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Recombinant adenovirus expression vector based on adenovirus HAd49 and construction method thereof

A technology of recombinant adenovirus and expression vector, applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problem of recombinant virus titer or purity can not reach clinical use, and achieve the effect of improving the success rate

Inactive Publication Date: 2019-12-13
广州佰芮慷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to differences in adenovirus subtypes or species, the titer and purity of the packaged recombinant virus cannot meet the standards for clinical use, and there is a certain gap with the packaged Ad5 virus vector

Method used

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  • Recombinant adenovirus expression vector based on adenovirus HAd49 and construction method thereof
  • Recombinant adenovirus expression vector based on adenovirus HAd49 and construction method thereof
  • Recombinant adenovirus expression vector based on adenovirus HAd49 and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Amplification and purification of adenovirus and extraction of adenovirus genome

[0088] The rare human serotype adenovirus HAd49 proliferates in HEK293 cells, and the purified virus is obtained by cesium chloride density gradient centrifugation, and then the viral genomic DNA is extracted, identified by double enzyme digestion with SrfI and KpnI, and subjected to agarose electrophoresis. For the experimental results, please refer to figure 1 , where Figure A is the result of cesium chloride purified virus, the white band clearly visible in the lower layer is the wild-type virus HAd49 with infectious activity, and Figure B is the result of SrfI and KpnI double enzyme digestion identification of HAd49 virus extracted, The strips are the right size.

Embodiment 2

[0089] Example 2 Construction of a replication-deficient adenoviral vector

[0090] according to Figure 5 Flowchart shown, PCR amplification of the LITR fragment (1-462) of the genome of HAd49. Using the pUC57-linker plasmid as a template, the Linker fragment was amplified by PCR. Using the DNA fragments of LITR and Linker as templates, the sequence LITR-Linker was obtained by OverlappingPCR amplification, and the target gene was connected to the vector pNEB193 using restriction sites PmeI and SpeI to obtain the plasmid pNEB193-LITR+linker;

[0091] PCR amplified HAd49 genome position 30736 to 32257 fragment is 3; PCR amplification Ad5 genome fragment E4orf6 is 4; PCR amplification HAd49 genome position 33389 to 35215 fragment is 5; with DNA fragments 3,4 and 5 is a template, OverlappingPCR amplifies to obtain fragment 345, inserts the target gene into the plasmid pNEB193-LITR+linker with the restriction sites of SwaI and PacI, and obtains the plasmid pNEB193-LITR+linker+34...

Embodiment 3

[0094] Example 3, construction of recombinant adenovirus Ad49-eGFP

[0095] The green fluorescent protein gene (eGFP) was cloned into the Ad49 vector, and the recombinant adenoviral vector Ad49-eGFP was identified by HindIII digestion, and the Ad49 empty vector digested by restriction enzyme was used as a control. Please refer to image 3 A, the size of the band is correct, and the exogenous gene eGFP is correctly connected after sequencing analysis.

[0096] After the recombinant vector Ad49-eGFP was identified by enzyme digestion and sequencing, it was transfected into HEK293 cells after linearization with AsiSI enzyme digestion, and the virus was packaged. Cytopathies appeared on the fifth day of transfection, and then the plaques of the virus gradually expanded, as shown in image 3 As shown in B, the formation of clear viral plaques can be seen, and a large amount of expression of green fluorescent protein can be observed under a fluorescent microscope.

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Abstract

The invention discloses a recombinant adenovirus expression vector based on human rare serotype adenovirus HAd49 and a construction method of the recombinant adenovirus expression vector. According tothe invention, a genome of wild adenovirus HAd49 is used as a basis, through a direct cloning method by genome, an E1 coding region and an E3 coding region are deleted, an I-CeuI enzyme cutting siteand a PI-SceI enzyme cutting site are inserted into an E1 deletion region, meanwhile, the E4orf6 of the human adenovirus Ad5 is used for replacing the E4orf6 of the HAd49, so that the success rate ofpackaging the recombinant virus in a human kidney embryo cell (HEK293) and the titer of the packaged recombinant virus are improved, and the recombinant adenovirus expression vector Ad49 with replication defects is obtained. The expression vector can be used for expressing various antigens, and provides a basis for research and development of vaccines and biological medicines.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant adenovirus vector used in the research and development of gene therapy and vaccine vectors; specifically, the invention relates to a recombinant adenovirus expression vector based on adenovirus HAd49 and a construction method thereof. Background technique [0002] The development of adenovirus (adenovirus, Ad) as a gene expression vector began in the early 1960s, when scientists discovered that the adenovirus genome can hybridize with the simian virus 40 (SV40) genome, indicating that the adenovirus genome can access heterologous Gene. Since then, adenovirus has gradually developed into an important vector system. Existing studies have proved that adenovirus vector vaccines can stimulate and induce specific T cell and B cell immune responses in different animals, but different subtypes of adenovirus have different infective and stimulating abilities. Adenoviruses in s...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/40A61K39/12A61P31/14
CPCA61K39/12A61P31/14C07K14/005C12N15/86C12N2710/10043C12N2770/24122C12N2770/24134Y02A50/30
Inventor 黎诚耀罗升学刘博超张攀丽
Owner 广州佰芮慷生物科技有限公司
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