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Construction method and application of targeting hepatoma oncolytic adenovirus

An oncolytic adenovirus and a construction method are applied in the field of construction and application of an adenovirus vector GD55 carrying a GP73 promoter targeting liver cancer, which can solve the problems of low cure rate, high recurrence rate and mortality rate, poor prognosis, etc. Ease of grasp, tumor growth inhibition, and safety-enhancing effects

Active Publication Date: 2014-08-13
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] For most types of cancer, traditional therapies such as surgery, radiotherapy, chemotherapy, etc. Helpless, mainly manifested in low cure rate, high recurrence rate and mortality rate, and poor prognosis

Method used

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  • Construction method and application of targeting hepatoma oncolytic adenovirus
  • Construction method and application of targeting hepatoma oncolytic adenovirus
  • Construction method and application of targeting hepatoma oncolytic adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Background transcription of embodiment 1, GP73 and AFP gene in different cell lines

[0061] Related primers:

[0062] GP73 gene primers:

[0063] Sense: GGATGTCCTCCAGTTTCAGA;

[0064] Anti-sense: TTCTCCCACTTCCTTGTCAC

[0065] AFP gene primers:

[0066] Sense: CTTACACAAAGAAAAGCCCC

[0067] Anti-sense: GTCCGATAATAATGTCAGCC

[0068] GAPDH gene primers:

[0069] Sense: ATTCCACGGCACAGTCAAGG

[0070] Anti-sense: ACATACTCAGCACCAGCATCG

[0071] The total RNA of normal liver cells QSG-7701 and liver cancer cells SMMC-7721, Huh7, and Hep3B in the logarithmic growth phase was extracted by TRIZOL method, and reverse-transcribed into cDNA. - The relative expression of AFP and GP73 was obtained by PCR.

[0072] details as follows:

[0073] Note: The Q-PCR reaction system and reaction conditions are:

[0074]

[0075] Put the loaded eight-tube tube into the ABI7300 fluorescent quantitative PCR instrument, set the Q-PCR program, and the cycle conditions are:

[0076] ...

Embodiment 2

[0080] Example 2, Evaluation of the activity of the novel GP73 promoter in cells

[0081] The reporter gene plasmid phRL-TK was purchased from Promega Company, expressing Renilla luciferase under the control of the TK promoter; the plasmids pGL3-Basic (without promoter and enhancer) and pGL3-Control (with SV40 promoter) was purchased from Promega Company.

[0082] 1) Related plasmid construction

[0083] First design the following primers:

[0084] p-19: (Hind III) ACT AAGCTT CCGGCCTCCGCAGCGGCAAG

[0085] p-2616:(MluI)CG ACGCGT TAGTCCCACCACTCTCGA

[0086] p-413:(MluI)CG ACGCGT ATAAAGAAGGTAACGTGA

[0087] p-613:(MluI)CG ACGCGT GAATCTTCACTTTTTCCGTTG

[0088] p-677:(MluI)CG ACGCGT TCATCTGAGGCGCTGTTTCA

[0089] p-729:(MluI)TG ACGCGT CTCCGGGAGGTACGGCCTCA

[0090] Remarks: The underline represents the enzyme cleavage site.

[0091] Using the specific primers p-19:(Hind III) and p-2616:(MluI) at both ends of the above-mentioned GP73 promoter, using the total ...

Embodiment 3

[0104] Example 3. Construction of dual-targeted liver cancer oncolytic adenovirus

[0105] 1) Construction of related plasmids (underlined are the sequences of restriction sites listed)

[0106] p-19:(SnaB I)TAG TACGTA CCGGCCTCCGCAGCGGCAAG

[0107] p-677:(Xho I)CG CTCGAG TCATCTGAGGCGCTGTTTC

[0108] GM-CSF (sense): (Hind III) CC AAGCTT ATGTGGCTGCAGAGCCTGCT

[0109] GM-CSF (Anti-sense): (BamH I) AT GGATCC TCACTCCTGGACTGGCTC

[0110] mGM-CSF (sense): (Hind III) CCC AAGCTT ATGTGGCTGCAGAATTTAC

[0111] mGM-CSF (Anti-sense): (BamH I) CG GGATCC TCATTTTTGGCCTGGTT

[0112] EGFP (sense): (BamHI) AT GGATCC ATGGTGAGCAAGGGCGAGGAG

[0113] EGFP (antisense): (HindIII) CCT AAGCTT TTATCTAGATCCGGTGGATC

[0114] Using the total genomic RNA extracted from the peripheral blood of healthy people as a template, cDNA was amplified by RT-PCR, and the GM-CSF gene was obtained by PCR with GM-CSF upstream and downstream primers. The PCR reaction system was:

[0115]

[0116] The PC...

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Abstract

The invention discloses a construction method of targeting hepatoma oncolytic adenovirus, which comprises the following steps: 1), pXC2-GP73 plasmid carrying GP73 core promoter is prepared; 2), pSD55-GP73-gene plasmid is prepared; 3), the pSD55-GP73-gene plasmid is linearized by PmeI and transformed into plasmid Adeasy-1 escherichia coli BJ5183 containing whole sequence adenovirus backbone DNA for recombination to produce an adenovirus backbone DNA plasmid vector, wherein E1A region of the adenovirus backbone DNA plasmid vector is controlled by the GP73 core promoter, and target genes carried by E1B55 and E3 regions of the adenovirus backbone DNA plasmid vector are deleted; 4), the recombinant plasmid, which is identified to be correct, is linearized by enzyme digestion with Pac I, and then transfected into 293A cells for virus packaging to obtain the targeting oncolytic adenovirus GD55-gene when the cell lesion is appeared. The invention also provides application of the oncolytic adenovirus GD55-gene in preparation drugs for curing liver cancer.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and gene therapy, and specifically relates to the screening and cloning of the core sequence of a liver cancer-specific promoter GP73 promoter, and the construction and construction of an adenovirus vector GD55 carrying a GP73 promoter targeting liver cancer. application. Background technique [0002] For most types of cancer, traditional therapies such as surgery, radiotherapy, chemotherapy, etc. are still used at present, but in the face of many advanced malignant tumors, traditional therapies are helpless, mainly manifested in low cure rate, high recurrence rate and high mortality , The prognosis is poor. Gene therapy is to correct or compensate diseases caused by gene defects and abnormalities by introducing exogenous normal genes into target cells. People are very enthusiastic about gene therapy for tumors, and 64.2% of gene therapy clinical trials are used for tumor treatment. So far,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861C12N15/66A61K48/00A61P35/00
Inventor 王毅刚刘涛黄芳马步云黄盼盼周秀梅刘新垣
Owner ZHEJIANG SCI-TECH UNIV
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