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Liver cancer-specific gp73 core promoter and its screening and construction method

A core promoter and construction method technology, applied in the field of construction and application of the adenovirus vector GD55 carrying the GP73 promoter targeting liver cancer, can solve the problems of poor prognosis, low cure rate, high recurrence rate and mortality, and achieve tumor suppression Growth, ease of handling, highly effective liver cancer-specific effects

Active Publication Date: 2016-04-27
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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  • Liver cancer-specific gp73 core promoter and its screening and construction method
  • Liver cancer-specific gp73 core promoter and its screening and construction method
  • Liver cancer-specific gp73 core promoter and its screening and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Background transcription of embodiment 1, GP73 and AFP gene in different cell lines

[0058] Related primers:

[0059] GP73 gene primers:

[0060] Sense: GGATGTCCTCCAGTTTCAGA;

[0061] Anti-sense: TTCTCCCACTTCCTTGTCAC

[0062] AFP gene primers:

[0063] Sense: CTTACACAAAGAAAAGCCCC

[0064] Anti-sense: GTCCGATAATAATGTCAGCC

[0065] GAPDH gene primers:

[0066] Sense: ATTCCACGGCACAGTCAAGG

[0067] Anti-sense: ACATACTCAGCACCAGCATCG

[0068] 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.

[0069] details as follows:

[0070] Note: The Q-PCR reaction system and reaction conditions are:

[0071]

[0072] Put the loaded eight-tube tube into the ABI7300 fluorescent quantitative PCR instrument, set the Q-PCR program, and the cycle conditions are:

[0073] ...

Embodiment 2

[0077] Example 2, Evaluation of the activity of the novel GP73 promoter in cells

[0078] 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.

[0079] 1) Related plasmid construction

[0080] First design the following primers:

[0081] p-19:(HindIII)ACT AAGCTT CCGGCCTCCGCAGCGGCAAG

[0082] p-2616:(MluI)CG ACGCGT TAGTCCCACCACTCTCGA

[0083] p-413:(MluI)CG ACGCGT ATAAAGAAGGTAACGTGA

[0084] p-613:(MluI)CG ACGCGT GAATCTTCACTTTTTCCGTTG

[0085] p-677:(MluI)CG ACGCGT TCATCTGAGGCGCTGTTTCA

[0086] p-729:(MluI)TG ACGCGT CTCCGGGAGGTACGGCCTCA

[0087] Remarks: The underline represents the enzyme cleavage site.

[0088] Using the specific primers p-19:(HindIII) and p-2616:(MluI) at both ends of the above-mentioned GP73 promoter, using the total DNA genome...

Embodiment 3

[0101] Example 3. Construction of dual-targeted liver cancer oncolytic adenovirus

[0102] 1) Construction of related plasmids (underlined are the sequences of restriction sites listed)

[0103] p-19:(SnaBI)TAG TACGTA CCGGCCTCCGCAGCGGCAAG

[0104] p-677:(XhoI)CG CTCGAG TCATCTGAGGCGCTGTTTC

[0105] GM-CSF (sense): (HindIII) CC AAGCTT ATGTGGCTGCAGAGCCTGCT

[0106] GM-CSF (Anti-sense): (BamHI) AT GGATCC TCACTCCTGGACTGGCTC

[0107] mGM-CSF (sense): (HindIII) CCC AAGCTT ATGTGGCTGCAGAATTTAC

[0108] mGM-CSF (Anti-sense): (BamHI) CG GGATCC TCATTTTTGGCCTGGTT

[0109] EGFP (sense): (BamHI) AT GGATCCATGGTGAGCAAGGGCGAGGAG

[0110] EGFP (antisense): (HindIII) CCT AAGCTT TTATCTAGATCCGGTGGATC

[0111] 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:

[0112]

[0113]

[0114] ...

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Abstract

The invention discloses a live cancer specific GP73 core promoter which is any of the following: p-19 / -413, p-19 / -613, p-19 / -677 and p-19 / -729. The invention also discloses a screening building method of the live cancer specific GP73 core promoter, and the method is as follows: using human hepatoma cell Huh7 total DNA genome as a template to obtain a product, namely a GP73 promoter with a total length sequence of p-19 / -2616, using Mlu I and HindIII for double enzyme digestion, connecting with pGL3-Basic plasmid which is also digested with the double enzymes Mlu I and HindIII to obtain pGL3-L2598 plasmid; 2) using the pGL3-L2598 as a template, and using different primers carrying Mlu I and Hind III enzyme digestion sites for PCR (polymerase chain reaction) to obtain plasmids containing different GP73 fragment lengths.

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 Patents(China)
IPC IPC(8): C12N15/113C12N15/10
Inventor 王毅刚刘涛马步云黄盼盼周秀梅刘新垣
Owner ZHEJIANG SCI-TECH UNIV
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