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Acidic mammal chitinase coding gene and application

A technology of chitinase and coding gene, applied in plant gene improvement, application, genetic engineering, etc., can solve the problems of low activity, large amount of acidic mammalian chitinase expression, low expression level, etc.

Active Publication Date: 2019-10-22
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although some studies have reported that acid chitinase is expressed heterologously in E. coli hosts, the expression level and activity are relatively low, which makes it have obvious disadvantages in industrial applications.
[0004] Through the search of existing technologies at home and abroad, no method for mass expression of acidic mammalian chitinase in the host has been found

Method used

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  • Acidic mammal chitinase coding gene and application
  • Acidic mammal chitinase coding gene and application
  • Acidic mammal chitinase coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, acidic mammalian chitinase gene sequence codon optimization

[0028] The gene sequence of mammalian chitinase derived from mouse stomach tissue was optimized to a certain extent by codon: a, reducing the occurrence probability of continuous A / T / G / C bases, and avoiding the generation of stem-loop structure; b, Throughout the gene sequence, some rare codons were added, especially at the initiation of translation, to reduce the rate of ribosome elongation. The optimized acidic mammalian chitinase gene sequence is shown in SEQ ID NO.2, and the optimization rate of the whole sequence is 23.4% compared with the original sequence.

Embodiment 2

[0029] Embodiment 2, construction of expression vector and protein expression

[0030] 1. Artificial synthesis of gene sequence

[0031] The nucleotide sequence shown in SEQ ID NO.2 was entrusted to Wuhan Jinkairui Bioengineering Co., Ltd. to artificially synthesize the gene according to the conventional technology in the field, and the gene was inserted into the plasmid vector pUC57, and stored for future use.

[0032] 2. Gene sequence amplification

[0033] Design primer pair (Dchit-F, Dchit-R) according to the nucleotide sequence shown in SEQ ID NO.2

[0034] The underlined part of the forward primer is the Cpo I restriction site, the underlined part of the reverse primer is the Not I restriction site, and the sequence design of this site conforms to the T 4 Cohesive ends produced by DNA polymerase trimming.

[0035] PCR reaction system:

[0036]

[0037]

[0038] PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 5 s, annealing...

Embodiment 3

[0050] Example 3, construction of cofactor genes and related vectors

[0051] 1. Cofactor gene amplification

[0052] Through the website https: / / www.ncbi.nlm.nih.gov / , find the Mxr1, Hac1 and Pdi1 gene sequences derived from Pichia pastoris GS115 and design primers for gene amplification (primer list), these genes were respectively constructed in the pGAPZB vector between the EcoR I and Age I restriction sites.

[0053] PCR reaction system:

[0054]

[0055] PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 5 s, annealing at 55°C for 5 s, extension at 72°C for 10 s, 30 cycles of amplification, and final extension at 72°C for 10 min.

[0056] The PCR product was detected by 0.7% agarose gel electrophoresis, and purified with a DNA purification kit (produced by GeneMark).

[0057] 2. Construction of recombinant expression vector

[0058] 1) The plasmid pGAPZB was double-digested with EcoR I and Age I, and the digested product was recov...

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PUM

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Abstract

The invention discloses an acidic mammal chitinase coding gene and application. The gene has the nucleotide sequence as shown in SEQ ID NO.2. A multi-copy method without antibiotic screening marks isconstructed in vitro, so that high expression of acidic mammal chitinase is realized; meanwhile, the expression quantity of the acidic mammal chitinase is further improved by virtue of co-expression of auxiliary factors Hac1.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to an acidic mammalian chitinase coding gene, a recombinant vector, a transformant and application. Background technique [0002] Chitin (chitin) is a straight-chain polymer connected by N-acetyl-β-D-glucosamine through β-1,4-glycosidic bonds. It is one of the most abundant natural polymers. Chitobiose, the product of chitin hydrolysis by chitinase, has high application value in the fields of medicine and health care. According to the different cleavage sites of chitinases, chitinases can be divided into two categories: endochitinases and exochitinases. Endochitinase cuts randomly inside the chitin chain to produce chitooligosaccharides with smaller molecular weight; exochitinase catalyzes the hydrolysis of the substrate and releases it from the reducing or non-reducing end of the chitin chain (GlcNAc) 2 , and β-N-acetylglucosaminidase that can decompose oligo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12P19/26C12P19/14C12R1/84
CPCC12N9/2442C12N15/815C12P19/26C12P19/14C12Y302/01014
Inventor 张桂敏杜超周玉玲何华华
Owner HUBEI UNIV
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