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V5 epitope fusion yeast expression vector and construction method thereof

A yeast expression vector and antigen epitope technology, applied in the field of genetic bioengineering

Inactive Publication Date: 2012-07-04
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the current difficulties in verifying the expression of exogenous genes in yeast cells, the present invention provides a yeast expression vector with V5 epitope fusion, which can effectively solve the problem of verifying the expression of exogenous genes in yeast cells

Method used

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  • V5 epitope fusion yeast expression vector and construction method thereof
  • V5 epitope fusion yeast expression vector and construction method thereof
  • V5 epitope fusion yeast expression vector and construction method thereof

Examples

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Embodiment 1

[0026] Example 1: The present invention verifies the expression of tetracycline repressor protein (TR) in yeast cells.

[0027] Tetracycline repressor protein (TR) is a protein for Escherichia coli tetracycline transposon, with a molecular weight of about 20kD, and there is no report of its expression in yeast cells so far. Using pcDNA / TR as a template, the open reading frame (ORF) of the TR gene was amplified by PCR, and the upstream primer was 5′-GTGgaattc ATGTCTAGATTAGATAAAAG-3' (EcoR I) (SEQ ID No. 5), downstream is 5'-AAGctcgagTTAATAAGATCTGAATTCCC-3' (Xhol I) (SEQ ID No. 6). After the PCR product is purified by the DNA kit, it is digested with EcoR I and Xhol I, and the excised TR is inserted into the V5 fusion yeast expression vector pGPDV5, and the flexible short peptide Gly (glycine)-Ile (isoleucine) The codon of -Leu (leucine), that is, GGA-ATT-CTG, was fused with the V5 epitope, and the constructed vector was named pGPDV5 / TR.

[0028] The vector pGPDV5 / TR was transf...

Embodiment 2

[0029] Example 2: Verification of the present invention for the expression and function of human androgen receptor (AR) in yeast cells

[0030] 1) Validation of human androgen receptor (AR) expression in yeast cells.

[0031] The molecular weight of human androgen receptor is about 110kd.

[0032] The AR ORF was amplified from pcDNA3.1 / AR by PCR, and the primers were designed as: upstream 5′-CAGgaattc ATGGAAGTGCAGTTAGGGCT-3' (SEQ ID No. 7), downstream is 5'-CTActcgagTCACTGGGTGTGGAAATAGA-3' (SEQ ID No. 8), each containing EcoR I and Xho I restriction sites (in lower case). The PCR product was recovered and purified with a kit, and inserted into the corresponding restriction site of pGPDV5 to construct pGPDV5 / AR. The V5 epitope is fused to AR through the codons of the flexible short peptide Gly (glycine)-Ile (isoleucine)-Gln (glutamine), namely GGA-ATT-CAA.

[0033] According to the method of Example 1, pGPDV5 / AR was transformed into W303-1A yeast cells, and the expression o...

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Abstract

The invention relates to a V5 epitope fusion yeast expression vector and a construction method thereof. The V5 epitope fusion yeast expression vector is a yeast expression vector containing a V5 tag, wherein the tag can be fused with downstream genes through a flexible small peptide Gly-Ile, and the biological activity of downstream expression proteins cannot be affected. The construction method comprises the following steps of: taking a pYestrp2 vector of Invitrogen as a basic framework, inserting a yeast strong promoter GPD into the pYestrp2 vector to construct a yeast expression vector pGPD, and inserting V5 epitope DNA with BamHI and EcoRI cohesive termini at 5' and 3' ends into corresponding endonuelease sites of the pGPD to construct a V5 fusion yeast expression vector pGPDV5, wherein the pGPDV5 can be fused with the downstream genes through flexible small peptides. According to the invention, as V5 antibodies are commercialized, the expression of exogenous genes in yeast cells can be conveniently, specifically and effectively verified, and the cost is relatively low.

Description

technical field [0001] The invention belongs to the field of genetic bioengineering, and in particular relates to a V5 antigen epitope fusion yeast expression vector and a construction method. Background technique [0002] Yeast is the most commonly used genetic bioengineering host cell. Yeast is a single-celled lower eukaryotic cell. It not only has the characteristics of easy cultivation, reproduction, and low requirements for growth conditions, but also has clear genetic background, gene structure, and gene regulation. , metabolism, and protein post-translational processing are similar to those of mammalian cells. Therefore, using yeast cells to express exogenous genes has good development and application prospects. [0003] To verify whether the exogenous gene is expressed in yeast, it is usually achieved by selecting a specific antibody for the exogenous gene expression product or by testing the function of the exogenous gene expression product. However, there are man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66
Inventor 李湘鸣罗方妮葛宜枝
Owner YANGZHOU UNIV
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