ING4 and IL-24 dual gene co-expression vector and application thereof

A CMV-IL-24, recombinant vector technology, applied in gene therapy, genetic engineering, plant genetic improvement and other directions, can solve problems such as inability to use, affecting the normal expression of dual genes, and no related reports.

Active Publication Date: 2009-12-02
SUZHOU UNIV
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Problems solved by technology

[0012] Constructing a double-gene co-expression recombinant transfer vector is one of the effective ways as a gene therapy drug for tumors, but so far, there is no research on the construction of ING4 using the IRES strategy at home and abroad and IL-24 double-gene vectors; in addition, using the strategy of constructing polyA+promoter double-promoters to construct double-gene recombination transfer vectors, although there are related studies in theory, there are no related reports in practice, and this method has the following difficulties: In the process of constructing a double-gene recombination transfer vector using the polyA+promoter double-promoter strategy, it was found that there was a pacI restriction site in polyA, which could not be linearized by pacI restriction and then packaged in 293A cells for adenovirus packaging, which affected the double gene normal expression of

Method used

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  • ING4 and IL-24 dual gene co-expression vector and application thereof
  • ING4 and IL-24 dual gene co-expression vector and application thereof
  • ING4 and IL-24 dual gene co-expression vector and application thereof

Examples

Experimental program
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Embodiment 1

[0109] Embodiment one, the construction of each group of recombinant transfer vectors mediated by IRES

[0110] 1.1 Construction of pAdTrack-CMV-IRES Transformation Transfer Empty Vector

[0111] (1) Obtain the IRES target fragment by PCR: compare the conserved sequences of IRES, design primers P1 and P2 (as shown in Table 1), use the pGEZ-Term plasmid containing the IRES fragment as a template to amplify the IRES target fragment by PCR, and introduce SalI at both ends , NotI restriction site, the obtained IRES PCR product;

[0112] (2) Subcloning the IRES fragment into the pAdTrack-CMV transfer plasmid: use the IRES PCR product purified by the DNA cleaning kit and the transfer plasmid pAdTrack-CMV extracted by the daily mini-plasmid extraction kit, respectively, with SalI and NotI at 37°C with double enzymes After cutting for 5 hours, tap the gel to recover the target fragment, and use T4 DNA ligase to connect it overnight at 4°C, and then transform the ligation product into...

Embodiment 2

[0121] Example 2, polyA Δ296～298 +Construction of each group of recombinant transfer vectors mediated by CMV

[0122] 2.1pAdTrack-CMV-polyA Δ296～298 + CMV transformation transfer empty vector construction

[0123] (1) According to the reporter sequence of pORF-mbcl-2α plasmid, design primers P3, P4, P5, P6 (as shown in Table 1). Using the pORF-mbcl-2α plasmid as a template, P3, P4; P5, P6 as primers, PCR amplifies polyA and CMV target gene fragments (the size is consistent with the expected polyA and CMV theoretical value (333bp; 555bp) ); and with polyA and CMV mixed PCR product as template, P3, P6 as primer SOE-PCR amplification polyA+CMV fusion fragment (polyA+CMV fusion fragment of about 888bp size;

[0124] (2) Assembly of pUcm-T-polyA-CMV plasmid: Since the pAdTrack-CMV plasmid itself contains multiple PacI restriction sites, it is necessary to use PacI enzyme to cut the PacI restriction site in polyA-CMV, and then polymerize with T4DNA The base deletion method of en...

Embodiment 3

[0135] Example 3, construction and identification of homologous recombination adenoviral vector

[0136] The above pAdTrack-CMV-IRES, pAdTrack-CMV-IRES-IL-24, pAdTrack-CMV-ING4-IRES and pAdTrack-CMV-ING4-IRES-IL-24; pAdTrack-CMV-polyA Δ296～298 +CMV, pAdTrack-CMV-polyA Δ296～298 +CMV-IL-24, pAdTrack-CMV-ING4-polyA Δ296～298 +CMV and pAdTrack-CMV-ING4-polyA Δ296～298 +CMV-IL-24 recombinant transfer plasmid was linearized with PmeI at 37°C for 2 hours, then co-transformed BJ5183 competent bacteria with pAdEasy-1 adenovirus backbone plasmid by calcium chloride method, and LB containing Kana (50 μg / ml) Agar plate screening picks single clones to extract plasmids, and preliminarily screens pAdEasy-1-pAdTrack-CMV-IRES (abbreviated as pAd-IRES), pAdEasy-1-pAdTrack-CMV-IRES-IL-24 (abbreviated as pAdEasy-1-pAdTrack-CMV-IRES-IL-24) pAd-IRES-IL-24), pAdEasy-1-pAdTrack-CMV-ING4-IRES (referred to as pAd-ING4-IRES) and pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24 (abbreviated as pAd -ING4-IRES-IL...

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Abstract

The invention relates to a dual gene recombinant transfer vector, namely pAdTrack-CMV-ING4-IRES-IL-24 and pAdTrack-CMV-ING4-polyA+CMV-IL-24. The dual gene vector perfectly and successfully combines ING4 gene and IL-24 gene to ensure that the two genes can acquire successful expression on the same vector; and through the combination action of a dual gene expressed product, the ING4 acting in a tumor cell associatively acts with IL-24 secreted out of the cell so as to play acts of tumor suppression synergy and radiotherapy synergy. The dual gene recombinant transfer vector has more obvious effect compared with the action of single ING4 or IL-24.

Description

technical field [0001] The invention belongs to the field of recombinant vectors, and in particular relates to the preparation and application of co-expression vectors of human ING4 gene and human IL-24 gene. Background technique [0002] Tumor is one of the main diseases that affect human health in today's society. The occurrence and development of tumors are regulated by various genes in the body. Tumor suppressor genes are important molecules involved in this regulation. Many tumors have certain tumor suppressor gene failures. Introducing a normal tumor suppressor gene into cells lacking a certain tumor suppressor gene can reverse the phenotype of tumor cells, inhibit cell proliferation, and induce cell apoptosis to achieve therapeutic purposes. For example, the human ING4 gene ( See: Chinese patent application with publication number CN101058809A) and human IL-24 gene. [0003] The human ING4 gene is located at 12p13-31, with a gene span of 13kb, consisting of 8 exons a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/24C12N15/861C12N7/01A61K48/00A61P35/00
Inventor 杨吉成谢宇锋
Owner SUZHOU UNIV
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