Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof

An oncolytic adenovirus and virus proliferation technology, applied in the fields of genetic engineering and virology, can solve the problems of short time, strong immunogenicity and poor clinical efficacy of foreign genes

Inactive Publication Date: 2010-07-21
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the proliferation-defective adenovirus has shown certain safety in vivo and in vitro, most of the adenoviral vectors used in clinical trials so far still have a sh

Method used

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  • Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof
  • Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof
  • Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1: Construction of the adenoviral vector pCLON9-F11 containing the coding sequence of the ciliary protein gene of type 11 adenovirus

[0050] Step 1: Synthesize the coding sequence of the ciliary protein gene containing adenovirus type 11 as shown in SEQ ID NO: 3, named Ad5F11, wherein: (1) bases 236-369: the coding sequence of the ciliary tail protein of adenovirus type 5; (2) Bases 367-1216: coding sequence of adenovirus type 11 cilium shaft and knob protein; (3) Other bases: genome sequence of type 5 adenovirus, see pBHGloxdeltaE1, 3Cre sequence of Microbix Biosystems.

[0051] Step 2: Synthesize the VT176 sequence (SEQ ID NO: 4) and the VT177 sequence (SEQ ID NO: 5) to construct the pCLON9 vector. The Ad5F11 sequence was digested with PacI+HindIII, and a 2656bp fragment was recovered, which was ligated with the pCLON9 / PacI+HindIII digested vector to construct pCLON9-F11, which was confirmed by enzyme digestion and sequencing. Build steps like figure 1 show...

Embodiment 2

[0052] Example 2: Homologous recombination in BJ5183 cells

[0053] Step 1: pCLON9-F11 was digested with HindIII+AatII, and a 5913bp fragment was recovered.

[0054] Step 2: Synthesize ADP sequence (SEQ ID NO: 6), such as figure 2 As shown, the adenovirus packaging plasmid pPE3 was constructed.

[0055] Step 3: The pPE3 vector is digested with PacI to recover the linearized vector. 4 microliters of pCLON9-F11 / HindIII+AatII and 2 microliters of pPE3 / PacI were used to co-transform competent Escherichia coli BJ5183 (Qbiogene). Plate culture, pick clones for enzyme digestion and identification, the correct one is named pPE3-F11, the large sample plasmid is extracted and purified, and stored in a deep low temperature refrigerator. The construction process of pPE3-F11 is as follows: image 3 shown.

Embodiment 3

[0056] Embodiment 3: Infectivity experiment of adenovirus containing Ad11 adenovirus ciliary protein gene to cultured cells in vitro

[0057] The first step: use human telomerase reverse transcriptase (hTERT) promoter to control type 5 adenovirus E1a, insert site NotI+SwaI, and construct adenovirus vector pQW-hTERT-Ep-HRE (see: patent ZL028138198, a Viruses specifically proliferating in tumor cells highly expressing anti-cancer genes and uses thereof; now renamed as pSG502). The hTERT promoter regulates the E1a gene, and the human hypoxia response element (HRE) regulates the E1b region gene (E1b-55kDa and E1b-19kDa genes). The core sequence of the hTERT promoter is shown in SEQ ID NO: 7, and the HRE sequence of the hypoxia response element is shown in SEQ ID NO: 8.

[0058] The second step: the EGFP gene (SEQ ID NO: 9) was inserted into pSG502 to construct pSG502-EGFP, and the insertion site of EGFP was EcoRI+SalI.

[0059] The second step: the adenovirus plasmid pSG502-EGFP...

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Abstract

The invention relates to gene engineering and virology, in particular to a proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene of a human B subset, a construction method and application thereof. The oncolytic adenovirus can be 11 type adenovirus or heterozygous adenovirus constructed by utilizing the coding sequence of 11-type adenovirus cilia protein to replace the coding sequence of non-11 type adenovirus cilia protein. The adenovirus has selective multiplication capacity by carrying out effective control or mutation on genes essential to proliferation. As a conditional proliferous type combination virus with wider spectrum of infection and improved infectious ability, the recombination oncolytic adenovirus of the invention can be used for gene treatment, in particular to tumor gene treatment.

Description

technical field [0001] The present invention relates to genetic engineering and virology, in particular to a proliferative recombinant oncolytic adenovirus containing human B subgroup 11 adenovirus ciliary protein gene, its construction method and its application. The adenovirus may be type 11 adenovirus, or may be a hybrid adenovirus formed by substituting the amino acid sequence of the ciliary head of the non-type 11 adenovirus with the amino acid sequence of the ciliary head of the type 11 adenovirus. The adenovirus has selective proliferation ability through effective control or mutation of genes necessary for its proliferation. As a conditionally proliferative recombinant virus with a wider infection spectrum and improved infection ability, the recombinant oncolytic adenovirus of the present invention can be used for gene therapy, especially gene therapy for tumors. Background technique [0002] Malignant tumors are a large class of diseases that seriously threaten hum...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/861A61K48/00A61P35/00C12R1/93
Inventor 钱其军苏长青李琳芳吴红平吴孟超
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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