Anthropogenic lysozyme encoding gene and expressing method and application thereof in pichia pastoris

A technology encoding a gene, lysozyme, applied in the direction of microorganism-based methods, applications, genetic engineering, etc., can solve the problem of low expression

Active Publication Date: 2019-04-16
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the existing human lysozyme is heterologously expressed in a variety of expression hosts, the expression level is generally relatively low, which makes it problematic in industrial application.

Method used

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  • Anthropogenic lysozyme encoding gene and expressing method and application thereof in pichia pastoris
  • Anthropogenic lysozyme encoding gene and expressing method and application thereof in pichia pastoris
  • Anthropogenic lysozyme encoding gene and expressing method and application thereof in pichia pastoris

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, human source lysozyme gene sequence codon optimization

[0023] The sequence derived from the human lysozyme gene was codon-optimized to a certain extent: a. Reduce the occurrence probability of continuous A / T / G / C bases and avoid the generation of stem-loop structure; b. In the entire gene sequence, increase Some rare codons, especially during translation initiation, reduce the rate of ribosome elongation. The optimized human lysozyme gene sequence is shown as SEQ ID NO.2, and the optimization rate of the entire sequence is 23.2% compared with the original sequence.

Embodiment 2

[0024] Embodiment 2, construction of expression vector and protein expression

[0025] 1. Artificial synthesis of gene sequence

[0026] The nucleotide sequence shown in SEQ ID NO.2 was entrusted to Shanghai 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.

[0027] 2. Gene sequence amplification

[0028] Design primer pair (hLYZ-F, hLYZ-R) according to the nucleotide sequence shown in SEQ ID NO.2

[0029] The underlined part of the forward primer is the Cpo I restriction site, and the underlined part of the reverse primer is the Not I restriction site. The sequence design of this site conforms to the sticky end produced by the method of cutting with T4 DNA polymerase.

[0030] PCR reaction system:

[0031]

[0032] PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C...

Embodiment 3

[0045] Example 3, construction of cofactor genes and related vectors

[0046] 1. Cofactor gene amplification

[0047] Through the website https: / / www.ncbi.nlm.nih.gov / , search for the Bip, Ero1 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. The underlined part of the forward primer is the restriction site of EcoR I and its homologous sequence at the left end of the vector, and the underlined part of the reverse primer is the restriction site of Age I and its homologous sequence at the right end of the vector. The sequence design conforms to the method of T5 exonuclease construction vector.

[0048] PCR reaction system:

[0049]

[0050] PCR reaction conditions: pre-denaturation at 98°C for 6min, denaturation at 95°C for 30s, annealing at 57°C for 30s, extension at 72°C (1kb / 30s), 30 cycles, and...

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Abstract

The invention relates to an anthropogenic lysozyme encoding gene and an expressing method and application thereof in pichia pastoris. The anthropogenic lysozyme encoding gene has the nucleotide sequence shown in SEQ ID NO2. Anthropogenic lysozyme regards the eucaryon (pichia pastoris GS115) as the expression host, and in combination with the gene copying number and the cofactor co-expression strategy, the obtained lysozyme product has the advantages of being high in expression amount, high in activity and the like, can serve as an antibiotic substitute and is widely applied in the fields of feed and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the mass expression and application of a human lysozyme and its coding gene in Pichia pastoris GS115. Background technique [0002] Lysozyme is an antibacterial enzyme with a molecular weight of 14.4kDa, which has a significant effect on sterilization. Lysozyme disrupts the cell wall structure of bacteria (mainly Gram-positive bacteria) by destroying the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, and promotes cell rupture. resulting in the death of bacteria. Because of its good bactericidal effect, it is mainly used in medical treatment, food, feed and scientific research. According to different molecular structures, sources and molecular weights, lysozymes can be divided into 6 categories: plant lysozymes, microbial lysozymes, bacteriophage lysozymes and 3 types of animal lysozymes (type c, type g and type...

Claims

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Application Information

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IPC IPC(8): C12N9/36C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2462C12N15/815C12Y302/01017Y02A50/30
Inventor 易犁何华华吴世杰张桂敏
Owner HUBEI UNIV
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