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

Inactive Publication Date: 2004-07-21
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

[0127] A eukaryotic expression system based on RNA transfection.

[0128] Such as figure 1 As shown, the 5'end of the full-length cDNA of Giardia lamblia virus (Giardia lamblia) was connected to the T7 promoter, and then at the 5'end at 670 bp and the 3'end at 2017 bp, the in vitro mutagenesis kit was used to produce NcoI. NcoI and XhoI restriction sites were used to cut the sites with NcoI and XhoI respectively. At the same time, NcoI and XhoI restriction sites were generated at both ends of the Cryptosporidium gp40 gene, and cut with NcoI and XhoI. Under the action of T4DNA ligase, the secretion The gp40 gene of sporeworm is connected with the gene of Giardia lamblia virus, and then transcribed in vitro under the action of T7 RNA polymerase. 100μg of the transcript is electroporated under the conditions of a voltage of 1000V / cm and a capacitance of 50μF. Giardia nourishes the body. After the electric shock, the electro-transducer cup is placed in an ice bath for 10 minutes, and...

Embodiment 2

[0130] Eukaryotic expression system by DNA transfection

[0131] Such as figure 2 As shown, the 5'end of the full-length cDNA (ppoly2 / sfinot GLV) of Giardia lamblia virus (GLV) loaded on the ppoly2 / sfinot plasmid vector is connected to the Giardia lamblia a microtubule promoter gene and signal peptide Then, at the 5'end of 670bp and 3'end of 2017bp, the NcoI and XhoI restriction sites were generated by in vitro mutagenesis kits, which were cut with NcoI and XhoI, and the Cryptosporidium gp40 gene was cut at both ends. Generate NcoI and XhoI restriction sites, cut with NcoI and XhoI, construct a recombinant plasmid ppoly2 / sfinot GLV-gp40 under the action of T4 DNA ligase, and transfer 50μg of the plasmid at a voltage of 400V / cm and a capacitance of 800μF. Infected with virus-containing Giardia lamblia nourish the body, place the electrotransformer in an ice bath for 10 minutes after the electric shock, then culture at 37°C, and detect the expression of gp40 gene in Giardia lamblia ...

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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 vector, 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 genetic engineering expression systems, prokaryotic expression systems and eukaryotic expression systems. In order to express foreign genes in prokaryotic or eukaryotic cells, gene expression vectors must be constructed first. There are many prokaryotic expression vectors in use, such as non-fusion expression protein vector PKK223-3, secretory cloning expression vector PINIII and fusion protein expression vector PGEX. Eukaryotic expression vectors include SV40 vector, adenovirus vector and vaccinia virus vector. However, the current problem is that the expression product formed by prokaryotic expression...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/33C12N15/85C12N15/86
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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