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Gene tiny chain carrier, construction method and application of the same

A construction method and chain vector technology, applied in the field of gene microchain vector and its construction, can solve the problems affecting the actual value of target gene expression, insufficient transfection efficiency, etc., and achieve convenient replacement, low immunogenicity, and high biological safety. sexual effect

Inactive Publication Date: 2008-05-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The resulting consequences will seriously affect the expression of the target gene and its practical value in gene therapy
Second, the presence of large amounts of DNA expression products, especially CpG sequences, derived from non-host vectors may trigger host immune responses
Kay MA from Stanford University in the United States developed minicircle DNA vectors (Minicircle DNA vectors) that contain almost no non-host DNA components, but the transfection efficiency of such minicircle vectors is still insufficient

Method used

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  • Gene tiny chain carrier, construction method and application of the same
  • Gene tiny chain carrier, construction method and application of the same
  • Gene tiny chain carrier, construction method and application of the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 The construction of the microchain vector containing GFP reporter gene and the measurement of transfection efficiency

[0065] 1. Materials and methods

[0066] (1) Materials

[0067] 1. Main materials pEGFP-N3 plasmid, Escherichia coli strain DH5α, cell lines 3T3, CNE2 and B95-8 were preserved in our laboratory, 293 cells were donated by Dr. Zou Juntao from the Human Anatomy Teaching and Research Office of Sun Yat-sen University, and the Cap sequence was provided by Shanghai Sangon Bioengineering Technology Ltd Synthetics.

[0068] 2. Tool enzymes and main reagents Restriction enzymes AseI and AflII were purchased from Jingmei Company, exonuclease ExoIII and T4 ligase were purchased from TaKaRa Company; Ligation high ligase was purchased from Japan TOYOBO BO Company, and agarose powder was from Shanghai YitoEnterprise Company; calf serum was purchased from Beijing Dingguo Biotechnology Co., Ltd.; cell transfection reagents were purchased from Ribo Biotechno...

Embodiment 2

[0142] Example 2 Construction and expression detection of MEKK1 gene microchain vector

[0143] 1. Materials and methods

[0144] (1) Materials

[0145] 1 The main materials pCMV-MEKK1 plasmid and B95-8 cells were kept in our laboratory, pMD18-T Vector was purchased from TaKaRa Company, pre-stained protein standard protein was purchased from Gene Company, and from New England biolabs; The primary antibody serum from patients with nasopharyngeal carcinoma was donated by Professor Wanli Liu from the Laboratory Department of Cancer Hospital Affiliated to Sun Yat-sen University; the secondary antibody was purchased from Boster Biological Co., Ltd., and the primary antibody rabbit anti-β-actin was purchased from Beijing Boaosen Biotechnology Co., Ltd.; Goat anti-rabbit-HRP was purchased from Boster Biological Co., Ltd.; PMSF and bovine serum albumin fraction five (BSA) were purchased from Beijing Dingguo Biotechnology Co., Ltd.; protein quantification reagent was Bio-Rad Protein f...

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Abstract

The invention discloses a genetic micro-chain carrier, which comprises a linear DNA sequence expression cassette and two symmetrically arranged Cap protection sequences; wherein the Cap protection sequences close both ends of the linear DNA sequence expression cassette and are modified forming different protection structures, based by the building of MEKK1 micro-chain carrier specifically expressing inside the tumour relative with EB virus. The invention simultaneously discloses a construction method of the genetic micro-chain carrier. The invention has the advantages of good versatility of loading foreign gene and convenient replacement, since the linear nucleic acid structure of the micro-chain carrier comprises a replaceable target gene expression cassette; the chance to be a popular carrier of gene therapy, providing a new thinking and direction for the carrier development of gene therapy.

Description

technical field [0001] The invention relates to the technical field of gene carriers, in particular to a gene microchain carrier and its construction method and application. Background technique [0002] At present, the difficulties of gene therapy are vector transfection, low expression efficiency and potential safety hazards. Safe and effective gene therapy vectors are the focus of current gene therapy research. The safety of gene therapy vectors makes the development of safe, reliable, and high transfection vectors the focus of current gene therapy research. [0003] The limitations of viral vectors as the main carrier of gene therapy are obvious: because viral vectors use their own infection process to introduce foreign target genes into host cells, the participation of intact viruses is required to maintain the infection process. The replication of these viral components in host cells can easily trigger the body to produce different degrees of immune responses, and the...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66A61K48/00A61P35/00
Inventor 杜军何玉文王红胜
Owner SUN YAT SEN UNIV
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