRE enzyme effects of incasing cells, while helper virus packaging signal Ψ is removed, induction auxiliary The expression of Cre genes entrained by virus itself, so as to improve the amount of CRE enzymes, the removal for making packaging signal Ψ is more thorough.Together When the genome structure of helper virus is adjusted, avoid helper virus with it is virus genomic heavy in incasing cells Group, so as to avoid the generation of wild-type virus.The pollution of helper virus is further reduced by the two aspects.So as to obtain Obtain the higher high capacity adenoviral of purity.

Description of the drawings

Fig. 1 is Ad helper1 structures and action principle figure.

Fig. 2 is that helper adenovirus packs the lesion picture to be formed.

Fig. 3 is helper adenovirus packaging signal removal effect testing result figure.

Fig. 4 is helper adenovirus pollution detection result figure.

The present invention is described in more detail with reference to the accompanying drawings and examples.

Specific embodiment

Applicant is sequentially inserted between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base Loxp sequences, adenoviral packaging signal Ψ, Cre gene expression frame, loxp sequences, so that in the adenovirus of expression CRE enzymes In incasing cells, helper adenovirus induces the expression of itself Cre gene, so as to carry while itself packaging signal Ψ is removed The amount of CRE enzymes in high incasing cells so that packaging signal Ψ removals are more thorough.Simultaneously by pIX bases in 5 type adenoviral gene groups Because expression cassette and IVa2 gene expression frames have been moved to distance the 27892nd, 5 ＇ ends base and the 30991st alkali from original position Between base, so as to avoid fractionated viral genome sequence weight present in helper adenovirus genes group and incasing cells genome Group generates the possibility of wild-type virus.It is caused by improving helper adenovirus above when high capacity adenoviral is assisted to pack Helper virus pollution further reduce.

It is the embodiment that inventor provides below, it should be noted that these embodiments are only the preferred implementations of the present invention Mode, the present invention is not limited to these Examples.

Embodiment 1：

For from the helper adenovirus Ad helper1 for removing packaging signal and pIX gene controlled expressions, the auxiliary gland Organization of viral genome is as follows：

Based on 5 type adenoviral gene groups, 5 type adenoviral gene group sequence NCBI numbers are AC_000008.Removal Between DNA sequence dna and 27893bp to 30990bp in 5 type adenoviral gene groups between distance 5 ＇ ends 193bp to 5196bp DNA sequence dna；Loxp sequences are sequentially inserted between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base Row, adenoviral packaging signal Ψ, Cre gene expression frame, loxp sequences, two loxp sequences directions of insertion are consistent；5 PIX gene expression frames and IVa2 are inserted between type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base Gene expression frame.

Adenoviral packaging signal Ψ is placed to be positive, and the 193rd, 5 ＇ ends of specially 5 type adenoviral gene group distance base arrives DNA sequence dna between 440th base.

DNA fragmentation of the Cre gene N-terminals sequence for 5 ＇ ends of Cre gene coding regions, 453 base length, Cre gene C terminal sequences DNA fragmentation for 3 ＇ ends of Cre gene coding regions, 600 base length.

The DNA sequence dna being inserted between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base is such as Under：

ATAACTTCGTATAATGTATGCTATACGAAGTTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTT GTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAA TAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCC AGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCTCGAC AAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAA CGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCAT TGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTT ACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTT ATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGC GATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGG TGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTG CTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAA GTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAA ACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTAC TGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAAT AGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGC GGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCC CGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAC CGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCA GCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGA ATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTC CCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCA TGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGC GCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAA TCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCAT TTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGT TTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAA CATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTAT AAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGA GAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGT TAT。

The pIX bases being inserted between 5 type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base Because expression cassette and IVa2 gene expression frames are as follows：

GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGAT GCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAA ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATATGAGCACCAAC TCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGAT GGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGC CGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTC CTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATT GGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGG CTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCT TGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTAT TTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGC ACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTA AAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGA TGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCC GGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGA AATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGG CCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCAT AGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTA CCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAAC GGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCC CGTAAATCACACCTATTACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCC ACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAG CAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAA GCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGT TGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGG GTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCT GGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGGCTGCAGGTCGAAAGGCCCGGAGATG AGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCGGAGGACCTTCGGGCG CCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAA GGAGCAAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTCCATCTGCACGA GACTAGCTAGTAGTGAGACGTGCTACTCCCATTTGTCACGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTT CCTGACTAGGGGAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATG TGGAATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAAGCGCATGCTCCAGAC TGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAT。

Shown in the Organization of viral genome and action principle as attached drawing 1.

