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Antineoplastic ad virus preparation

An adenovirus, tumor technology, applied in the fields of biotechnology and medicine, can solve the problems of safety, low transduction efficiency, and limited in vivo application

Inactive Publication Date: 2009-01-21
ZHENGZHOU VIRIGE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the intracellular release of chemically synthesized siRNA or plasmids mostly adopts the method of cationic liposome transfection, and the transfection with cationic liposome is successful in vitro, but in vivo, due to its low transduction efficiency and dose-related Toxic side effects limit its in vivo application
Viruses are considered to be more efficient transgenic carriers than non-viral vectors in vivo and in vitro, however, most viruses have safety issues when used for in vivo transfection

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0091] The present invention has no particular limitation on the preparation method of siRNA, including but not limited to: chemical synthesis method, in vitro transcription method, RNase III (such as Silencer siRNA Construction Kit, Ambion Company) or Dicer enzyme digestion dsRNA method, etc. It should be understood that those skilled in the art can conveniently prepare or express the siRNA in various ways after knowing the sequence of the siRNA provided by the present invention. For example, in a preferred embodiment of the present invention, the siRNA is chemically synthesized.

[0092] siRNA can be prepared in the form of double-stranded nucleic acid, which contains a sense strand and an antisense strand, and these two strands form a double strand only under hybridization conditions, and are not linked under other conditions. A double-stranded RNA complex can be prepared from separate sense and antisense strands. Thus, for example, complementary sense and antisense strand...

Embodiment 1

[0126] Example 1 Design and synthesis of human effective PDX-1 siRNA fragments

[0127] 1. Determination of target sequence

[0128] The human PDX-1 siRNA target was designed by the Dharmacon siDesign tool (Dharmacon, USA), based on the human PDX-1 mRNA sequence (GenBank accession number NM_000209). Select several candidate human PDX-1 siRNA targets with 30-50% GC content, and use NCBI BLAST to search for any PDX-1 siRNA that is complementary to other gene sequences other than human PDX-1, excluding those that are complementary to human PDX- PDX-1 siRNA targets complementary to gene sequences other than 1.

[0129] After repeated tests and screening, four siRNA target sequences targeting human PDX-1 were determined, which are as follows:

[0130] 5'AGTTCCTATTCAACAAGTA 3' (SEQ ID No: 3), corresponding to the 590th-608th position of the nucleotide sequence SEQ ID No: 1. See SEQ ID No: 1 for the nucleotide sequence of human PDX-1, and see SEQ ID No: 2 for the encoded human PDX...

Embodiment 2W

[0143] Example 2 Western blot

[0144]Cells in logarithmic growth phase were lysed with RIPA lysis buffer (consisting of 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA and 1% NP-40 with a mixture of protein inhibitors). Protein concentrations in the lysates were determined using the BSA protein assay kit (purchased from Pierce, Rockford, IL). 50 mg of protein lysates from each sample were mixed with 5× loading buffer (composed of 0.25M Tris-HCl, pH 6.8, 50% glycerol, 5% SDS, %5 2-β-mercaptoethanol, and 2.5% bromophenol blue) . The samples were incubated at 98°C for 5 minutes, on ice for 5 minutes, and centrifuged at maximum speed in a small centrifuge at room temperature. Proteins were resolved in 10% SDS / polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in TBST (consisting of 10 mM Tris-HCL, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk for 1 hour. Blocked membranes were probed for two hours with primary antibodies dilut...

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Abstract

The invention discloses a siRNA which inhibits tumor through inhibiting the expression of PDX-1, and a recombinant adenovirus designed on the basis of the sequence of the siRNA. The invention also provides a drug composition containing the adenovirus. The adenovirus provided by the invention has the advantages of high titration, absence of integration into the genome of the host, and high safety.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine; more particularly, the invention relates to a new preparation for tumor treatment. Background technique [0002] Malignant tumors are a serious disease that seriously endangers human health. The main obstacle to current drug treatment is the lack of specific targets for tumor treatment and effective drug carriers in vivo. That is, many anti-tumor drugs kill normal cells while killing tumor cells. ; Some specific biomacromolecule anti-tumor preparations, such as antisense nucleic acid and siRNA, lack effective release carriers in vivo. Therefore, the key to developing biomacromolecular antitumor drugs is to discover tumor-specific targets and develop effective drug carriers. [0003] Pancreas duodenum homeobox-1 (Pancreas duodenum homeobox-1, PDX-1) is a transcription factor that plays a crucial role in the development of pancreas in embryonic stage and the maintenance of normal β-cell ...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N15/861A61K48/00A61P35/00
Inventor 韩志强
Owner ZHENGZHOU VIRIGE BIOTECH
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