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In-vitro efficiently-constructed multi-copy yarrowia lipolytica expression vector and application method thereof

A technology of Yarrowia lipolytica and expression vector, which is applied in the field of genetic engineering, can solve the problems that the stability needs to be further improved, the expression level of the expression system is not very ideal, etc., and achieve the goal of avoiding inhibition, high safety, and increasing expression level Effect

Inactive Publication Date: 2014-12-24
HUBEI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, this expression system also has the problems that the expression level is not very ideal and the stability needs to be further improved.

Method used

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  • In-vitro efficiently-constructed multi-copy yarrowia lipolytica expression vector and application method thereof
  • In-vitro efficiently-constructed multi-copy yarrowia lipolytica expression vector and application method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of recombinant vector pINA1296I.

[0029] 1) Find the promoter and terminator regions of the pINA1296 vector and other elements that constitute the vector according to the product catalog of NEB company. Among them, the same tail enzymes that can be used are added upstream of the promoter and downstream of the terminator respectively, and then 4 primers (5' agcaccAAGCTTGCTAGCgccgccgcaaggaatggt 3', 5'gaattcGGATCCTCTAGAggacacgggcatctcacttgca3', 5'TCTAGAGGATCCgaattctcatgtttgacagcttatcatcg3', 5'GCTAGCAAGCTTggtgctcaac 3'ccacctcaagCTTggtgctcaacggtca. The size of the fragment and the small fragment are about 1065bp and 6164bp respectively. After obtaining two fragments of different sizes by PCR, the product was recovered and purified by agarose gel electrophoresis, and then digested with DpnI at 37°C for 3 hours to eliminate the plasmid background. The two fragments were mixed and transformed into the large intestine by enzyme-free cloning. Bacillus XL10-G...

Embodiment 2

[0035] 1. Select the target gene to be expressed-mannanase.

[0036] 2. Recombinant plasmid. Select from Aspergillus niger ( Aspergillus niger ) The mannanase gene was cloned into the modified pINA1296I vector.

[0037] First, use restriction enzymes Sfi I and restriction endonuclease Kpn I digested the pINA1296I vector, and the product was a linear fragment with sticky ends. The digested product was recovered and purified by agarose gel. Secondly, use restriction enzymes Sfi I and restriction endonuclease Kpn I double-enzyme digestion of the PCR product of the mannanase gene will produce a cohesive end complementary to the pINA1296I vector after enzymatic digestion, and the fragment after the enzyme treatment is recovered. Finally, the pINA1296I vector and the mannanase gene fragment treated with two enzymes were mixed and treated with TaKaRa's Ligase Kit Solution I for 4 h at 16°C, and then transformed into E. coli XL10-Gold at 37° C culture overnight, pick 17 transforman...

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Abstract

The invention provides an in-vitro efficiently-constructed multi-copy yarrowia lipolytica expression vector and an application method thereof. The expression vector is recombined through the following steps: designing four primers added on the upstream of a promoter and a downstream of a terminator of a pINA1296 carrier respectively, respectively enabling the primers to be respectively subjected to PCR amplification, including small fragments taking the promoter and the terminator as regions and large fragments taking other frames of the carrier as regions, transforming Escherichia coli XL10-Gold after recovery and purification, and verifying the recombinant to obtain the multi-copy yarrowia lipolytica pINA1296I. The application method comprises the following steps: selecting the target genes to be expressed, inserting the target genes into the pINA1296I carrier to construct the single-copy recombinant plasmid pINA1296I-xxx-A1 containing the target genes. The in-vitro series connection of the multi-copy target genes such as pINA1296I-xxx-A2, pINA1296I-xxx-A3, and pINA1296I-xxx-A4 is achieved by the biologic bricking method, The method is safe, simple and easy to operate, and can improve the expression quantity of the target protein.

Description

technical field [0001] The invention relates to high-efficiency construction of a multi-copy Yarrowia lipolytica expression vector in vitro and an application method thereof, belonging to the technical field of genetic engineering. Background technique [0002] With the rapid development of genetic engineering technology, how to improve the expression and stability of the target protein in the host bacteria has received great attention at home and abroad. In particular, some biologically active small molecule peptides such as antimicrobial peptides have received high attention. However, due to its small molecular weight, low expression level, and poor stability in cells, it is easily degraded into inactive oligopeptides by host proteases. In addition, the product is toxic to the host. For example, the antimicrobial peptide usually inhibits the growth of the host bacteria or even directly kills the host bacteria during the expression process. These limiting factors are consi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12R1/645
Inventor 马立新赵西选陈羽西李晔星张玲姚永兰
Owner HUBEI UNIV