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ING4 and OSM double-gene co-expression vector and application thereof

A technology of genes and vectors, applied in the field of adenovirus vectors, can solve the problem of co-expression plasmid vectors and adenovirus vectors of ING4 and OSM double genes, and achieve the effect of down-regulating expression

Active Publication Date: 2012-02-15
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, there are no reports on the construction and co-expression of ING4 and OSM double-gene co-expression plasmid vectors and adenoviral vectors in the prior art. Whether the treatment can exert a synergistic or superimposed tumor suppressor effect by inhibiting the growth of tumor cells and their transplanted tumors and inducing tumor cell apoptosis
In particular, whether the combination of the two can play a radiosensitizing effect and enhance the effect of radiotherapy on tumor radiotherapy has not been reported at home and abroad so far. For this reason, it has become a research hotspot in today's tumor treatment, and it is also an important way to improve tumor treatment in tumor treatment. Efficacy of treatment is an urgent problem to be solved

Method used

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  • ING4 and OSM double-gene co-expression vector and application thereof
  • ING4 and OSM double-gene co-expression vector and application thereof
  • ING4 and OSM double-gene co-expression vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1: Construction and detection of gene recombinant adenovirus vector

[0066] Materials: Bgl II, Sal I, Not I, Hind III restriction enzymes, T4 DNA ligase, Taq DNA polymerase, dNTPmix Oligo d(T) 18 , DL2000marker, Ribonuclease Inhibitor were purchased from TaKaRa Company; Reverse Transcriptase MMLV, Lambda DNA / HindIII marker were purchased from MBI Company; PmeI, PacI were purchased from New England Biolads Company; daily miniature plasmid extraction kit, PCR product cleaning kit, DNA gel recovery kit was purchased from Hangzhou Weitejie Biochemical Technology Co., Ltd.; pAdTrack-CMV-ING4-IRES-hOSM plasmid, pAdTrack-CMV-poly-A-promoter plasmid, QBI-293A packaging cells, pAdTrack-CMV-polyA +promoter -OSM plasmid, pAdEasy-1 backbone plasmid, Escherichia coli DH5α, BJ5183 bacteria, empty vector adenovirus Ad-GFP, etc. are all preserved in our laboratory; primers: β-actin, ING4, OSM gene primers (as shown in Table 1) Synthesized by Shanghai Sangon; RPMI-1640 was pur...

Embodiment 2

[0102] Example 2: Experimental research on the growth inhibitory effect of Ad-ING4-OSM on human laryngeal cancer Hep-2 cells;

[0103] Reagents: constructed Ad-ING4, Ad-OSM, Ad-ING4-OSM and empty viral vector Ad-GFP, RPMI-1640 medium was purchased from GIBCO; 6-well, 24-well and 96-well cell culture plates were purchased from CORNING Company; MTT was purchased from Sigma Company of the United States; newborn calf serum was purchased from Hangzhou Saile Biotechnology Company; Trizol kit was purchased from Invitrogen Company; MMLV reverse transcriptase, TaqDNA polymerase, dNTPmix, Oligo(dT) 18 , DNA marker were purchased from TaKaRa; β-actin antibody was purchased from Beyontien Institute of Biotechnology; mouse anti-human ING4 and mouse anti-human OSM antibodies were purchased from abcam; nitrocellulose membrane (NC membrane), Western blot chemiluminescence Kits and cassettes were purchased from Shanghai Pulilai Gene Technology Co., Ltd.; HRP-labeled rabbit anti-mouse IgG secon...

Embodiment 3

[0151] Example 3: Experimental research on the growth inhibitory effect of Ad-ING4-OSM on human laryngeal carcinoma xenografts in nude mice

[0152] Reagents and experimental animals: Ad-GFP, Ad-ING4, Ad-OSM and Ad-ING4-OSM adenovirus vectors; RPMI-1640 medium was purchased from GIBCO; calf serum was purchased from Hangzhou Sijiqing Company; adenovirus amplification Cell QBI-293A was donated by Professor Zhong Jiang from the Department of Microbiology, School of Life Sciences, Fudan University; human laryngeal carcinoma Hep-2 cells were preserved in this laboratory; 96-well cell culture plates were purchased from CORNING Company; BALB / c nude mice, female, 4- 5 weeks old, 18-22 g were purchased from Shanghai Slack Experimental Animal Co., Ltd.; ING4, OSM, P21, P27, Cox-2, Bcl-2, Survivin, Bax, Caspase-3, CD34 antibodies were purchased from Fuzhou Maixin Biotechnology Company; ready-to-use immunohistochemical ultrasensitive UltraSensitiveTM SP kit (mouse / rabbit) was purchased ...

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Abstract

The invention belongs to the field of adenovirus vectors and in particular discloses a recombinant adenovirus vector Ad-ING4-OSM and a preparation method thereof. The preparation method comprises the following steps: on the basis of a transfer plasmid pAdTrack-CWV-polyA-promoter, inserting an ING4 segment between BglII and SalI polyclonal sites of the transfer plasmid and inserting an OSM segment between NOtI and HindIII restriction sites of the transfer plasmid so as to prepare a recombinant transfer plasmid pAdTrack-CWV-ING4-poly-A-promoter-OSM; carrying out co-transformation on the recombinant transfer plasmid and a pAsEasy-1 adenovirus skeleton plasmid to BJ5183 competence bacteria with a calcium chloride method; screening, carrying out PacI restriction, and transfecting QBI-293A cells with a liposome; and carrying out multi-time infection and amplification so as to obtain the recombinant adenovirus vector Ad-ING4-OSM. Compared with the Ad-ING4 and Ad-OSM single genomes, the recombinant adenovirus vector Ad-ING4-OSM shows obvious tumor-inhibiting synergy and radiotherapy sensitizing synergistic effect on human laryngocarcinoma Hep-2 cells and nude mouse transplanted tumor thereof and shows superimposed anticancer synergy for inhibiting growth of SPC-A1 pulmonary adenocarcinoma cells and inducing apoptosis.

Description

technical field [0001] The invention belongs to the field of adenovirus vectors, and specifically relates to an adenovirus vector prepared by inserting ING4 and OSM genes into a vector to form a recombinant vector and the application of the obtained adenovirus vector. Background technique [0002] In the prior art, exploring the occurrence, development mechanism and treatment methods of laryngeal cancer from the aspect of genes has become one of the key directions of cancer research in recent years. Tumor gene therapy is to introduce the target gene into target cells by gene transfer technology, so that it can perform specific functions, and then execute the killing and inhibiting effect on tumors, or protect normal cells from the damage of chemotherapy and radiation therapy. Since Rosenberg et al first used retroviral vector gene transfer technology to treat advanced cancer in 1990, tumor gene therapy has developed by leaps and bounds, and more and more tumor gene therapy d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/861A61K48/00A61P35/00
Inventor 杨吉成马士崟
Owner SUZHOU UNIV
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