Double-reporter-gene recombinant adenovirus vector, construction method and application thereof

A recombinant adenovirus and vector technology, applied in the field of genetic engineering, can solve the problem of difficult to meet the requirements of in vivo optical and magnetic resonance dual-modal imaging tracking at the same time, so as to increase biological safety, improve adaptability and stability, and achieve transformation. High dyeing efficiency

Inactive Publication Date: 2013-06-19
郜发宝
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, most of the reporter gene imaging using adenovirus as a carrier is a single reporter gene, that is, using adenovirus as a carrier to introduce a single reporter gene into the body for gene expression, so it is difficult to meet the requirements of in vivo optical and magnetic resonance dual-modal imaging at the same time. Require

Method used

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  • Double-reporter-gene recombinant adenovirus vector, construction method and application thereof
  • Double-reporter-gene recombinant adenovirus vector, construction method and application thereof
  • Double-reporter-gene recombinant adenovirus vector, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of recombinant adenoviral vector

[0033] 1. Materials and methods

[0034] 1.1 Target gene, vector plasmid, cell

[0035] The original cDNA plasmid of the human transferrin receptor gene (TFRC): GeneCopoeia, USA; the shuttle vector pShuttle-CMV-Luciferase and the chemically competent cell DH5α strain: Shanghai Hanheng Company; the backbone vector pAdenoviral Vector (pAdeno for short): Qbiogene, USA; human embryonic kidney cell line HEK293 cells: Cell Center, Institute of Basic Medical Sciences, Peking Union Medical College, China.

[0036] 1.2 Reagents

[0037] The restriction endonucleases used were all purchased from New England Biolabs; T4DNA ligase was MBI (Fermentas) product of Jingmei Biological Company; CIP enzyme (Alkaline Phosphatase, Calf Intestinal) was purchased from Promega; pfx DNA polymerase was purchased from Invitrogen; Small / medium-volume extraction and purification kits, DNA purification kits (column centrifugal type), DNA ...

Embodiment 2

[0077] Example 2: Infection and expression experiment of recombinant adenovirus vector Ad-CMV-TFRC-CMV-Luciferase on human colorectal cancer tumor cell LOVO

[0078] Human colorectal cancer tumor cell LOVO came from the Cell Center of Institute of Basic Medical Sciences, Peking Union Medical College, China, and the control Ad-GFP adenovirus came from Shanghai Hanheng Company (GFP stands for green fluorescent protein). Proceed as follows:

[0079] (1) Human colorectal cancer tumor cell LOVO was divided into 1×10 6 Inoculate each well into a 6-well plate, continue culturing for 24 hours, and replace the culture medium before infection;

[0080] (2) Add the recombinant adenovirus vector Ad-CMV-TFRC-CMV-Luciferase prepared in Example 1 and the control Ad-GFP adenovirus to infect human colorectal cancer tumor cell LOVO respectively, according to 1×10 10 / ml titer, that is, adding 5 μl of recombinant adenovirus vector Ad-CMV-TFRC-CMV-Luciferase or control Ad-GFP adenovirus solutio...

Embodiment 3

[0083] Example 3: In vivo tumor tracing experiment of recombinant adenovirus vector Ad-CMV-TFRC-CMV-Luciferase

[0084] 1.1 Instrument

[0085] In vivo fluorescence imaging system: IVIS, Caliper; 7.0T micro-MR imaging system: Bruker, Germany.

[0086] 1.2 Experimental animals

[0087] BALB / c nude mice, 4-5 weeks old, weighing 15-18g, 15 were purchased from Chengdu Dashuo Experimental Animal Technology Co., Ltd., and were raised in SPF (Specific Pathogen Free) environment breeding room in the Animal Experiment Center of Sichuan University. All experimental procedures were approved by the Management Committee for the Use of Experimental Animals of the Institute of Experimental Animals.

[0088] 1.3 Experimental method

[0089] The recombinant adenovirus vector Ad-CMV-TFRC-CMV-Luciferase successfully constructed in Example 1 was used to infect human colorectal cancer tumor cells LOVO at an optimal multiplicity of infection of MOI=50, and the recombinant adenovirus was inoculat...

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Abstract

The invention provides a recombinant adenovirus vector which contains a human transferrin receptor gene and a firefly luciferase gene and can simultaneously express a transferrin receptor protein and a luciferase protein in a eukaryotic cell. The construction method of a double-reporter-gene recombinant adenovirus vector is characterized by comprising the following steps in sequence: step (1), using a polymerase chain reaction (PCR) method to amplify the human transferrin receptor gene; step (2), inserting the human transferrin receptor gene segment amplified in step (1) in a shuttle vector containing a firefly luciferase gene segment to obtain a recombinant shuttle vector, and conducting sequencing analysis; step (3), connecting the recombinant shuttle vector which is verified correctly on a framework vector to obtain a recombinant adenovirus plasmid; step (4), conducting XhoI enzyme digestion determination on the recombinant adenovirus plasmid; and step (5), using a human embryo kidney cell, namly an HEK 203 cell, to pack the recombinant adenovirus plasmid to obtain the recombinant adenovirus vector. As indicated in an experiment, the recombinant adenovirus vector can be applied to magnetic resonance and optics bimodal imaging and used as a tumor tracer agent.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a recombinant adenoviral vector containing double reporter genes and its construction method and application. Background technique [0002] Malignant tumors are refractory diseases that seriously threaten human health and life. Whether early diagnosis, metastasis warning and curative effect prediction of malignant tumors can be realized is directly related to the success or failure of its clinical treatment, and it is also the three keys for human beings to finally overcome the difficulties of malignant tumors breaking point. For decades, scientific researchers have been deeply studying the law of the occurrence and development of malignant tumors, trying to explore effective ways to break through these three key problems. [0003] The occurrence of tumor generally goes through the process of gene mutation-biological macromolecule change-metabolic abnormality-functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/12A61K49/00A61K49/06
Inventor 郜发宝王一帆刘婷
Owner 郜发宝
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