Helper adenovirus Ad helper1 building process is as follows：

(1) structure of the helper adenovirus E3 areas shuttle vector of pIX and IVa2 expression cassettes is carried

In Huada gene company synthetic primer, sequence is as follows：

P1EPXN for：AATTTTAATTAATTCTCGAGAATCGATAAGCGGCCGC；

P2EPXN reverse：AGCTGCGGCCGCTTATCGATTCTCGAGAATTAATTAA；

EPXN linker (EcoRI M-PacI-XhoI-ClaI-NotI- will be formed after above two primer annealings HindIIIM), EPXN linker with the pUC19 carriers by EcoRI and HindIII digestions processing are connect, obtains pUC19/ EPXN。

By Huada gene company synthetic primer, sequence is as follows：

P3E3up PacI for：ATTAATTAAatcgacccgcgagcttagaaacag；

P4E3up XhoI reverse：ACTCGAGtttcaggcgcagttgctctgcctc；

P5E3down ClaI for：AATCGATgtttcctcctgttcctgtccatc；

P6E3down NotI reverse：AGCGGCCGCtgagctgcccggggagtttatt；

Respectively using P3/P4 and P5/P6 as primer, 5 type adenoviral gene group position templates, PCR amplification amplification obtain E3 up and E3 down.E3up and E3 down are handled with PacI/XhoI and ClaI/NotI digestions respectively and recycle segment, with being connected into successively The pUC19/EPXN carriers handled with similary digestion obtain phelperE3 shuttle.

Following primer is synthesized by Huada gene company：

CMV XhoI for：ACTCGAGGTTACATAACTTACGGTAAATGGC；

CMV pIX overlap reverse：

ttccatcaaacgagttggtgctcatATCTGACGGTTCACTAAACGAGCTCT；

pIX for：atgagcaccaactcgtttgatggaa；

IVa2reverse：atggaaaccagagggcgaagaccggcagcgct；

EF1αClaI for：AATCGATAAGGATCTGCGATCGCTCCGGTGCCCG；

EF1αIVa2overlap reverse：

AGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGTGGCGTCTAGCGTAGGCGC。

Using 5 type adenoviral gene groups, CMV promoter, EF1 α promoters as template, CMV- is obtained by overlapping PCR method PIX-IVa2-EF1 α segments with XhoI and ClaI digestions and recycle CMV-pIX-IVa2-EF1 α segments, with passing through similary digestion The phelperE3shuttle connections of processing obtain phelperE3/CMV-pIX-IVa2-EF1 α.

(2) structure of the adenovirus E 1 area shuttle vector of ITR-loxp- Ψ-cre-loxp elements is carried

Following primer is synthesized by Huada gene company：

helperE1down ClaII for：AATCGATctacggcatctcgatccagcata；

helperE1down NotI reverse：AGCGGCCGCgggccaggtgaatatcaaatc.

Using 5 type adenoviral gene groups as template, PCR amplification obtains helperE1 down segments, with ClaI and NotI digestions And helperE1down segments are recycled, it is connect with the pUC19/EPXN carriers by similary digestion processing, obtains helper adenovirus E1 areas shuttle vector phelperE1 shuttle.

Following DNA fragmentation ITR-loxp- Ψ-cre-loxp are synthesized by Huada gene company, sequence is as follows：

TTAATTAAcatcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgt ggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacat gtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgATAACTTCGTATAATGTATGCTATACGAAG TTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGAT TTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATAT TTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTC CGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACC TGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGC ATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACC GACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGA AGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCG GCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGC GAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTC TTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTA CCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACT CCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCC AAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATG TACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTG ATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTA TGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGT TATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCC TGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGT TTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTG CTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTG CAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGT TGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAG GCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAG CTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGAC CAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAG GGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTAT TCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCT ACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATC TTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTA TATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACC AATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGTTATCTCGAG。

ITR-loxp- Ψ-cre-loxp are handled with PacI and XhoI digestions, with passing through similary digestion processing PhelperE1 shuttle carriers connect, and obtain the adenovirus E 1 area shuttle load for carrying ITR-loxp- Ψ-cre-loxp elements Body phelperE1/ITR-loxp- Ψ-cre-loxp.

