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Eucaryon expression system using jiadi flagllate virus as gene expression carrier

A Giardia and expression system technology, applied in the field of genetic engineering, can solve the problems of gene pollution and low expression efficiency, and achieve the effect of small genome, small cytotoxicity and easy purification

Inactive Publication Date: 2007-11-28
MILITARY SUPPLIES UNIV THE CHINESE PLA
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Problems solved by technology

[0003] The invention provides a eukaryotic expression system using giardia dsRNA virus as a gene expression carrier, which overcomes the problems of low expression efficiency and easy occurrence of gene pollution in the current eukaryotic gene expression system
Overcome the problems of low expression efficiency and prone to gene contamination in the current eukaryotic gene expression system

Method used

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  • Eucaryon expression system using jiadi flagllate virus as gene expression carrier
  • Eucaryon expression system using jiadi flagllate virus as gene expression carrier

Examples

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Embodiment 1

[0018] Eukaryotic expression system by RNA transfection.

[0019] As shown in Figure 1, the 5' end of the full-length cDNA of Giardia lamblia virus (Giardia lamblia) was connected to the T7 promoter, and then 670 bp at the 5' end and 2017 bp at the 3' end were induced with an in vitro mutagenesis kit. Change to produce NcoI and XhoI restriction sites, use NcoI and XhoI to digest respectively, and at the same time generate NcoI and XhoI restriction sites at both ends of the Cryptosporidium gp40 gene, use NcoI and XhoI to digest, under the action of T4DNA ligase , the Cryptosporidium gp40 gene was connected with the Giardia lamblia virus gene, and then transcribed in vitro under the action of T7 RNA polymerase, and 100 μg of transcripts were electroshocked under the conditions of voltage 1000V / cm and capacitance 50μF, and transfected with virus The Giardia lamblia was nourished in the body. After the electric shock, the electroporation cup was placed in an ice bath for 10 minute...

Embodiment 2

[0021] Eukaryotic expression system by DNA transfection

[0022] As shown in Figure 2, the 5' end of the full-length cDNA (ppoly2 / sfinot GLV) of Giardia lamblia virus (GLV) loaded on the ppoly2 / sfinot plasmid vector was connected to the Giardia lamblia a microtubule promoter gene and the signal peptide gene, and at the 670bp of the 5' end and 2017bp of the 3' end, use an in vitro mutagenesis kit to generate NcoI and XhoI restriction sites, which are digested with NcoI and XhoI respectively, and at the same time Cryptosporidium gp40 Generate NcoI and XhoI restriction sites at both ends of the gene, digest with NcoI and XhoI, construct the recombinant plasmid ppoly2 / sfinot GLV-gp40 under the action of T4 DNA ligase, and put 50 μg of the plasmid under the conditions of voltage 400V / cm and capacitance 800μF Electric shock, transfected Giardia lamblia trophoblasts containing virus, after electric shock, put the electroporation cup in ice bath for 10min, then culture at 37°C, detect...

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Abstract

An eucaryotic expression system using Giardia virus as gene expression carrier is disclosed, which can be the one using DNA transfection mode and the other using RNA transfection mode. Said Giardia virus can effectively infect its trophont and has lower toxin and more number of copies in gene duplication. Said Giardia is easily cultured. Its advantages are high expression efficiency and no pollution to gene.

Description

Technical field: [0001] The invention provides a eukaryotic expression system using giardia dsRNA virus as a gene expression carrier, belonging to the technical field of genetic engineering. Background technique: [0002] The ultimate goal of genetic engineering is to efficiently express foreign genes in a suitable system to produce valuable protein products. There are two types of expression systems for genetic engineering: prokaryotic expression systems and eukaryotic expression systems. To express foreign genes in prokaryotic or eukaryotic cells, gene expression vectors must first be constructed. There are many prokaryotic expression vectors currently in use, such as non-fusion protein expression vector PKK223-3, secreted cloning expression vector PINIII and fusion protein expression vector PGEX. Eukaryotic cell expression vectors include SV40 vectors, adenovirus vectors, and vaccinia virus vectors, etc. However, there are currently problems that the expression products...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/86C12N15/12C12N15/33
Inventor 张西臣田宗成李建华尹继刚杨举
Owner MILITARY SUPPLIES UNIV THE CHINESE PLA
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