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Expression plasmid, cell strain for packing capacity-increased bibasic adenovirus and application of cell strain

A technology for expressing plasmids and packaging capacity, applied to cells, viruses, and viral peptides modified by the introduction of foreign genetic material, to achieve high specificity, simple operation, and less consumables

Active Publication Date: 2020-01-07
JIAXING ANYU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the first generation of non-proliferating adenoviral vectors can only load about 8kb of foreign genes at most, which is still far from enough for many gene therapy programs

Method used

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  • Expression plasmid, cell strain for packing capacity-increased bibasic adenovirus and application of cell strain
  • Expression plasmid, cell strain for packing capacity-increased bibasic adenovirus and application of cell strain
  • Expression plasmid, cell strain for packing capacity-increased bibasic adenovirus and application of cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The construction of embodiment 1pcDNA3.1+(hyg)-ORF6-IRES-DBP-6his plasmid

[0059] 1. Primer synthesis

[0060] According to the sequence of human adenovirus type 5 (hAd5) in NCBI, primers (sequence number AC_000008.1) were designed, PCR amplified the two sequences encoding the ORF6 protein of hAd5-E4 and the DBP protein of hAd5-E2a, and added the 6his tag sequence , the translation of the two proteins is initiated by the IRES sequence, and the two gene co-expression plasmid pcDNA3.1+(hyg)-ORF6-IRES-DBP-6His based on the IRES sequence is constructed. The primer sequences are shown in the table below:

[0061] Table 1 Primer Sequence

[0062]

[0063]

[0064] 2. Plasmid construction

[0065] (1) Construction of pcDNA3.1-DBP-6his

[0066] ①Amplification of the target fragment

[0067] PCR template: adenovirus hAd5 genome;

[0068] Reaction system: 25 μL of high-fidelity Q5 DNA polymerase, 1 μL of DBP-6HIS sequence upstream and downstream primers, 1 μL of PC...

Embodiment 2

[0099] The construction of embodiment 2 cell lines

[0100] 1. Stable transfection of cells

[0101] Inoculate HEK293 cells in 6-well plates according to 8×10 5 Density of viable cells / mL, when the cell confluency is around 80%, mix 2 μg of pcDNA3.1+(hyg)-ORF6-6His-IRES-DBP-6His plasmid and 5 μL of PEI, and transfect cells into 6-well plates medium, placed at 37°C, 5% CO 2 Static cultivation.

[0102] 2. Screen positive clones

[0103] After 2 days of transfection, change the resistant medium, DMEM containing 125 μg / mL hygromycin (hygromycin) in 10% FBS, and then change the medium once every 3 or 4 days, until 99% of the control blank cells are completely dead, use 0.05% pancreatic Cells were digested with protease and transferred to a 10cm dish at a dilution of 1:20. Continue to change the selection medium every 3 days until visible clones are formed, and expand the cell clones into 24-well plates through the cloning ring.

[0104] 3. Target gene and protein Western b...

Embodiment 3A

[0106] Example 3 Analysis of AY293-TD-6\24\37\40\55 Packaging Adenovirus Ability

[0107] The logarithmically grown AY293-TD-6\24\37\40\55 and HEK293 cells were divided into 6×10 5 Inoculate a 6-well plate at a density of 1 / mL. When the cell confluency is 60-70%, inactivate 2 μg DNA of the adenovirus vector pAd5△E4△E2a-EGFP by linearization with PacI endonuclease, among which, pAd5△E4△E2a -EGFP linearized electrophoresis results such as Figure 9 As indicated, the linearized pAd5ΔE4ΔE2a-EGFP was named pAd5-ΔE4ΔE2a-PacI. Then, the linearized pAd5-△E4△E2a-PacI was transfected into the above cells, and after 72 hours of culture, the cell suspension was collected, and after three times of freezing and thawing at -80°C and 37°C, centrifuged at 500g and 4°C After 5 minutes, the supernatant was taken to inoculate cells at the same density again, and after 3 passages, it was found that AY293-TD-37 cells could express a large amount of green fluorescent protein, and obvious cytopat...

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Abstract

The invention discloses an expression plasmid, a cell strain for packing capacity-increased bibasic adenovirus and an application of the cell strain. The cell strain is preserved in China Center for Type Culture Collection at May 08, 2019, wherein the preservation number is CCTCC NO:C201996, and the classification designation is human embryo kidney transformant AY293-TD-37. The cell strain comprises E2a-DBP gene and E4-ORF6 gene of adenovirus, can be used for packing the E2a-DBP gene and bibasic adenovirus wit E4 gene deletion to form complete bibasic adenovirus granules having infection. Compared with the bibasic adenovirus, a first generation adenovirus has the advantages that the emergence probability of RCA is greatly reduced, foundation is established for preparation of a live load vaccine, and due to simultaneous deletion of the E2a-DBP gene and the E4 gene, the package capacity is improved once again compared with the bibasic adenovirus with E2a mutation or E4 deletion, the insertion quantity of adenovirus vector exogenous genes is further increased, and the cell strain has important significance for increment of the application level of an adenovirus vector.

Description

technical field [0001] The invention belongs to the fields of gene therapy and recombinant vaccines, and specifically relates to an expression plasmid, a cell strain of the second-generation adenovirus with increased packaging capacity and application thereof. Background technique [0002] Adenovirus vectors have the advantages of wide host range, high gene transfer efficiency, stability, safety, and easy operation, and are one of the most important viral vectors in the field of gene transfer and gene therapy. The adenovirus genome contains four early transcription elements, E1 (E1a and E1b), E2 (E2a and E2b), E3, and E4, which encode viral regulatory proteins, and five late transcription elements L1-L5, which encode late-expressed Viral structural protein. The genome of adenovirus is about 36000bp, and the whole genome is divided into 100 gene map distance units. There are 100-150bp inverted terminal repeats (ITR) at the 5' end and 3' end of the genome, and at its left end...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
CPCC07K14/005C12N15/85C12N2710/10022Y02A50/30
Inventor 陈平李娜王暄沈亚钕
Owner JIAXING ANYU BIOTECH CO LTD
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