(3) structure of helper adenovirus Ad helper1

PhelperE3/CMV-pIX-IVa2-EF1 α are handled with PacI digestions, SwaI is introduced with SwaI digestions processing E3 areas The adenoviral backbone carrier pAd5 backbone in site (carry the adenopathy of Zinc finger nuclease Expression element and donor dna in patent Its disclosed preparation method in poison and its construction method and application), will treated plasmid cotransformation to E.coli BJ5183 Middle carry out homologous recombination obtains pAd5/CMV-pIX-IVa2-EF1 α.

PAd5/CMV-pIX-IVa2-EF1 α and phelperE1/ITR-loxp- Ψ-cre-loxp are handled with PacI digestions, Later by the two cotransfection to HEK293 cells, visible lesion is formed after 7-8 days, which packs the lesion to be formed See attached drawing 2, illustrate that helper adenovirus Ad helper1 are successfully packed.By amplification repeatedly, expand to 10 150mm cells and train After supporting ware, virus is collected, uses Cscl₂Density gradient centrifugation purified virus.

Stablize the adenovirus packaging cell HEK293-cre of expression CRE enzymes with the Ad helper1 infection of 8MOI, use simultaneously Traditional helper adenovirus Ad helper of 8MOI infect another disk HEK293-cre, collect cell after 48 hours, are examined by PCR The removal effect of packaging signal is surveyed, as a result sees attached drawing 3, after smaller electrophoretic band is packaging signal removal in experimental result PCR product, and the PCR product that larger electrophoretic band is packaging signal when not removing, the packaging of visible Ad helper1 in figure Signal removal is more thorough.

With 4MOI Ad helper1 and 4MOI Ad eGFP (normal adenovirus for carrying eGFP genes) coinfection HEK293-cre cells, while with another disk HEK293-cre cells of 4MOI Ad helper and 4MOI Ad eGFP coinfections, Virus infection U87 cells are collected after 48 hours, infection collects U87 cell extraction genomic DNAs after 24 hours, passes through Realtime PCR detects the pollution of helper virus, as a result sees attached drawing 4, and Ad helper1 pollute lower than Ad helper as seen from Figure 4.

Helper adenovirus Ad helper1 application methods are as follows：

By high capacity adenoviral vector pHCAD PacI linearization for enzyme restriction, 60mm is transfected by coprecipitation of calcium phosphate and is put down HEK293-cre cells in ware, with after 4MOI helper adenovirus Ad helper1 infection pHCAD transfections after 18 hours After HEK293-cre, 48-72 hours, cell is collected, by 3 multigelations, supernatant is collected, obtains virus.Take 1/4 collection Employing virus cracking liquid and 4MOI helper adenovirus Ad helper1 coinfections collect cell acquisition after HEK293-cre, 48-72 hours Virus.Such repeated amplification virus passes through Cscl to the cell of 20 150mm culture dishes after collecting virus₂Density gradient centrifugation, Purified virus.Obtain high capacity adenoviral.

Embodiment 2：

For from the helper adenovirus Ad helper2 for removing packaging signal and pIX gene controlled expressions, the auxiliary gland Organization of viral genome is characterized as that the N-terminal sequence of Cre genes is the DNA fragmentation of 5 ＇ ends of Cre gene coding regions, 4 base length, DNA fragmentation of the Cre gene Cs terminal sequence for 3 ＇ ends of Cre gene coding regions, 1049 base length, adenoviral packaging signal is reversely puts It puts, the DNA sequence dna being inserted between adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base is as follows：

ATAACTTCGTATAATGTATGCTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAA AACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACAC AAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAAT TTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCTCGAC AAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACGCATGGTGGCATCTGACGGTTCAC TAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTAC GACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAAT CCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAA TACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGT CATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATAC TCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCG GGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCT CATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCC GGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTG AAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT CACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC ATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTA CGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCG GGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAG CGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGC GCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCA TCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGC CAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGT AACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGA ACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTC GGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTT AGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATC TTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCA TTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTT GCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGCTGTGGA GAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGT TAT。

Other structures are identical with the Ad helper1 that embodiment 1 is previously mentioned.

