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Monkey type 1 adenovirus (SAdV-1) vector system and application thereof

A vector system, monkey adenovirus technology, applied in the direction of virus/bacteriophage, virus, vector, etc., can solve the problems affecting the gene transfer efficiency of adenovirus vector

Pending Publication Date: 2021-11-12
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The results of preclinical studies or clinical trials show that serum neutralizing antibodies seriously affect the gene transfer efficiency of adenoviral vectors

Method used

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  • Monkey type 1 adenovirus (SAdV-1) vector system and application thereof
  • Monkey type 1 adenovirus (SAdV-1) vector system and application thereof
  • Monkey type 1 adenovirus (SAdV-1) vector system and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, SAdV-1 genome is cloned to plasmid

[0052] The wild-type SAdV-1 genome sequence is known (Genbank accession number: AY771780). SAdV-1 was amplified in 293 cells, and SAdV-1 genomic DNA was extracted using the Hirt method [15] . Under standard polymerase chain reaction (PCR) conditions, use pKFAV4GFP plasmid as template, 2001KSAV1p2: tgatgatgat ttaaatccaa gtcgacgatcccgagcggta tcagctc, 2001KSAV1p3: tgatgattta aatggttggc gcgcctggaa caacactcaaccctatcg as primer to amplify a fragment of 2514 bp 2001KSAV1p1: gtttccagaataaggtatat tattgatgat gatttaaatc caagtcgac, 2001KSAV1p4: gtttccagaa taaggtatattattgatgat gatttaaatg gttggcgcgc c was used to amplify the 2563 bp fragment Kan-Ori with primers. The 31 bp at both ends of the fragment is identical to the end sequence of the ITR of the SAdV-1 genome. The Kan-Ori fragment recovered by electrophoresis was mixed with wild-type FAdV-4 genomic DNA, and then an equal volume of DNA assembly reagent (Gibson assembly) was a...

Embodiment 2

[0053] Example 2, Construction of Adenoviral Plasmid pKSAV1DE3 Deleted in E3 Region

[0054] figure 1The construction process of adenoviral plasmid pKSAV1DE3 is shown. A strategy of isolating an intermediate plasmid containing the E3 region from pKSAV1, deleting the E3 region in the intermediate plasmid, and then restoring the transformed intermediate plasmid to pKSAV1 was adopted. 利用2对引物(2001KSAV1p5:gatagggttg agtgttgttc caggcgcgcc aaccatttaa atcatcat,2001KSAV1p6:gcgcgccaac catttaaatc atcatcaata atatacctta attaagac,2001KSAV1p7:cgccgctggc ggcagaggag tttgtcttaa ttaaggtata ttattgat,2001KSAV1p8:cgctgaaaccggaccacagg gcgcgccgct ggcggcagag gagtttgt)进行重叠延伸PCR合成长117bp连接区linker-AscI;AscI The pKSAV1 plasmid was digested, and the small fragment pKSAV1-AscI-FS (13290bp) and the large fragment pKSAV1-AscI-FL (23661bp) were recovered after electrophoresis; the small fragment was mixed with linker-AscI, and the DNA was assembled to obtain the intermediate plasmid pKSAV1-AscI. 再以pKSAV1-AscI为模...

Embodiment 3

[0055] Example 3, Construction of E1 / E3 Region Deleted Adenoviral Plasmid pKSAV1-EG

[0056] figure 2 The construction process of the adenoviral plasmid pKSAV1-EG is shown. The pKSAV1DE3 plasmid (441, 11188, 23039 bp) was digested with SalI, and the smaller fragment pKSAV1DE3-SalI-FS (11188 bp) and the larger fragment pKSAV1DE3-SalI-FL (23039 bp) were recovered by electrophoresis. 再以pKSAV1为模板,以2001KSAV1p19:gacgctccatggcctcgtag aagtccacgg cgaagttgaa aaattg和2001KSAV1p20:aatcatcatc aataatataccttattaatt aacgctttcc tagagaagtt ctcggatc为引物,扩增SalI-SalI片段(529 bp);两条引物2001KSAV1p21:gcgttaatta ataaggtata ttattgatga tgatttaaat ccaagtcgac和2001KSAV1p22:gtgagctgat accgctcggg atcgtcgact tggatttaaa tcatcatcaa self-annealed and extended to obtain the linker-SalI fragment (73 bp); these two fragments were mixed with pKSAV1DE3-SalI-FS for DNA assembly to obtain the intermediate plasmid pKSAV1ME1-SalI (11704 bp). AscI / EcoRV double enzyme digestion, 8163 bp fragment was recovered after electrophor...

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Abstract

The invention provides a monkey type 1 adenovirus SAdV-1 vector system. The monkey type 1 adenovirus SAdV-1 vector system comprises an adenovirus plasmid pKSAV1-EG and a packaging cell line 293SE13. A target gene coding sequence can be inserted into a SpeI restriction enzyme cleavage site of the pKSAV1-EG plasmid through DNA (Deoxyribonucleic Acid) assembly or restriction enzyme cleavage-ligation cloning to generate an adenovirus plasmid carrying the target gene; and an exogenous gene expression cassette containing an expression regulatory sequence can be similarly inserted into an FseI site of pKSAV1-EG, and an adenovirus plasmid carrying the exogenous gene expression cassette is generated. By transfecting a 293SE13 cell with the adenovirus plasmid subjected to SwaI linearization, and a recombinant virus is rescued; and the 293SE13 is also used for amplifying the recombinant virus. The generated SADV-1 virus genome is deleted in an E1 / E3 part, and the SADV-1 virus is a replication-defective virus. The human lacks the pre-stored immunity to the SADV-1, and the target cells of the SADV-1 are different from those of a common HAdV-5 vector, so that the vector system is predicted to have a wide application prospect in the fields of gene therapy and vector vaccines.

Description

technical field [0001] The invention belongs to the field of recombinant vaccines, and in particular relates to a recombinant monkey adenovirus SAdV-1 vector system and its application. Background technique [0002] Adenovirus has no envelope, and the virus particles are icosahedral in shape, with a diameter of about 80nm. The genome is a linear double-stranded DNA with a length of 26-48kb. Adenoviridae (Adenoviridae) is divided into five genera (genus), of which mammalian adenoviruses infect mammals. Adenovirus infection has been found in various mammalian hosts, including humans, monkeys, cattle, horses, pigs, sheep, mice, etc. Human adenovirus (humanadenovirus, HAdV) is divided into seven species A-G, and each species includes multiple types. In general, the pathogenicity of human adenoviruses is weak, and wild-type adenoviruses generally do not cause serious diseases in people with normal immune function. Types 2 and 5 of HAdV-C are the most well-studied, and HAdV-5 ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/65C12N5/10
CPCC12N15/86C12N15/65C12N5/0686C12N2710/10343C12N2800/107C12N2510/00Y02A50/30
Inventor 鲁茁壮郭小娟谭文杰邓瑶尹丰彩
Owner 中国疾病预防控制中心病毒病预防控制所
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