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Recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of recombinant adeno-associated virus

A virus and transduction technology, applied to recombinant adeno-associated virus and its application field, can solve the problems of low infection efficiency, poor local action specificity, difficult to achieve therapeutic effect, etc., and achieves increased transduction efficiency, increased targeting, The effect of good application prospects

Active Publication Date: 2012-08-01
ANLONG BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, plasmids are often used to carry genes, but the biggest drawback of this strategy is the low infection efficiency, poor specificity of local action, and it is difficult to achieve the desired therapeutic effect.

Method used

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  • Recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of recombinant adeno-associated virus
  • Recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of recombinant adeno-associated virus
  • Recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of recombinant adeno-associated virus

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Experimental program
Comparison scheme
Effect test

Embodiment

[0151] Construction and identification of embodiment recombinant four-plasmid adeno-associated virus (FLAG-AAV.Luc)

[0152] 1. Construction and identification of recombinant expression plasmid p3×FLAG-CMV-VP2

[0153] 1.1 Using pAAV-RC as a template, amplify the VP2 sequence by PCR, and the primers are:

[0154] Table 1 Primer Sequence

[0155]

[0156] The two parts in italics are the artificially added EcoR V and BamH I restriction sites respectively.

[0157] 1.2 Use EcoR V and BamH I to cut the empty vector p3×FLAG-CMV-10 and the VP2 sequence amplified by PCR respectively, and connect the VP2 sequence to the p3×FLAG-CMV-10 vector in the correct reading frame to obtain the expression of FLAG Plasmid p3xFLAG-CMV-VP2 fusion protein with VP2.

[0158] 1.3 Identification of the obtained recombinant plasmid vector.

[0159] Obtain the VP2 sequence of AAV virus by PCR amplification ( figure 1 ), figure 1 The middle arrow points to the target band of the corresponding se...

experiment example 2

[0206] Experimental example 2 Application of the four-plasmid recombinant virus with FLAG tag of the present invention in absorbable materials

[0207] 1.PLGA

[0208] experimental method:

[0209] Poly(lactic-co-glycolic acid) (poly(lactic-co-glycolic acid), PLGA) (purchased from Shandong Medical Instrument Factory) was used as solid phase material, coated with 1 mg / ml collagen activated by EDAC, and dried at room temperature. 20mM SPDP was incubated at room temperature for 4 hours, and antibody immobilization was performed after washing with PBS. Use non-specific antibody IgG (50mg / ml) as negative control group, Anti-FLAG monoclonal antibody (50mg / ml) as positive control group, and incubate overnight at 4°C. After washing with PBS, AAV.LacZ and FLAG-AAV.LacZ were incubated at room temperature for 2 hours. After washing with PBS, Hela cell suspension was added, after 2 hours of attachment, 10% FBS was added to incubate for 48 hours, stained with β-galactosidase in situ sta...

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Abstract

The invention discloses recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of the recombinant adeno-associated virus. The recombinant adeno-associated virus contains modified VP2 protein of the adeno-associated virus and primary VP1 protein, primary VP2 protein and primary VP3 protein of the adeno-associated virus, wherein the modified VP2 protein of the adeno-associated virus is formed in the way that a section of FLAG tag sequence is inserted into N terminus of the VP2 capsid protein. The invention further discloses a construction method of the recombinant adeno-associated virus and the application of the recombinant adeno-associated virus in gene therapy and in preparation of tissue engineering scaffold materials. The recombinant adeno-associated virus disclosed by the invention contains FLAG epitope, thus being capable of effectively infecting target cells and enhancing transduction efficiency. Compared with the conventional virus vector, the recombinant adeno-associated virus has higher transduction efficiency and better targeting performance, thus being applicable to multiple aspects of the gene therapy, construction of virus delivery systems, the preparation of the tissue engineering scaffold materials, and the like.

Description

technical field [0001] The present invention relates to a recombinant adeno-associated virus, in particular to a recombinant four-plasmid adeno-associated virus that increases the targeted transduction efficiency of adeno-associated virus (adeno-associated virus). The application in tissue engineering belongs to the field of recombinant adeno-associated virus and its application. Background technique [0002] From the perspective of genes, gene therapy is a method of replacing or supplementing defective genes with normal and functional genes. From the perspective of treatment, it is to transfer new genetic material into the cells of an individual to obtain therapeutic effects. As a new means of treating diseases, gene therapy is attracting more and more attention and attention. From the late 1980s, scientists such as French Anderson, Michael Blaese, and Steven Rosenberg of the National Institutes of Health of the United States jointly proposed clinical trial applications fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864C12N15/34C12N15/63A61K48/00A61L27/54C12R1/93
Inventor 丁卫石文洁郑少鹏牛静吴巍
Owner ANLONG BIOPHARMACEUTICAL CO LTD
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