Embodiment 3：

For from the helper adenovirus Ad helper3 for removing packaging signal and pIX gene controlled expressions, the auxiliary gland Organization of viral genome is characterized as that the N-terminal sequence of Cre genes is the DNA pieces of 5 ＇ ends of Cre gene coding regions, 1049 base length Section, DNA fragmentation of the Cre gene Cs terminal sequence for 3 ＇ ends of Cre gene coding regions, 4 base length, adenoviral packaging signal is reversely puts It puts, the DNA sequence dna being inserted between adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base is as follows：

ATAACTTCGTATAGCATACATTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAA AACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACAC AAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAAT TTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCTCGAC AAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACTCGCCATCTTCCAGCAGGCGCACC ATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCAT GATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCA GGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGA GTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATC ATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTG CGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACC CTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTG GCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCAC GTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGC AGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTG AAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCG CCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATC AGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGA CACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCG TACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCC AAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTG ATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCA TAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATAT GATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATT GGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATT TACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGC TGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGT GTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAG GTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAAC TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACT GCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAACTGTGGA GAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAGCATACATTATACGAAGT TAT。

Other structures are identical with the Ad helper1 that embodiment 1 is previously mentioned.

Nucleotide or amino acid sequence table

<110>Shaanxi Normal University

<120>A kind of helper adenovirus for being used to improve high capacity adenoviral packaging efficiency

<160>

<210> 1

<211> 2467

<212>What embodiment 1 was inserted between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base Sequence

<213> DNA

<220>

<400>

ATAACTTCGTATAATGTATGCTATACGAAGTTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGA TGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATC TGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTC GCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCT CGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTT CGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACA GCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGT TTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCC GGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCC TGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATT TTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGC TCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTG GAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAG TCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGAT GTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGT CAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCAT TGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTT GGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGC GCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTG TGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATA ATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAA ATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGA TCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAG TTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATC TCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAA TCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACA CCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGAC CAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGA TTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTG TTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTG TGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACG AAGTTAT。

<210> 2

<211> 2882

<212>Embodiment 1 interleaves 5 type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base The pIX gene expression frames and IVa2 gene expression frames entered

<213> DNA

<220>

<400>

GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCAC TTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATT GACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATATGAGCAC CAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATG TGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGA ACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGC TTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCAC AATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTG AAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGT GTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGT GTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGG TAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTG CCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCT GGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCC CTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGA AGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAA TGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCG TCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTA GTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGA AAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTG GGCCCGTAAATCACACCTATTACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGG GGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCG ATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGA CCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGC GGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCG CAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGA GGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGGCTGCAGGTCGAAAGGCCCGGA GATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCGGAGGACCTTCG GGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAG CGAAGGAGCAAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTCCATCTGC ACGAGACTAGCTAGTAGTGAGACGTGCTACTCCCATTTGTCACGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGA ACTTCCTGACTAGGGGAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTG GATGTGGAATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAAGCGCATGCTCC AGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAT。

<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 P3 E3 up PacI for

<213> DNA

<220>

<400>

ATTAATTAAatcgacccgcgagcttagaaacag。

<210> 6

<211> 31

<212>Primer P4 E3 up XhoI reverse

<213> DNA

<220>

<400>

ACTCGAGtttcaggcgcagttgctctgcctc。

<210> 7

<211> 30

<212>Primer P5 E3 down ClaI for

<213> DNA

<220>

<400>

AATCGATgtttcctcctgttcctgtccatc。

<210> 8

<211> 31

<212>Primer P6 E3 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 I va2 reverse

