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Common expression CTLA4Ig and CTLA4 recombinant adenovirus carrier, recombinant adenovirus and uses thereof

A technology of recombinant adenovirus and expression vector, which is applied in the direction of virus/bacteriophage, application, and resistance to vector-borne diseases, etc., and can solve problems such as restricting the development of organ transplantation and affecting the long-term quality of life of patients

Inactive Publication Date: 2009-01-14
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the application of immunosuppressants has promoted the development of organ transplantation. However, various toxic and side effects caused by long-term application of immunosuppressants not only seriously affect the long-term quality of life of patients, but also greatly limit the development of organ transplantation.

Method used

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  • Common expression CTLA4Ig and CTLA4 recombinant adenovirus carrier, recombinant adenovirus and uses thereof
  • Common expression CTLA4Ig and CTLA4 recombinant adenovirus carrier, recombinant adenovirus and uses thereof
  • Common expression CTLA4Ig and CTLA4 recombinant adenovirus carrier, recombinant adenovirus and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, Construction of adenoviral plasmid vector pAdeasy-CIC for secretory expression of CTLA4Ig and membrane-bound expression of CTLA4

[0034] 1. Acquisition of rat CTLA4 full-length, CTLA4 extracellular segment and IgG Fc segment gene fragments

[0035]Male Wistar rats (purchased from the Animal Center of the Academy of Military Medical Sciences) were taken and killed by pulling the neck. The spleen was removed under sterile conditions, the spleen tissue was squeezed with ophthalmic curved forceps, and the spleen cell suspension was collected through a 400-mesh sieve and centrifuged in 50 mL. In the tube, centrifuge, discard the supernatant, wash the cell pellet, and resuspend the cells with RPMI1640 (Sigma company product) culture medium containing 10% fetal bovine serum (pre-added penicillin-streptomycin 100U / mL). The isolated splenocytes were divided into 2×10 6 / cm 2 Concanavalin A (ConA, purchased from Sigma Company) was added to the 50mL plastic culture b...

Embodiment 2

[0060] Example 2. Liposome-mediated recombinant adenovirus plasmid pAdeasy-CIC transfected human embryonic kidney 293A cells for virus packaging, amplification and titer determination

[0061] 1. Liposome-mediated transfection of recombinant adenovirus plasmid pAdeasy-CIC into human embryonic kidney 293A cells for virus packaging

[0062] 1 day before transfection, inoculate 7.5×10 5 Human embryonic kidney 293A cells (purchased from Nanjing Kaiji Biological Co., Ltd.)) were placed at 37°C in CO 2 cultured in an incubator. Change the medium 3-4h before transfection to ensure that the cells grow exponentially (add 5mL medium); prepare for transfection with the linearized plasmid pAdeasy-CIC obtained in Example 1 and liposome Lipofectamine 2000 (purchased from Invitrogen Company) (1) Dilute 15-20 μg linearized pAdeasy-CIC plasmid DNA with 500 μl serum-free DMEM (purchased from Sigma); (2) Dilute 20 μl Lipofectamine 2000 with 500 μl serum-free DMEM, mix gently, No more than 5mi...

Embodiment 3

[0067] Example 3, Detection of transfection efficiency and expression of recombinant adenovirus Ad-CIC in vitro transfection of target cells

[0068] Taking rat bone marrow mesenchymal stem cells (BMDMSCs) as an example, the infection efficiency of recombinant adenovirus Ad-CIC of the present invention infecting target cells (mammalian cells) in vitro and the expression identification of target genes are detected. The specific detection methods are as follows:

[0069] 1. Isolation of rat bone marrow mesenchymal stem cells (BMDMSCs)

[0070] Male LEWIS rats (purchased from Beijing Weitong Lihua Experimental Animal Center) were killed by neck pulling and immersed in 75% alcohol for 10 minutes. Rat femurs were separated with surgical instruments such as sterile scissors, and low-sugar DMEM culture solution (purchased from Sigma) was sucked into a syringe to repeatedly rinse the bone marrow cavity until the color turned white (about 3-4 times). Filter the rinse solution with a 4...

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Abstract

The invention discloses an IRES-mediated recombinant adenovirus vector and the recombinant adenovirus for co-expressing CTLA4Ig and CTLA4. The preparing method thereof comprises the steps: a CTLA4Ig gene, an IRES gene and a CTLA4 gene are inserted sequentially into the downstream of a giant cell virus promoter in a pAdTrack-CMV shuttle vector, a recombinant shuttle vector carrying the CTLA4Ig gene, the IRES gene and the CTLA4 gene is then obtained, and the recombinant shuttle vector is recombined in homologous with a virus backbone vector pAdeasy-1 to obtain the recombinant adenovirus vector for co-expressing the CTLA4Ig and the CTLA4; and the recombinant adenovirus expression vector transfects packaging cells (293 cells of human embryo kidney) to obtain recombinant adenovirus, and mammalian cells are infected by the recombinant adenovirus in vitro, thereby ensuring purpose cells with immunological suppression function. The recombinant adenovirus can play an important role in the medical field, especially in the organ transplanting field, thereby having extensive application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant adenovirus, in particular to a recombinant adenovirus vector for co-expression of CTLA4Ig and CTLA4 mediated by IRES, a recombinant adenovirus and applications thereof. Background technique [0002] Transplantation is a radical treatment for end-stage tissue and organ failure. Rejection of grafts by recipients is a major obstacle to successful transplantation. In recent years, the application of immunosuppressants has promoted the development of organ transplantation. However, various toxic and side effects caused by long-term application of immunosuppressants not only seriously affect the long-term quality of life of patients, but also greatly limit the development of organ transplantation. The induction of donor-specific immune tolerance brings hope to finally get rid of the toxic side effects of non-specific immunosuppressants and improve the quality of l...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/12C12N7/01C12N5/10A61K38/17A61P37/06
CPCY02A50/30
Inventor 裴雪涛罗海英王韫芳翟树森管利东
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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