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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 low infection efficiency and high production cost of gene-transfected pigskin, and achieve the effects of improving transfection efficiency, improving gene transfection efficiency and reducing rejection reaction.

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

[0005] However, the infection efficiency of Ad5 adenovirus vector to pig skin tissue cells is low, and a higher titer of virus infection is required to exert its effect, which makes the production cost of gene transfected pig skin higher

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

[0022] Embodiment 1: Construction of Ad5F35-CTLA4Ig adenoviral vector

[0023] Specifically include the following steps:

[0024] 1. Construction of pUC19-CTLA4Ig plasmid

[0025] 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.

[0026] 2. Construction of pDC316-CTLA4Ig plasmid

[0027] The schematic diagram of the pDC316-CTLA4Ig plasmid is shown in figure 1 shown. The specific construction steps are as follows:

[0028] 1) HindIII+BamHI (TAKARA, JAPANESE) double-digested the vector plasmid pcDNA3 (invitrogen, USA), and recovered the 5.4kb fragment of the digested product of the pcDNA3 plasmid.

[0029] 2), HindIII+BamHI double-enzyme digestion with the target gene plasmid pUC19-CTLA4Ig, and recover the 1200bp fragment of the pUC19-CTLA4Ig ...

Embodiment 2

[0058] Example 2: In vitro transfection of Ad5F35-CTLA4Ig chimeric adenovirus vector into pigskin tissue and its detection

[0059] Specific steps are as follows:

[0060] 1. Preparation of pigskin slices: In the ultra-clean workbench, cut the pigskin into 2cm×2cm small pieces, disinfect with 5% povidone iodine for 10 minutes, wash with a large amount of sterilized normal saline, and set aside.

[0061] 2. Transfection: adjust the titer of Ad5F35-CTLA4Ig chimeric adenovirus vector 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.

[0062] 3. Real-time quantitative PCR detection ...

Embodiment 3

[0097] Example 3 Application of Ad5F35-CTLA4Ig chimeric adenovirus vector to transfect pigskin on burn wounds of Wistar rats

[0098] The two kinds of transfected pig skins and non-transfected normal control skins in Example 2 were respectively transplanted on the 2 cm x 2 cm blade-thick skin wounds on the back of burned Wistar rats. The experiment found that the average survival time of pig skin transfected with Ad5F35-CTLA4Ig chimeric adenovirus vector was 20 days, and the average survival time of pig skin transfected with Ad5-CTLA4Ig adenovirus vector was 17 days, while the survival time of the control group was 8 to 9 days. sky.

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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 Ad5F35 mosaic type adenoviral vector containing a CTLA41g recombination gene which is effectively connected with a promoter. The invention successfully constructs the Ad5F35 mosaic type adenoviral vector containing the CTLA41g recombination gene; by using the adenoviral vector to transfect the pig skin tissue, the transfection efficiency can be improved by 5-7 times in comparison with an Ad5-CTLA41g adenoviral vector, thereby the efficiency for gene transfection is 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): C12N15/861C12N15/13C12N15/12A61F2/10
Inventor 魏泓葛良鹏熊菲孔勇
Owner CHONGQING ZONGSHEN JUNHUI BIOTECH