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Mosaic type adenoviral vector for transfecting pig skin and use thereof

An adenovirus and chimeric technology, applied in the field of biomedicine, can solve the problems of not meeting long-term coverage of wounds, slowing down humoral immune rejection, etc., and achieve the effect of improving transplant survival time and transfection efficiency

Inactive Publication Date: 2009-12-16
CHONGQING ZONGSHEN JUNHUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, simply blocking T cell activation by expressing CTLA4Ig protein can slow down the occurrence of cellular immune rejection, but it cannot slow down humoral immune rejection, so prolonging the survival time of transplanted skin is limited and cannot meet the needs of long-term wound coverage

Method used

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  • Mosaic type adenoviral vector for transfecting pig skin and use thereof
  • Mosaic type adenoviral vector for transfecting pig skin and use thereof
  • Mosaic type adenoviral vector for transfecting pig skin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of Ad5F35-CTLA4Ig-CD40Ig chimeric adenovirus vector

[0027] Step 1: Construction of CTLA4Ig fusion protein expression vector

[0028] The pUC19-CTLA4Ig plasmid was constructed according to Example 1 disclosed in the patent application No. 00103528 and the title of the invention is "a kind of allogeneic skin transfected with recombinant gene". The CTLA4Ig recombinant gene expression series is shown in SEQ ID NO:1.

[0029] Step 2: Construction of CD40Ig fusion protein expression vector

[0030] 1), routine method is isolated mononuclear cell from the peripheral blood of people;

[0031] 2), conventional methods extract the RNA of mononuclear cells; and reverse transcribe into cDNA;

[0032] 3), the extracellular region of CD40 is amplified by PCR with specific primers; and the product is cloned into T vector;

[0033] The sequence of primer 1 is shown in SEQ ID NO:5, and the sequence of primer 2 is shown in SEQ ID NO:6.

[0034] Primer 1: 5'-...

Embodiment 2

[0063] Example 2: Real-time quantitative PCR detection of target gene transfected with porcine skin tissue by Ad5F35-CTLA4Ig-CD40Ig chimeric adenovirus vector in vitro

[0064] 1. Preparation of pigskin slices: In the ultra-clean workbench, cut the pigskin into small pieces of 2cm×2cm, disinfect with 5% povidone iodine for 10 minutes, wash with a large amount of sterilized normal saline, and set aside.

[0065] 2 Transfection: adjust the Ad5F35-CTLA4Ig-CD40Ig chimeric adenovirus vector titer to 10 with DMEM medium 8 PFU, Ad5-CTLA4Ig adenoviral vector titer to 5×10 8 PFU, take the same area of ​​pig skin slices (6-month-old Bama pigs), add the same volume of 10 8 PFU Ad5F35-CTLA4Ig chimeric adenovirus solution and 5×10 8 PFU Ad5-CTLA4Ig adenovirus solution was placed in a cell culture incubator at 37°C for culture. Another pig skin with the same area and the same source was transfected and cultured in a virus-free medium as a control.

[0066] 3. Real-time quantitative PCR ...

Embodiment 3

[0085] Example 3. Detection of Ad5F35-CTLA4Ig-CD40Ig chimeric adenovirus vector-mediated immunosuppressive function in vitro

[0086] Pig fibroblasts were 1.0×10 4 cell / well (96-well plate) was subcultured in 100 μl high-glucose DMEM+10% fetal bovine serum medium, and the culture conditions were 37°C, 5% CO 2 , 4-12 hours after the cells adhered to the wall, wash 3 times with 0.01M / LPBS, Ad5F35-CTLA4Ig-CD40Ig chimeric adenovirus vector, Ad5-CTLA4Ig adenovirus vector were transfected at MOI=100, 100μl for 2 hours, blotted transfection solution. Lymphocytes were separated using human lymphocyte separation medium. Resuspend 2.0×10 in 200 μl RPMI 1640 complete medium containing 10% calf serum 5 cells / well (96-well plate) were co-cultured with fibroblasts. After 48h, centrifuge at 1000rpm×5min, add 20μl of 0.5mg / ml MTT solution, continue to incubate for 4h, add 100μlDMSO after suction and incubate on an air-bath shaker at room temperature for 10min, measure the OD value at 570n...

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Abstract

The invention relates to a mosaic type adenoviral vector for transfecting pig skin and the use thereof. The mosaic type adenoviral vector is an Ad5F35 mosaic type adenoviral vector containing a CTLA4Ig recombination gene and a CD40Ig recombination gene which are both effectively connected with a promoter. The invention successfully constructs the Ad5F35 mosaic type adenoviral vector containing the CD40Ig-CTLA41g recombination genes, and the adenviral vector can block a CD40 / CD40L passage and a B7 / CD28 passage simultaneously, and restricts exclusion of cell immune as well as exclusion of humoral immunity. By using the adenoviral vector to transfect the pig skin tissue, the transplant survival time and transfection efficiency of pig skin tissue are greatly improved. The mosaic type adenoviral vector can be used for preparing transfected pig skin for covering burning surface of a wound for a clinical patient.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric adenovirus vector for transfecting pig skin and its application. Background technique [0002] Burns are a special type of trauma with a high incidence and disability rate. The fundamental problem is the wound surface. If the burn wound is not covered and sealed in time, it will definitely cause the disorder of the internal environment of the patient, infection and The occurrence of multiple organ dysfunction; in addition, non-physiological healing of the wound surface may occur, resulting in increased scar formation and aggravated disability. Therefore, the study of skin grafting on burn wounds is an urgent need for modern burn treatment; it is also a hot spot and difficulty in burn research. [0003] Deep burn wounds need to be healed by skin transplantation, while the source of autologous skin is seriously insufficient in patients with large-area and super-larg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/62A61F2/10
Inventor 魏泓葛良鹏黄正根吴军
Owner CHONGQING ZONGSHEN JUNHUI BIOTECH
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