IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application

A technology of recombinant adenovirus and recombinant plasmid, which is applied in application, gene therapy, genetic engineering, etc., can solve the problems of low inhibitory effect and limit the application of gene therapy technology, and achieve the effect of improving the effect of radiotherapy

Inactive Publication Date: 2012-07-11
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]Although there are many advantages in gene therapy for skin malignant melanoma and nasopharyngeal carcinoma, at present, the homologous recombination of IL-24 gene or OSM gene with adenovirus is The inhibitory effect of tumor cells is not high, which limits the application of gene therapy technology in skin malignant melanoma and nasopharyngeal carcinoma

Method used

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  • IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application
  • IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application
  • IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Map of pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene co-expression recombinant plasmid

[0077] In the application number 200810244301.3 Chinese Invention Patent Application specification discloses polyA Δ296～298 -promoter (polyA-promoter for short) sequence. The double-gene co-expression recombinant plasmid of the present invention is based on the pAdTrack-CMV-polyA-promoter transfer plasmid, inserts the IL-24 fragment between the Bgl II and Sal I restriction sites, and inserts an IL-24 fragment between the Not I and Xho I restriction sites. The OSM fragment is inserted between the sites, and the pAdTrack-CMV-polyA-promoter plasmid map is shown in figure 1 .

Embodiment 2

[0078] Embodiment 2: Cloning of IL-24 and OSM target gene

[0079] Compare the conserved sequence of IL-24, design upstream primer IL-24-F, downstream primer IL-24-R (see Table 1), and introduce Bgl II and Sal I restriction sites at both ends; The pAdTrack-CMV-IL-24-IRES plasmid containing the IL-24 fragment was used as a template, and the target fragment of IL-24 obtained by PCR amplification was identified by agarose electrophoresis to be consistent with the expected size.

[0080] Compare the conserved sequence of OSM, design upstream primer OSM-F, downstream primer OSM-R (see Table 1), and introduce Not I and Xho I restriction sites at both ends, and use the pAdTrack that the applicant has constructed containing the OSM fragment - The CMV-polyA-promoter-OSM plasmid was used as a template, and the target fragment of OSM obtained by PCR amplification was identified by agarose electrophoresis to be consistent with the expected size.

[0081] Table 1 PCR amplification primers...

Embodiment 3

[0083] Example 3: Construction of pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene co-expression recombinant plasmid

[0084] 1. Construction of pAdTrack-CMV-IL-24-polyA-promoter single gene recombinant plasmid

[0085] The PCR product of the IL-24 gene purified by the DNA cleaning kit and the transfer plasmid pAdTrack-CMV-polyA-promoter extracted by the mini-plasmid extraction kit were digested with Bgl II and Sal I at 37°C for 5 hours, and recovered by tapping rubber For the target fragment, follow the gel recovery method and steps of the kit, and then use T4 DNA ligase to ligate it overnight at 4°C, and then transform the ligated product into Escherichia coli DH5α, and select it on a Kana (50 μg / ml) resistant plate Positive single clones were identified by PCR, double enzyme digestion and DNA sequence determination, and the positive clones were stored in a -20°C refrigerator for later use.

[0086] CaCl 2 Method for preparing DH5α E. coli competent cells and the transfor...

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Abstract

The invention relates to the field of biology, and discloses a double-gene co-expression recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM with the preservation number as China center for type culture connection number (CCTTCC NO.): M2011378, and a recombinant adenovirus Ad-IL-24-polyA-promoter-OSM prepared by the plasmid. An IL-24 gene and an OSM gene are subcloned between Ad-polyA-promoter empty carriers to construct the IL-24 and OSM double-gene co-expression recombinant plasmid, homologous recombination is performed between the recombinant plasmid and an adenovirus pAdEasy-1 to generate the recombinant adenovirus in packaging mode in a QBI-293A cell. The recombinant adenovirus has synergistic effect on inhibition of melanoma and cancer cells of nasopharyngeal darcinoma, and simultaneously has synergistic effect on radiosensitivity of the cancer cells of the nasopharyngeal darcinoma.

Description

technical field [0001] The invention relates to the field of biology, in particular to a recombinant plasmid for co-expression of IL-24 and OSM double genes, a recombinant adenovirus and its application. Background technique [0002] In recent years, the incidence of skin malignant melanoma and nasopharyngeal carcinoma among malignant tumors has been increasing year by year. Among them, cutaneous malignant melanoma (cutaneous malignant melanoma, CMM) is a highly malignant skin tumor, and its occurrence often begins in those parts of pigmentation, which is usually caused by the formation of melanin inlaid in the basal cells of the epidermis. Cells derived from cells that are not controlled by the cell proliferation machinery. Some recent reports have shown that the incidence rate in our country has a tendency to increase gradually. Due to insufficient understanding of its severity, it is often late when the doctor seeks treatment. Nasopharyngeal carcinoma (NPC) is one of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/861A61K48/00A61P35/00
Inventor 杨吉成缪竞诚刘济生盛伟华
Owner SUZHOU UNIV
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