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Pichia pastoris expression vector taking chloramphenicol as screening marker and application thereof

An expression vector, Pichia pastoris technology, applied in the direction of using the vector to introduce foreign genetic material, recombinant DNA technology, fungi, etc., to achieve the effect of reducing experimental or production costs, facilitating preparation and related operations, and avoiding cumbersome medium configuration

Inactive Publication Date: 2015-03-04
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Based on the fact that Pichia pastoris cannot grow under the chloramphenicol-resistant plate solid medium, the purpose of the present invention is to solve the problem of how to use chloramphenicol resistance to screen Pichia transformants; a novel Pichia pastoris is provided Expression vectors, that is, Pichia pastoris expression vectors using chloramphenicol as a selection marker, such as pPIC3.5C, pPIC9C vectors, the expression vectors are replaced by the chloramphenicol resistance gene CAT on the existing Pichia pastoris expression vectors Nuclear cells screen the gene to form an expression vector containing the chloramphenicol resistance gene CAT; after the Pichia expression vector is transformed into Pichia, chloramphenicol can be used to screen recombinant Pichia transformants on YPG medium

Method used

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  • Pichia pastoris expression vector taking chloramphenicol as screening marker and application thereof
  • Pichia pastoris expression vector taking chloramphenicol as screening marker and application thereof
  • Pichia pastoris expression vector taking chloramphenicol as screening marker and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Preparation of Pichia pastoris GS115 electroporation competent

[0024] The present invention requires a special preparation method for electrotransformation competence. This method is different from the classic electrotransformation competent preparation method. The main difference lies in the culture medium and some reagents used. The specific steps are as follows:

[0025] 1) Cultivate Pichia pastoris in a 50ml Erlenmeyer flask containing 5ml of YPG medium (peptone 2g / 100ml, yeast extract 1g / 100ml, glycerol 3g / 100ml), overnight at 30°C;

[0026] 2) Take 1-5ml of the overnight culture, inoculate it into a 2L shake flask containing 500ml of fresh YPG medium, and grow to OD overnight 600 Between 1.3-1.5;

[0027] 3) Collect the cells by centrifugation at 1500g for 5 minutes at 4°C, and suspend the cells with 500ml pre-cooled sterile water;

[0028] 4) Collect the cells by centrifugation at 1500g for 5 minutes at 4°C, and suspend the cells with 250ml pre-c...

Embodiment 2

[0032] Example 2: Sensitivity detection of Pichia pastoris to chloramphenicol

[0033]The present invention finds for the first time that chloramphenicol has an inhibitory effect on Pichia pastoris. On YPG solid medium (peptone 2 g / 100ml, yeast extract 1 g / 100ml, glycerin 3g / 100ml, Agar 2g / 100ml), chlorine Pichia pastoris could not grow on the resistant plate solid medium of Mycin (purchased from Sangon Biotechnology Company, 0692C503), but it grew normally on the non-resistant plate solid medium ( image 3 ). The present invention further explored the optimal resistance concentration of Pichia pastoris GS115 (purchased from Novagen, K1750-01), added different concentrations of chloramphenicol to the YPG medium, and found that the inhibitory effect of chloramphenicol on GS115 has a dosage dependency ( Figure 4 ), and at a concentration of 2 mg / ml and above, Pichia GS115 was basically unable to grow.

Embodiment 3

[0034] Example 3: Transformation of the chloramphenicol resistance gene and verification of its release from the inhibitory effect of chloramphenicol

[0035] In order to verify that the inhibitory effect of chloramphenicol on Pichia can be relieved by degrading chloramphenicol through gene expression in the presence of a chloramphenicol resistance gene (CAT), the present invention obtained from the pACYC184 plasmid (purchased from New England

[0036]

[0037] 0.25pm / μL of swimming primers, 0.4mmol / μL of dNTP, 2 ng / μL of DNA template, 5 U / 100 μL of taq DNA polymerase, 1×taq buffer, the total volume is 25 μL. Perform the reaction as follows: 93°C, 3min pre-denaturation; 93°C, 30s; 50°C, 40s; 72°C, 1min reaction for 30 cycles; finally 72°C extension for 10min) to obtain the chloramphenicol resistance gene CAT, by Bam HI (purchased from TAKARA, 1001A), not Ⅰ (purchased from TAKARA, 1166A) was connected to Pichia pastoris expression vector pPIC3.5K (purchased from Novagen...

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Abstract

The invention discloses a pichia pastoris expression vector taking chloramphenicol as a screening marker, particularly a pichia pastoris screening system based on chloramphenicol. The invention reveals a fact that chloramphenicol has an obvious inhibitory effect on pichia pastoris GS115 so that pichia pastoris cannot grow on a YPG resistant solid medium containing chloramphenicol; and based on the fact, a pichia pastoris screening system using chloramphenicol as a screening antibiotic is established. Pichia pastoris is screened by using a vector containing chloramphenicol resistance gene CAT, as well as pichia pastoris electroporation transformation competence prepared by a method disclosed by the invention and a chloramphenicol screening solid medium; EGFP is used as a reporter gene; after pichia pastoris is transformed, green fluorescence can be observed under a fluorescence microscope, which means that the vector and the screening system can be effectively used for transformation and screening of pichia pastoris and expression of foreign protein.

Description

technical field [0001] The invention belongs to the technical field of yeast gene engineering, and in particular relates to a Pichia expression vector using chloramphenicol as a selection marker and an application thereof. Background technique [0002] Pichia pastoris is a methanolotrophic yeast that can use methanol as the sole carbon source and was developed as a protein expression system in the 1980s-1990s. Compared with the prokaryotic expression system, Pichia pastoris has the advantage of glycosylation modification, but it does not have the common hyperglycosylation problem of Saccharomyces cerevisiae. At the same time, compared with other eukaryotic expression systems such as insect expression system and Chinese hamster ovary cell (CHO) expression system, it has the advantage of large-scale fermentation and culture. In addition, in mammalian cell expression systems, exogenous genes cannot be expressed permanently, and problems such as cost and technical background al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/65C12N1/19
Inventor 井申荣马贵兴黄芬曾韦锟
Owner KUNMING UNIV OF SCI & TECH
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