Recombinant contagious ecthyma oncolytic virus and preparation method and application thereof

An oncolytic virus, infectious technology, applied in the field of genetic engineering, to achieve the effect of shortening the screening time and easy operation

Active Publication Date: 2018-06-29
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, p53 is neither a cell surface protein nor an enzyme, so recent antibodies and low-molecular-weight enzyme inhibitors used in targeted molecular therapies

Method used

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  • Recombinant contagious ecthyma oncolytic virus and preparation method and application thereof
  • Recombinant contagious ecthyma oncolytic virus and preparation method and application thereof
  • Recombinant contagious ecthyma oncolytic virus and preparation method and application thereof

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preparation example Construction

[0064] The preparation method of the recombinant infectious pustular oncolytic virus of the present invention comprises the following steps:

[0065] (1) overlap PCR amplifies the gene p53 of the protein described in claim 7;

[0066] (2) Connect to the pSPV-EGFP vector, insert the p53 gene into a recombinant plasmid to obtain the recombinant plasmid p53-pSPV-EGFP, and then insert the left and right sides of the ORFV132 gene to construct the shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF; Recombinant technology inserts p53 and EGFP genes into position 132 of the viral genome, thereby knocking out the ORFV132 gene; PCR and double digestion of ORFV132LF-p53-pSPV-EGFP-ORFV132RF: the recombinant plasmid band is at 6500bp, PCR amplification The band is at 700bp, and the two bands are 5600bp and 700bp respectively after digestion; the predicted ORFV132LF-p53-pSPV-EGFP-ORFV132RF is 6392bp, and ORFV132RF is 676bp, and the two bands after double digestion are 5649bp and 5649bp respe...

Embodiment 1

[0081] Construction of recombinant plasmid p53-pSPV-EGFP

[0082] (1) Amplification and identification of the target gene p53

[0083] According to the requirements of the seamless cloning kit, using p53 as a template, use the Primer Premier5 software to design PCR primers (the underlined part is the restriction site):

[0084] Hp53-Fw:

[0085] 5'-CAGTGACGCCTCGAG GAATTC ATGGAGGAGCCGCAGTCAGA-3' (EcoRI); SEQ ID No.1

[0086] Hp53-Rv:

[0087] 5'-GCTCACCATGGTGGC GAATTC GTCTGAGTCAGGCCCTTCTGTCTT-3' (EcoRI). SEQ ID No. 2

[0088] Perform PCR reaction (50 μL) according to the following system as shown in Table 1:

[0089] Table 1

[0090]

[0091] Reaction conditions:

[0092]

[0093] The PCR products were identified by agarose electrophoresis and recovered by tapping the gel.

[0094] (2) pSPV-EGFP digestion and rubber tapping recovery are shown in Table 2:

[0095] Table 2

[0096]

[0097] Digest at 37°C for 2 hours according to the above system, and recover...

Embodiment 2

[0107] Construction of recombinant plasmid ORFV132LF-p53-pSPV-EGFP

[0108] The method for constructing the recombinant plasmid ORFV132LF-p53-pSPV-EGFP is the same as 1. The upstream and downstream primers are respectively (the underlined part is the restriction site):

[0109] NA1 / 11-ORFV132L-Fw: 5'-AGTAGGCCTGCGCGCAAGCTTCGTCTTCTCCCGCTGGATAAA-3'(HindⅢ) SEQ ID No.3

[0110] NA1 / 11-ORFV132L-Rv: 5'-GACCTGCAGGCATGCAAGCTTGCCTCACCCTTAAAAGTTGG-3'(HindⅢ) SEQ ID No.4;

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Abstract

The invention discloses ORFV (Orf virus) oncolytic virus and a preparation method and application thereof. The recombinant contagious ecthyma oncolytic virus preparation method disclosed by the invention comprises the following steps: (1) overlap PCR amplifying gene p53 of protein shown in claim 7; (2) connecting to a pSPV-EGFP carrier, inserting into the p53 gene to obtain recombinant plasmid p53-pSPV-EGFP and then inserting into the left side and the right side of ORFV132 gene to construct shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF; (3) converting host bacteria TOP10 to obtain positive recombinant bacteria; (4) performing transfection on OFTu cells and performing fluorescent identification on fusion expression of the p53 and the EGFP to prepare the purified recombinant contagiousecthyma oncolytic virus. The prepared recombinant contagious ecthyma oncolytic virus can be duplicated and multiplicated in OFTu and varieties of tumor cells and can effectively inhibit growth of varieties of tumor cell. The recombinant contagious ecthyma oncolytic virus disclosed by the invention can be observed in a microscope; thus, easiness in operation is achieved, convenience and quickness are achieved, and screening time is greatly shortened.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant infectious pustular oncolytic virus and its preparation method and application. Background technique [0002] Oncolytic virus is a kind of virus that can specifically infect and kill tumor cells. In recent decades, oncolytic virus therapy has attracted widespread attention, and related research has made great progress. The most studied oncolytic viruses currently include adenovirus, type I herpes simplex virus (HSV), vaccinia virus, etc. They specifically recognize and infect tumor cells, and eventually cause cell swelling to destroy tumor cells, but cannot Normal body cells replicate without killing effect, theoretically have higher anti-tumor effect and lower side effects. Oncolytic viruses have the ability to target and infect and kill tumor cells. Based on the differences in the susceptibility of different viruses to tumor cells, different virus pa...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/62C12N15/66C07K19/00A61K48/00A61K38/17A61K35/76A61P35/00
CPCA61K38/1758A61K48/005A61K35/768C12N15/66C07K14/4746C07K2319/60
Inventor 郝文波罗树红陈达香陈瑜
Owner SOUTHERN MEDICAL UNIVERSITY
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