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Pichia pastoris gene knockout and resistance gene recovery carrier, and construction method and application thereof

A technology of Pichia pastoris and gene knockout, which is applied in the field of genetic engineering, can solve the problems such as difficulty in taking into account the efficiency of screening marker recovery for resistance knockout efficiency, heavy screening workload of target clones in the knockout process, and long homologous arms. Achieve the effect of improving knockout efficiency, facilitating knockout of multiple genes, and improving efficiency

Active Publication Date: 2018-05-08
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the low success rate of Pichia pastoris gene knockout in the prior art, it is difficult to balance the efficiency of resistance knockout and the recovery efficiency of screening markers after gene knockout, the long homology arms used, and the workload of screening target clones in the knockout process Large technical defects or improvement needs, the present invention provides a Pichia pastoris gene knockout and resistance gene recovery vector and its construction method and application

Method used

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  • Pichia pastoris gene knockout and resistance gene recovery carrier, and construction method and application thereof
  • Pichia pastoris gene knockout and resistance gene recovery carrier, and construction method and application thereof
  • Pichia pastoris gene knockout and resistance gene recovery carrier, and construction method and application thereof

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Embodiment 1

[0055] Example 1: A template vector pAOXZ-mazf for gene knockout of Pichia pastoris, such as figure 1 As shown, the template vector includes the mazf gene expression cassette and the bleomycin resistance gene expression cassette regulated by the Pichia methanol-type promoter AOX1;

[0056] The 5' end of the mazf expression cassette contains a restriction site A for inserting a homologous fragment downstream of the gene to be knocked out, and the 5' end of the restriction site contains a lox66 site recognized by the Cre-specific recombination system;

[0057] The 3' end of the mazf expression cassette contains a restriction site B for inserting the upstream homologous fragment of the gene to be knocked out, and the 3' end of the restriction site contains the lox71 site recognized by the Cre-specific recombination system;

[0058] The bleomycin resistance gene expression cassette is located between the 5' end of the lox66 site and the 3' end of the lox71 site; the restriction si...

Embodiment 2

[0059] Embodiment 2: a kind of Pichia pastoris gene resistance gene recovery carrier pHGAPCre-mazf, such as figure 2 As shown, the recovered vector includes: the mazf gene expression cassette AOX1-mazf-AOX1TT regulated by the Pichia methanolic promoter AOX1, the Cre gene expression cassette GAP-Cre-AOX1TT constitutively expressed by the GAP promoter, and HygB resistance The gene expression cassette TEF1-HygB-CYC TT; the mazf gene expression cassette and the Cre gene expression cassette are in opposite directions on the pHGAPCre-mazf recovery vector; the GAP promoter contains at least one recovery vector in the pHGAPCre-mazf The only enzyme cleavage site present.

Embodiment 3

[0060] Example 3: Construction of the template vector pAOXZ-mazf for Pichia pastoris gene knockout

[0061] (1) Using Escherichia coli DH5α cells as a template, using primers mazf-F and mazf-R to amplify the mazf gene by colony PCR, introducing EcoR I and Sal I restriction sites at both ends of the gene, agarose gel Recovery and purification after electrophoresis;

[0062] (2) Purify and recover after double digestion with EcoR I and Sal I, and then digest the commercialized vector pPICZA with EcoR I and Sal I (removing the Xho I restriction site of the vector itself) and gel the recovered vector in 3 :1 mixed, transformed into Escherichia coli DH5α competent cells through Takara's ligation kit Solution I at 16°C for 1 h, the clones were screened on an LLB plate containing 25 μg / mL zeocin, and the grown clones were randomly selected to extract plasmids After enzyme digestion verification, it was named pPICZA-mazf after sequencing verification;

[0063] (3) Using pPICZA-mazf ...

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Abstract

The invention discloses a Pichia pastoris gene knockout and resistance gene recovery carrier, and a construction method and application thereof, and belongs to the technical field of microbial geneticengineering. A template carrier of the Pichia pastoris gene knockout comprises a mazf gene expression cassette regulated by a methanol type promoter AOX1 of the Pichia pastoris, specific recombinaserecognition sites of lox71 and lox66 at two ends and a bleomycin resistance gene expression cassette; the Pichia pastoris gene resistance gene recovery carrier comprises the mazf gene expression cassette regulated by the methanol type promoter AOX1 of the Pichia pastoris, a cre gene expression cassette constitutively expressed by using a GAP promoter and a HygB resistance gene expression cassette.The Pichia pastoris gene knockout and the resistance gene recovery carrier and the construction method thereof greatly improve the knockout efficiency of a Pichia pastoris gene. The invention provides a method for recovering antibiotic markers after a gene knockout is conducted, the method for recovering the antibiotic markers after the gene knockout is conducted provides great convenience for efficient knockout of multiple genes, and has high knockout efficiency, and a used homology arm is short.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a method for gene knockout of Pichia pastoris. Background technique [0002] Pichia pastoris is a kind of methanolotrophic yeast that can grow with methanol as the only carbon and energy source. Pichia pastoris contains a strong promoter of alcohol oxidase AOX1, which is one of the promoters with the strictest regulatory mechanism. When methanol is the only carbon source, the expression of the gene can be strictly regulated by methanol. The expression system of Pichia pastoris is widely used in the expression of various exogenous proteins because of its fast growth rate, easy genetic transformation operation, a variety of strong promoters to choose from, high expression efficiency, and high-density fermentation. And a higher protein expression level was obtained. [0003] There is no natural plasmid in Pichia pastoris, and the vector is generally integrated in...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66
CPCC12N15/66C12N15/815
Inventor 闫云君焦梁成周清华宿智新乔阳歌杨凯欣
Owner HUAZHONG UNIV OF SCI & TECH
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