Humanization modified rat ING4 gene and adenovirus expression vectors thereof

A humanized, adenovirus technology, applied in the fields of biotechnology and medicine, can solve problems such as reports about the application of adenovirus vectors in anti-tumor gene therapy, etc.

Active Publication Date: 2007-10-24
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, no humanized mouse ING4 gene has been seen at home and abroad, and there are no reports on the construction of an adenoviral vector of the ING4 gene and its application in anti-tumor gene therapy

Method used

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  • Humanization modified rat ING4 gene and adenovirus expression vectors thereof
  • Humanization modified rat ING4 gene and adenovirus expression vectors thereof
  • Humanization modified rat ING4 gene and adenovirus expression vectors thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Point mutation method of humanized transformation mouse ING4 gene

[0122] According to the amino acid sequence of human ING4, the mouse ING4 gene was humanized by point mutation technology (Fig. 1A). Design two pairs of mutant primers P1, P2, P3, P4 and full-length ING4 upstream and downstream primers P5, P6, which change the 66th amino acid coding sequence aga into aaa, and the 156th amino acid coding sequence gct into act in mouse ING4; use pcDNA3.0 -mING-4 plasmid as a template, the first round of PCR was performed with P1 and P6, P2 and P5 respectively, and mixed after gel recovery, then the mixed PCR product was used as a template, and the second PCR was performed with primers P5 and P6, after gel recovery Then use this PCR product as a template, carry out the third round of PCR with primers P3 and P6, P4 and P5 respectively, mix after gel recovery, and then use this mixed PCR product as a template to carry out the fourth round of PCR with primers P5 and...

Embodiment 2

[0124] Example 2 Construction of recombinant adenovirus vector

[0125] (1) Construction of pAdTrack-CMV-ING4 (with GFP marker)

[0126] After the purified fourth-round PCR product and the pAdTrack-CMV plasmid with the GFP marker gene were digested by SalI and HindIII, the target fragment was recovered by the gel extraction kit, and then ligated by T4 DNA ligase overnight, and the calcium chloride method was used. The DH5α competent cells were transformed, and the positive clones of the pAdTrack-CMV-ING4 transfer plasmid were screened and identified by sequencing. The correctly sequenced pAdTrack-CMV-ING4 plasmid and the pAdTrack-CMV empty plasmid were linearized with PmeI single enzyme digestion, gel recovery and pAdEasy-1 adenovirus vector calcium chloride method were used to co-transform BJ5183 competent, and positive clones were selected Plasmids were extracted, and after identification by enzyme digestion, a large number of transformants were amplified.

[0127] Plasmid...

Embodiment 3

[0131] Example 3 Obtaining and Identification of Recombinant Viruses Ad-ING4-GFP and Ad-ING4-ΔGFP

[0132] The pAd-ING4, pAd-ING4-ΔGFP and pAd homologous recombinant adenoviral plasmids constructed above were linearized with Pac I, and then 70% of adherent QBI-293A cells were transfected according to the operation instructions of Lipofectamine Reagent. Observe the fluorescence under a fluorescent microscope for 3-5 days, collect the cell suspension on 7-10 days, centrifuge at 2000r / min for 5min, suspend the cell pellet with sterile PBS, freeze and thaw the cell suspension three times, centrifuge at 2000r / min for 5min, and take In the supernatant, high-titer Ad-ING4-GFP, Ad-ING4-ΔGFP and Ad-GFP adenoviruses obtained after multiple rounds of infection and amplification were stored at -80°C. The light microscope morphology and fluorescence observation results of QBI-293A cells infected with ING4 recombinant adenovirus are shown in Figures 5A-5D.

[0133] Trypsinize the QBI-293A ...

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Abstract

The invention relates to a human reforming mouse ING4 (tumor growth inhibition factor) gene and recombinant adenovirus (adenovirus hominis 5 shape Ad-ING4-DeltaGFP), wherein preservation cooperation is Chinese typical culture preservation centre, preservation number is CCTCC-V200701. The invention also relates to a human reforming gene mutation technique, adenovirus transition carrier removing GFP and homologous recombinant adenovirus carrier. The ING4 gene and recombinant adenovirus carrier provide new immunodiagnosis and target therapeutic approach for treating neoplastic disease, development disease, immune disease, the other disease owing to abnormal expression of tumour growth inhibition factor ING4, special cancer of the lungs and leukocythemia, which is provided with a giant application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to the humanized modified mouse ING4 (tumor growth inhibitory factor 4) gene and the ING4 recombinant adenoviral vector transformed by removing the GFP vector, and the recombinant adenovirus vector containing the gene Vectors and host cells. The present invention also relates to the preparation method and application of the gene, recombinant vector and host cell of the present invention. Background technique [0002] Tumor is one of the major diseases affecting human health in today's society. Modern cell biology studies have shown that in vivo cells are regulated by two types of positive and negative regulatory signals in the process of proliferation, differentiation and apoptosis. Disruption of the balance between positive signals (oncogenes, growth factors) and negative signals (tumor suppressor genes, growth suppressors) leads to tumorigenesis. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/47C12N15/861C12N1/21A61K48/00A61K35/74A61P35/00A61P35/02
Inventor 杨吉成张海峰
Owner SUZHOU UNIV
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