<213> DNA

<220>

<400>

Atggaaaccagagggcgaagaccggcagcgct。

<210> 13

<211> 34

<212>Primer EF1 α ClaI for

<213> DNA

<220>

<400>

AATCGATAAGGATCTGCGATCGCTCCGGTGCCCG

<210> 14

<211> 53

<212>Primer EF1 α Iva2 overlap reverse

<213> DNA

<220>

<400>

AGCGCTGCCGGTCTTCGCCCTCTGGTTTCCATGGTGGCGTCTAGCGTAGGCGC。

<210> 15

<211> 30

<212>Primer helperE1 down ClaII for

<213> DNA

<220>

<400>

AATCGATctacggcatctcgatccagcata。

<210> 16

<211> 30

<212>Primer helperE1 down NotI reverse

<213> DNA

<220>

<400>

AGCGGCCGCgggccaggtgaatatcaaatc。

<210> 17

<211> 2674

<212>ITR-loxp- Ψ-cre-loxp segments

<213> DNA

<220>

<400>

TTAATTAAcatcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtg acgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaac acatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgATAACTTCGTATAATGTATGCTATAC GAAGTTATTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTA AGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTA ATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC GTTCCGGGTCAAAGTTGGCGTTTTAAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTA AACCTGTCTTGTAACCTTGATACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCAC CGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCG GACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATA TAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACG ACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTC TTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTT CCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACG CCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACA AACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTAC TGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGT CATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACA CTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTT ACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGT AAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGC GCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAG AGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTA CTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTT ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTC TAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCA GCAGGCGCACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCA CCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCC AGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTT CCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAG TTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTT CTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGA TATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTA CGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAA GACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACGAAGTTATCTCGAG。

<210> 18

<211> 2467

<212>Embodiment 2 is sequentially inserted between adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base Sequence

<213> DNA

<220>

<400>

ATAACTTCGTATAATGTATGCTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGA GAAAAACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTA ACACAAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCC AAATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCT CGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACGCATGGTGGCATCTGACGGT TCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTG TTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGG AAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGA CTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTA CCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAA ATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATG GGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCG ACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCT TACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGC AGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAA CCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCT TATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCCAG GTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAG CGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCC TTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGCTGGCTGGTGGCAGA TGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAA TCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTT CTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTAT ACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCT TTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCAT GTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAA CATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGA AGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACT CGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGCTG TGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAATGTATGCTATACG AAGTTAT。

<210> 19

<211> 2467

<212>Embodiment 3 is sequentially inserted between adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base Sequence

<213> DNA

<220>

<400>

ATAACTTCGTATAGCATACATTATACGAAGTTATAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGA GAAAAACACCTGGGCGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTA ACACAAAATTATTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCC AAATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTAAAACGCAAGAGTCTTCTCTGTCT CGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGATACTTACTCGCCATCTTCCAGCAGGCG CACCATTGCCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTT GCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAG GCCAGGTATCTCTGACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGC GCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCG GATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACA CCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTT AACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATAT CCTGGCAGCGATCGCTATTTTCCATGAGTGAACGAACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTT GCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCG TGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCG CCTGAAGATATAGAAGATAATCGCGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCAT GCCGCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTC CATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAAT TGGACACCTTCCTCTTCTTCTTGGGCATGGTGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCC ACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGG TGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCC ATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGT CCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGC ATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCC TATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGC CATTTACCGTAAGTTATGTAACGCCACCGCGGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACAC CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC ATGTGTCAAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGT AGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGT TAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTT CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAACTG TGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCATATAACTTCGTATAGCATACATTATACG AAGTTAT。

Claims (8)

1. a kind of helper adenovirus for being used to improve high capacity adenoviral packaging efficiency, which is characterized in that with 5 type adenoviral genes Based on group, the DNA sequence dna and 27893bp that eliminate between 5 type adenoviral gene group distance, 5 ＇ ends 193bp to 5196bp arrive DNA sequence dna between 30990bp；Between 5 type adenoviral gene group distance the 192nd, 5 ＇ ends base and the 5197th base according to Secondary insertion loxp sequences, adenoviral packaging signal Ψ, Cre gene expression frame, loxp sequences, two loxp sequences directions of insertion It is consistent；PIX genes are inserted between 5 type adenoviral gene group distance the 27892nd, 5 ＇ ends base and the 30991st base Expression cassette and IVa2 gene expression frames.

