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Adenovirus vector carrying inducible co-stimulater gene, and construction method and application of adenovirus vector

A co-stimulatory molecule and construction method technology, applied in the fields of application, gene therapy, genetic engineering, etc., can solve the problems of low expression, low efficiency of target cell infection, low specificity, etc., achieve high gene expression level, improve infection Efficiency, the effect of high infection efficiency

Inactive Publication Date: 2012-07-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it also has shortcomings: 1. It can cause host cell immunity and humoral immunity, so that adenovirus and foreign gene expression products are easily eliminated; 2. It cannot fully infect some cell types with clinical importance; 3. It cannot be specialized 1. Kill target cells with low specificity
51 serotypes of human adenovirus have been found, which are divided into six subgroups: A, B, C, D, E, and F. Ad5 belongs to subgroup C. This kind of adenovirus with CAR as its receptor has the following problems: 1. The infection efficiency of target cells is not high; 2. It will cause serious toxic and side effects in the body; 3. Neutralizing antibodies to adenovirus type 5 widely exist in the human body, causing the virus to be quickly cleared by the immune system in the body; 4. The virus’s effect on cells Infection efficiency depends on the expression level of CAR receptors on the cell surface, while the expression level of CAR on the surface of important cells such as hematopoietic system cells, skeletal muscle cells, stem cells, dendritic cells, and primary tumor cells is very low
[0010] There are no related reports on adenoviral vectors carrying inducible co-stimulatory molecule genes

Method used

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  • Adenovirus vector carrying inducible co-stimulater gene, and construction method and application of adenovirus vector
  • Adenovirus vector carrying inducible co-stimulater gene, and construction method and application of adenovirus vector
  • Adenovirus vector carrying inducible co-stimulater gene, and construction method and application of adenovirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Construction of non-propagating adenovirus Ad5 / F11b-mICOS-EGFP carrying mICOS gene.

[0083] 1. Construction of the PDC318-mICOS vector: the PCRPLAN program in the PCGENE software was used to set various parameters: the optimum Tm=66, the allowable range of Tm was 50-80, the primer length was 15-25, the G-C content was 40%-60%. The maximum complementarity is 3, the G-C addition at the 3' end is 1, and the maximum homology between the primer and other sites of the template is 60%. After calculation, better primer sequences GT144 and GT145 were obtained.

[0084] Upstream primer GT144 of mICOS gene:

[0085] 5' CCG GAA TT CAC CAT GAA GCC GTA CTT C 3' (SEQ ID NO: 1)

[0086] Downstream primer GT145 of mICOS gene:

[0087] 5' GCG TCG ACT TAT CAG GGG CTG TTG GTG 3' (SEQ ID NO: 2)

[0088] 2. Acquisition of mICOS target gene: pEGFP-N1-mICOS plasmid was amplified by PCR, primers were GT144 and GT145, and the amplified product was subjected to 0.8% agarose gel el...

Embodiment 2

[0095] Example 2: Recombination and identification of Ad5 / F11b-mICOS-EGFP virus.

[0096] 1. Combine the plasmid pDC318-mICOS with the plasmid containing the right arm of adenovirus type 5

[0097] pPE3-F11B-EGFP was co-transfected into 293 cells by Lipofectamine 2000, and the mICOS gene was inserted into the E1 region of the adenovirus genome. Viral plaques appeared in the cells 9 to 14 days after co-transfection. After 3 times of viral plaque purification, the adenovirus DNA was extracted with QIAamp DNA Blood Mini Kit and identified by PCR. The identified adenovirus was named Ad5 / F11b-mICOS - EGFP, a non-proliferating adenovirus carrying the mICOS gene.

[0098] 2. Virus amplification, titer determination and purification:

[0099] (1) Recovery and subculture of 293 cells: Thaw frozen 293 cells in a 37°C water bath with gentle shaking, add 10 mL of 10% FBS / DMEM, centrifuge at 1000×g for 5 minutes, and discard the supernatant. Resuspend the cells with 10mL 10% FBS / DMEM, m...

Embodiment 3

[0104] Example 3: Ad5 / F11b-mICOS-EGFP transfected mouse bone marrow-derived MSCs.

[0105] (1) MSC cell passage: Discard the culture medium, wash twice with 2ml PBS, add an appropriate amount of 0.05% trypsin-0.02% EDTA digestion solution to digest the cells, and see that most of the cells become round and fall off under the inverted microscope, add twice the volume Neutralize the trypsin in the culture medium, discard the supernatant after low-speed centrifugation, resuspend in the fresh mMSCs maintenance medium, and inoculate them on a petri dish.

[0106] (2) Divide cells into 1×10 5 Inoculate in a 24-well plate at a density of / well. After the cells are completely adhered to the wall, remove the culture medium, add 0.5ml of fresh DMEM complete medium to each well, and take 1ul, 2ul, 5ul, 10ul, 20ul, 50ul of Ad5 / F11b -mICOS-EGFP virus solution was added to each well respectively, and mixed slowly in a cross shape, 37°C, 5% CO 2 Culture in the incubator, replace the fresh ...

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Abstract

The invention relates to the field of biotechnology. Hematopoietic stem cell transplantation is a unique way of curing malignant hematologic diseases, but the complications in the hematopoietic stem cell transplantation process are still key factors causing death of the patient, and the most prominent is graft-versus-host disease (FVHD). How to prevent and treat the GVHD is a severe challenge to hematopoietic stem cell transplantation. The invention provides an adenovirus vector carrying inducible co-stimulater (ICOS) gene, and a construction method for the adenovirus vector. After the adenovirus vector transfects mesenchymal stem cells (MSCs), an aim of preventing and treating the GVHD is fulfilled by a method of combining the effect of the ICOS in the T lymphocyte activating process and the effect of the MSCs in the immune adjusting process.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an adenovirus vector carrying an inducible co-stimulatory molecule gene, a construction method thereof, and an application of the adenovirus vector in preparation, prevention and treatment of graft-versus-host disease. Background technique [0002] Gene therapy refers to the use of DNA transfer to treat or prevent human diseases. It was gradually brewed in the 1960s with the development of multiple disciplines. Gene therapy was originally used to treat single-gene genetic diseases, but now it has been widely used in the treatment of tumors and other diseases. Gene therapy involves three aspects: target gene, carrier and recipient cells. In the process of gene therapy, how to introduce normal genes into target cells is the key to whether the therapeutic purpose can be achieved. [0003] There are two types of approaches to gene therapy. One is the in vivo approach, that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N5/10A61K48/00
Inventor 王健民杨丹王利平包晓辰周虹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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