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DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of DNA and pichia pastoris expression vector

A constitutive promoter and yeast expression vector technology, applied in the field of DNA sequences, can solve the problems of limited application, the ability to express foreign proteins, and the lack of widespread application, and achieve the effect of high transcription activity and high expression.

Active Publication Date: 2013-03-27
林影 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This expression system needs to use flammable, explosive and toxic methanol in the fermentation process, which has certain risks, and also limits the application of Pichia pastoris expression system in food-grade protein production
In recent years, some endogenous constitutive strong promoters of Pichia pastoris have been discovered one after another. The more famous ones are the promoter of GAPDH gene and the promoter of TEF1 gene. The expression system of Pichia pastoris developed by using these promoters does not require methanol. can be induced to express foreign proteins, but compared with the expression system of AOX1 promoter, their ability to express foreign proteins still has a considerable gap, so it has not been widely used so far

Method used

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  • DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of DNA and pichia pastoris expression vector
  • DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of DNA and pichia pastoris expression vector
  • DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of DNA and pichia pastoris expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0057] 1. Cloning of DNA fragments

[0058] 1. Extract the genomic DNA of Pichia pastoris GS115

[0059] Genomic DNA of Pichia pastoris GS115 was extracted using the Yeast Genomic DNA Extraction Kit from OMEGA Company. For detailed methods, see the kit instructions.

[0060] 2. Clone the DNA fragment (SEQ NO.1) of the present invention from the genomic DNA of Pichia pastoris GS115

[0061] Primer: Primer PGCW14 -F (SEQ NO. 2) and P GCW14 -R (SEQ NO. 3).

[0062] Template: Genomic DNA of Pichia pastoris GS115.

[0063] DNA polymerase: PrimeSTAR HS (Takara)

[0064] Reaction system: Genomic DNA 10ng, 10μM P GCW14 -F and 10 μM P GCW14 -R 0.2 μM each, Premix PrimeSTAR HS 25 μL, ddH 2 O to make up 50 μL.

[0065] Reaction conditions: Denaturation at 98°C for 10s, annealing at 55°C for 5s, extension at 72°C for 1min, 30 cycles.

[0066] Results: A DNA fragment of 822 bp upstream of the 5' end of the chr1-4_0586 gene of GS115 genomic DNA was amplified by PCR method. Primer...

Embodiment 2

[0116] Construction of the expression vector pPGCW14ZαA using the DNA fragment of Example 1 as a promoter

[0117] In order to better understand the expression vector of the present invention, a specific expression vector will be provided below, which is transformed from the commercial Pichia pastoris expression vector pPICZαA (purchased from Invitrogen Company), that is, the activation of pPICZαA Substitute 5'AOX is replaced with DNA described in the present invention, and its plasmid map is as follows figure 2 As shown, the details are as follows:

[0118] A. Functional fragment:

[0119] (1) 5'GCW14 promoter region (5'GCW14promoter region): 1-834;

[0120] (2) α signal peptide (α-factor signal sequence): 835-1101

[0121] (3) c-myc epitope (c-myc epitope): 1169-1198

[0122] (4) 6×His tag (6×His tag): 1214-1231

[0123] (5) AOX1 transcription termination region: 1235-1576

[0124] (6) TEF1 promoter (TEF1promoter): 1577-1987

[0125] (7) EM7 promoter (EM7promoter) 19...

Embodiment 3

[0163] Construction of recombinant expression vector pPGCW14ZαA-EGFP

[0164] 1. Experimental materials

[0165] (1) The vector pPGCW14ZαA of the present invention: prepared according to the method in Example 2;

[0166](2) EGFP: enhanced green fluorescent protein, the gene of which is shown in Seq No.11.

[0167] (3) Escherichia coli Top10.

[0168] 2. Construction of recombinant expression vector pPGCW14ZαA-EGFP

[0169] (1) PCR method to obtain EGFP gene

[0170] Primers: E-F (Seq No.12) and E-R (Seq No.13).

[0171] Template: pEGFPN1 (purchased from clonetech company), the EGFP gene fragment was amplified from the plasmid pEGFPN1 (purchased from clonetech company), and EcoR I and XbaI restriction sites were added to the 5' end and 3' end of the gene, respectively.

[0172] DNA polymerase: Premix PrimeSTAR HS (Takara)

[0173] Reaction system: plasmid pEGFPN1 10ng, 10μM E-F and E-RμM each 0.2μM, Premix PrimeSTAR HS 25μL, ddH 2 O to make up 50 μL.

[0174] Reaction c...

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Abstract

The invention discloses a DNA (Deoxyribose Nucleic Acid) with constitutive promoter activity, application of the DNA and a pichia pastoris expression vector. The DNA has a base sequence as shown in SEQ No.1 (Sequence Number); and the application of the DNA relates to the application of the DNA in construction of the pichia pastoris (Pinchia Pastoris) expression vector. The DNA disclosed by the invention has the constitutive promoter activity, and can activate the transcription of a downstream structural gene without an inductor; the transcriptional activity shows little change in four different culture mediums, namely, ethanol, methanol, glucose and glycerol; the promoter activity is efficient, and the efficiency of the initiation transcription is four times more than the pichia pastoris GAPDH (Reduced Glyceraldehyde-phosphate Dehydrogenase) promoter. The pichia pastoris expression vector, constructed by the DNA disclosed by the invention, can efficiently express the extrinsic protein without methanol induction, and the efficiency of expressing the extrinsic protein (Enhanced Green Fluorescent Protein) is about 6 to 8 times that of the expression system of the GAPDH promoter and 1.5 to 2 times that of the expression system of a TEF1 (Transcription Enhancer Factor 1) promoter.

Description

technical field [0001] The invention relates to a DNA sequence, in particular to the DNA sequence containing a promoter and its application, and the constructed Pichia pastoris expression vector. Background technique [0002] Gene expression includes two stages of transcription and translation. Transcription refers to the process of copying an RNA single strand with the same sequence as the DNA strand (except T is replaced by U), which is the core step of gene expression. The basic process of transcription includes template recognition, transcription initiation, extension and termination through the promoter, and the key to this process is the interaction between RNA polymerase and the promoter. The promoter is a DNA sequence located in the upstream region of the 5' end of the structural gene, which can activate RNA polymerase to accurately combine with the template DNA and have the specificity of transcription initiation. The structure of a promoter affects its affinity f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/81C12N15/66C12R1/84
Inventor 林影叶燕锐邹承娟张轩薇卢俊裕韩双艳郑穗平
Owner 林影
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