2. the helper adenovirus as described in claim 1 for being used to improve adenovirus packaging efficiency, which is characterized in that the gland Viral packaging signal Ψ is the packaging signal of 5 type adenovirus, the 193rd, 5 ＇ ends of specially 5 type adenoviral gene group distance base DNA sequence dna between the 440th base, direction is forward or backwards.

3. the helper adenovirus as described in claim 1 for being used to improve adenovirus packaging efficiency, which is characterized in that described Cre gene expression frames are made of promoter, Cre genes, terminator；Wherein, Cre gene orders include N-terminal sequence, C-terminal sequence, Introne shearing donor sequences, introne shearing receptor sequence, N-terminal sequence is Cre gene coding regions 4,5 ＇ ends base to 1049 The DNA fragmentation of a base length, C-terminal sequence are the DNA fragmentation that Cre gene coding regions 4,3 ＇ ends base is grown to 1049 bases, One section of introne shearing donor sequences is inserted at 3 ＇ ends of N-terminal sequence, and one section of introne shearing receptor is inserted at 5 ＇ ends of C-terminal sequence Sequence, introne shearing donor sequences and shearing receptor sequence total length are no more than 500bp；Introne shearing donor sequences, Cre Gene N-terminal sequence, promoter, terminator, Cre gene Cs terminal sequence, introne shear receptor sequence successively according to 3 ＇ to the side of 5 ＇ To the 3 ＇ ends for being inserted into adenoviral packaging signal Ψ.

4. the helper adenovirus for being used to improve adenovirus packaging efficiency as described in claim 1 or 3, which is characterized in that described Cre gene expression frame sequences it is as follows：

AAACGCAAGAGTCTTCTCTGTCTCGACAAGCCCAGTTTCTATTGGTCTCCTTAAACCTGTCTTGTAACCTTGA TACTTACCTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTT CGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTT TAGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCCTGAAGATATAGAAGATAATCGCGAACAT CTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGCAAACGGACAGAAGC ATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGT TGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACACCTTCCTCTTCTTCTTGGGCATGG TGGCATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCA ATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATG GGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCAT GGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCC AGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGG GCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCAT TATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCACCGC GGGGACTAGAGTCGACCTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGC TCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAAGAGGTTTTCACCGTCATCACC GAAACGCGCGAGGCAGCCGGGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCC ACACCTCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTA CAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAAC TCATCAATGTATCTTATCATGTCTGGATCTCCCCGCGGTGCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCCCT GTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGG TATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCT GACCAGAGTCATCCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGTTGATAGC TGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTAC ACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTTACCCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAA CCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTTAACCCTGATCCTG GCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATC GCTATTTTCCATGAGTGAACGAACCTGTGGAGAGAAAGGCAAAGTGGATGTCAGTAAGACCAATAGGTGCCTATCAT 。

5. the helper adenovirus as described in claim 1 for being used to improve adenovirus packaging efficiency, which is characterized in that described PIX gene expression frames are to be separately added into promoter and terminator at 5 ＇ ends of the pIX gene coding regions of 5 type adenovirus and 3 ＇ ends； The IVa2 gene expressions are to be separately added into promoter and end at 5 ＇ ends of the IVa2 gene coding regions of 5 type adenovirus and 3 ＇ ends It is only sub.

6. the helper adenovirus for being used to improve adenovirus packaging efficiency as described in claim 3 or 5, which is characterized in that described Promoter be eukaryotic promoter, the terminator is eucaryon terminator.

7. the helper adenovirus for being used to improve adenovirus packaging efficiency as described in claim 3 or 5, which is characterized in that described Promoter be any one in CMV, EF1 α, PGK, SV40, CAG, Tet on, Tet off, the terminator is Any one in SV40pA, BGHpA, TKpA, hGHpA.

8. the helper adenovirus as claimed in claim 6 for being used to improve adenovirus packaging efficiency, which is characterized in that described opens Mover is any one in CMV, EF1 α, PGK, SV40, CAG, Tet on, Tet off, the terminator be SV40pA, Any one in BGHpA, TKpA, hGHpA.