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2A peptide, bicistronic expression vector, recombinant protein expression system and application

A technology of recombinant protein and expression vector, which is applied in the field of genetic engineering, can solve the problems of low expression level of target gene and inability to translate downstream genes, etc., achieve high-efficiency expression, increase expression amount, and reduce production cost

Pending Publication Date: 2021-04-02
河南普诺易生物制品研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2A peptide is an oligopeptide between two proteins, usually 18-22 amino acids. When the ribosome reaches the last two amino acids Gly-Pro at the C-terminal of 2A, it cannot be translated due to the change of the internal structure of the ribosomal amide center Downstream genes, at this time, the upstream nascent peptide is hydrolyzed to release the ribosome, and then the ribosome translates the downstream gene. In theory, the equal expression of the two genes can be achieved, so the 2A peptide is more suitable for the construction of a bicistronic vector, but The target gene expression level of the currently reported 2A peptide-mediated bicistronic vector is low

Method used

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  • 2A peptide, bicistronic expression vector, recombinant protein expression system and application
  • 2A peptide, bicistronic expression vector, recombinant protein expression system and application
  • 2A peptide, bicistronic expression vector, recombinant protein expression system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Synthesis of 2A peptide gene

[0033] This example provides the gene sequence for the 2A peptide.

[0034] According to the amino acid sequence of foot-and-mouth disease virus (F2A) shown in SEQ ID NO.1, and the equine rhinitis A virus (equine rhinitis A virus, E2A) shown in SEQ ID NO.3, such as SEQ ID Brown winged moth virus (Thosea asigna virus, T2A) shown in NO.5 and porcine Teschovirus (porcine teschovirus, P2A) amino acid sequence shown in SEQ ID NO.7; According to CHO cell codon preference, Design the 2A peptide gene sequence, as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.

Embodiment 2

[0035] Example 2 Construction of recombinant expression vector containing 2A peptide gene

[0036] This embodiment provides a method for constructing a recombinant expression vector comprising a gene encoding a 2A peptide, comprising the following steps:

[0037] Artificially synthesized SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8. In order to realize directional cloning, StuI (AGGCCT) and AgeI were introduced at the 5' end and 3' end of the synthetic sequence respectively. (ACCGGT) restriction site.

[0038] The synthesized 2A sequence was double-digested with AgeI / StuI respectively, and the pIRES-Neo plasmid DNA vector was double-digested with AgeI / StuI at the same time. The digestion results were identified by agarose gel electrophoresis, and the digested 2A sequence fragment and pIRES-Neo linear plasmid DNA were recovered from the gel.

[0039]The double enzyme digestion system for 2A sequence is: 10 μL of 2A sequence (1 μg / μL), 3.0 μL of 10×CutSmart Buffer, 1...

Embodiment 3

[0042] Example 3 Construction of EGFP recombinant expression vector

[0043] The present embodiment provides the construction method of the engineered bacterium that contains recombinant expression vector, comprises the following steps:

[0044] 1) PCR amplification of EGFP gene

[0045] Referring to the Enhanced green fluorescent protein (EGFP) gene sequence of the pEGFP-C1 vector (GenBank: U55763.1, bases 613-1332), design primers P1 and P2 (for amplifying 720bp EGFP gene DNA), primer 5 EcoRV and NheI restriction sites were introduced at the ' ends respectively, and the primer sequences were as follows (the restriction sites are underlined):

[0046] P1: 5′-CCG GATATC ATGGTGAGCAAGGGCGAGGAG-3′;

[0047] P2: 5′-CTA ACCGGT GGACTTGTACAGCTCGTCCATGC-3'.

[0048] Using the pEGFP-C1 plasmid (purchased from Clontech, USA) as a template, primers P1 and P2 were used to amplify the EGFP gene. The reaction system is shown in Table 1 below.

[0049] Table 1 PCR amplification syste...

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Abstract

The invention relates to a 2A peptide, a bicistronic expression vector, a recombinant protein expression system and application. The invention provides the 2A peptide (SEQ ID NO: 1 or SEQ ID NO: 3) and provides the bicistronic expression vector containing an encoding gene of the 2A peptide and a construction method for the bicistronic expression vector; and by applying the 2A peptide to expressionof a recombinant protein through constructing the recombinant protein expression system containing the 2A peptide, efficient expression of 2 target genes in cells of mammals can be achieved. Tests prove that on an equal footing, compared with other 2A peptides, the E2A-and-F2A-based recombinant protein expression system provided by the invention can be used for remarkably improving a stable expression level of recombinant proteins of mammalian cells and can be extensively applied to preparation of various target proteins, particularly antibodies. A foundation is settled for improving an expression system for the mammalian cells, further increasing the expression level of the recombinant proteins, improving stability of cell lines and reducing production cost.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a 2A peptide, a bicistronic expression vector, a recombinant protein expression system and applications. Background technique [0002] Recombinant protein is a protein produced by applying genetic engineering technology. In recent years, recombinant protein has become an important part of biological products and plays an important role in biopharmaceuticals, clinical diagnosis, and scientific research. The production of recombinant proteins mainly includes four major systems: prokaryotic protein expression, yeast protein expression, insect cell protein expression and mammalian cell protein expression. Recombinant proteins produced by mammalian cells can undergo correct folding, assembly and post-translational modification, and have the advantage of being closer to the molecular structure of human protein. Therefore, mammalian cells have become the main expr...

Claims

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Application Information

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IPC IPC(8): C07K14/09C07K14/095C12N15/85C07K16/24
CPCC07K14/005C12N15/85C07K16/241C12N2770/32122C12N2770/32722C12N2800/107C12N2800/22C12N2830/20C12N2830/60
Inventor 王天云林艳张俊河张继红樊振林米春柳杨献军
Owner 河南普诺易生物制品研究院有限公司
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