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Alpha-galactosidase gene and application thereof

A galactosidase and gene technology, applied in the α-galactosidase gene and its application fields, can solve the problems of heterologous genes that are difficult to obtain high-efficiency expression, and achieve the effects of reducing complexity, increasing expression, and efficient degradation

Inactive Publication Date: 2017-08-01
WUHAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, due to the codon usage preference of Pichia pastoris for heterologous genes, the complexity of the secondary structure of mRNA, the abundance of A / T or G / C segments in genes, protease cleavage sites, and the abundance of genes in the host Influenced by factors such as the degree of concentration, it is difficult to obtain high-efficiency expression of heterologous genes in Pichia pastoris

Method used

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  • Alpha-galactosidase gene and application thereof
  • Alpha-galactosidase gene and application thereof
  • Alpha-galactosidase gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] To construct a bacterial strain containing a single-copy vector of α-galactosidase, the method is as follows.

[0072] 1.1 According to the artificial design of the existing α-galactosidase gene (from Aspergillus niger) sequence, a new α-galactosidase gene sequence is designed, and the α-galactosidase gene sequence is obtained by artificial synthesis. - a galactosidase gene fragment whose base sequence is shown in SEQ ID NO: 1, named Gal A. The amino acid sequence of the alpha-galactosidase encoded by the alpha-galactosidase gene is shown in SEQ ID NO:2.

[0073] In order to increase the expression level of the α-galactosidase gene in Pichia pastoris, in this example, the sequence of the α-galactosidase gene was designed in silico with the assistance of software such as DNA 2.0. The following factors were mainly considered during the design process:

[0074] (1) Replace the original low-frequency codons with high-frequency codons in P. pastoris;

[0075] (2) reduced ...

Embodiment 2

[0086] Construction of recombinant expression vector with multiple copies of α-galactosidase gene

[0087] 2.1 The expression cassette fragment containing the α-galactosidase gene was obtained by double-enzyme digestion with the same tail enzyme, and the expression cassette fragment was connected to the single-copy expression vector of α-galactosidase to obtain a two-copy expression vector.

[0088] 2.1.1 After the pAO-Gal A recombinant expression vector obtained in Example 1 was digested with Bgl II and BamH I, the small fragment (ie, the AOX-Gal A expression cassette) was recovered from the gel, and the enzyme digestion system: 30 μL of pAO-Gal A expression vector, 1.5 μL of BamH I, 1.5 μL of Bgl II, 20 μL of 10×Buffer K, 20 μL of BSA, 20 μL of Triton X-100, ddH 2 Make up to 200 μL with O, and digest for about 4 hours at 37°C.

[0089] 2.1.2 Digest the new pAO-Gal A expression vector with BamH I and recover it from the gel, and connect it with the AOX-Gal A fragment obtained ...

Embodiment 3

[0102] Construction of co-expression vector of α-galactosidase gene and endoplasmic reticulum secreted protein-related genes

[0103] 3.1 Co-express genes related to endoplasmic reticulum secreted proteins such as HAC1, PDI, Ero1, Hsp40 and α-galactosidase gene respectively.

[0104] 3.1.1 HAC1, PDI, Ero1, and Hsp40 genes were respectively connected to the pAO815 vector to construct a recombinant vector of the endoplasmic reticulum secreted protein-related genes (the method is the same as step 1.2).

[0105] 3.1.2 Connect the expression cassette containing the α-galactosidase gene (the AOX-Gal A expression cassette obtained in step 2.1.1) to the recombinant vectors of the endoplasmic reticulum secreted protein-related genes obtained above to obtain Co-express the recombinant vector (for the method, refer to step 2.1). Wherein, the nucleotide sequence of the HAC1 gene is shown in SEQ ID NO: 3, the nucleotide sequence of the PDI gene is shown in SEQ ID NO: 4, the nucleotide seq...

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Abstract

The invention discloses an alpha-galactosidase gene and application thereof, and relates to the technical field of biology. The alpha-galactosidase gene is suitable for encoding alpha-galactosidase, and a nucleotide sequence is shown as SEQ ID NO:1. The artificially designed alpha-galactosidase gene has the advantages that the high-frequency codon in pichia pastoris is used for replacing the original low-frequency codon, so that the free energy of mRNA (messenger ribonucleic acid) and the complexity of mRNA secondary structure are decreased; by selecting the secondary high-frequency codon, the areas containing AT (adenine and thymine) or GC (guanine and cytosine) in the gene or the protease action site are eliminated, and the expression amount of the artificially synthesized alpha-galactosidase gene in the pichia pastoris cell is obviously increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an alpha-galactosidase gene and its application. Background technique [0002] α-Galactosidase (α-Galactosidase), also known as melibiase, is an exoglycoside hydrolase that can catalyze the hydrolysis of various non-reducing terminal α-galactoside compounds bound by α bonds, such as Melibiose, raffinose family oligosaccharides (raffinose, stachyose, etc.), lactose (galactose)-mannan, galactose. [0003] In the feed industry, soybean meal is the most widely used vegetable protein raw material, but the content of α-galactoside sugars in soybean meal is relatively high, such as raffinose, stachyose and verbascose, etc., which are anti-nutrients in feed. one of the factors. Adding α-galactosidase to the feed can effectively degrade the anti-nutritional factors in the feed, reduce flatulence, indigestion and other symptoms, and improve the nutrient utilization rate of the feed. [0004...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/40C12N15/81C12N1/19C12P19/14
CPCC12N9/2465C12N15/815C12P19/14C12Y302/01022
Inventor 杨江科张继文
Owner WUHAN POLYTECHNIC UNIVERSITY
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