Codon optimized recombinant human lysozyme gene and application thereof

A technology of codon optimization and human lysozyme, which is applied in the fields of cell engineering and molecular biology, can solve the problems of low recombinant protein expression, affecting mRNA stability and translation efficiency, and high purification and production costs.

Active Publication Date: 2018-10-09
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of bioreactor research, when the exogenous gene contains low-frequency codons used by host cell genes or the preferred codons are inconsistent, it will often affect the stability and translation efficiency of the mRNA of the exogenous gene, resulting in low expression of recombinant protein
Preliminary studies have found that the expression of recombinant human lysozyme in transgenic sheep and bovine mammary gland bioreactors is not ideal compared with other recombinant milk proteins, resulting in high production costs for subsequent purification

Method used

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  • Codon optimized recombinant human lysozyme gene and application thereof
  • Codon optimized recombinant human lysozyme gene and application thereof
  • Codon optimized recombinant human lysozyme gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Codon usage bias analysis of bovine dominant milk protein

[0046] The codon usage bias analysis of bovine dominant milk protein gene using CodonW and EMBOSS software showed that the codon preference of bovine milk protein gene ended with GC, while the codon preference of human lysozyme gene ended with AT.

[0047] In this example, the applicant selected αs1, αs2, β, K casein, β-lactoglobulin, whey albumin, and lactoferrin according to the content of various proteins in the mammary gland. High dominant milk protein was used as the analysis object, and the sequence of the CDS region of the protein-coding gene above was obtained by searching NCBI. For the specific milk protein gene name, GenBank accession number, and CDS sequence length, please refer to Attached Table 1.

[0048] Attached Table 1: Sequence information of bovine dominant milk protein gene

[0049]

[0050] The relative probability of codon usage (RSCU) analysis was carried out on 59 codons ...

Embodiment 2

[0055] Embodiment 2: Codon optimization design of human lysozyme gene

[0056] According to the analysis results of the above-mentioned CodonW software, the third base of the codon of human lysozyme ends with AT (the average content of the third base GC3s is 0.41), while the third codon of the bovine dominant milk protein gene ends with GC (The average content of the third base GC3s is 0.54). According to the use of high-frequency codons and low-frequency codons of milk protein in the attached table 2, the inventors optimized the codon design of human lysozyme. Among them, lyzop optimized all the codons of the human lysozyme gene, while Lyzop23 only optimized the first 23 codons of the human lysozyme gene (Supplementary Table 3).

[0057] Attached Table 3: Codon Optimization of Human Lysozyme Gene

[0058]

[0059] Note: The bold italic part indicates the optimized codons of the recombinant human lysozyme gene according to the cow milk protein preference codons.

[0060]...

Embodiment 3

[0063] Example 3: Detection of impact of codon optimization design on human lysozyme mRNA transcription level

[0064] Lyzcw, Lyzop23, and Lyzop were synthesized according to the sequence in Appendix 3, and HindIII and XhoI restriction sites were added.

[0065] Further, HindIII and XhoI digested Lyzcw, Lyzop23, Lyzop and PcDNA3.1 (+) vectors respectively, recovered the above fragments by gel, and used T4 DNA enzyme to connect and construct three human lysozymes: PcDNA-Lyzcw, PcDNA-Lyzop23 and PcDNA-Lyzop respectively expression plasmid (see figure 1 ).

[0066] After the above three plasmids were transfected with DH5α, single-clonal shaking bacteria, extracted plasmids, HindIII and XhoI double enzyme digestion and identification, the successfully constructed PcDNA-Lyzcw, PcDNA-Lyzop23 and PcDNA-Lyzop plasmids were selected for cell transfection.

[0067] When the confluence of cultured bovine fetal fibroblasts (BFFC), bovine mammary epithelial cells (BMEC), and mouse mammar...

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Abstract

The invention relates to codon optimized recombinant human lysozyme gene and application thereof. The codon optimized recombinant human lysozyme gene is characterized in that human lysozyme codons aresubjected to global optimization (Lyzop) by comparing bovine mammary superior milk protein high-frequency codons and low-frequency codons, and optimization (Lyzop23) is performed by interpreting initial post 23-bit codons. The optimized codons obviously improve the human lysozyme mRNA stability and the mRNA expression quantity in bovine mammary cells. Western immunoblotting experiments prove thatthe expression quantity of the codon optimized recombinant human lysozyme gene in the bovine mammary cells is 2.18 times of that of wild types. Tool gene is provided for efficiently producing the recombinant human lysozyme by a cell bioreactor and a bovine mammary bioreactor in future. Meanwhile, the codon optimized recombinant human lysozyme gene and luciferase are fused for expression; by analyzing the luciferase expression quantity, the fast and accurate evaluation on the codon optimizing effect is realized; a novel reliable evaluation method is provided for developing the gene codon optimization in the future.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and cell engineering, and in particular relates to a codon-optimized recombinant human lysozyme gene and application thereof. Background technique [0002] Lysozyme, also known as muramidase or N-acetyl murein hydrolase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria. It has broad-spectrum antibacterial, anti-inflammatory, antiviral and other functions. As a natural protein, human lysozyme is widely distributed in body fluids, cells, tissues and organs in the human body, and plays an important role in antibacterial, antiviral, and immune regulation. At present, human lysozyme has been widely used in clinical disease treatment, dairy product additive, food preservation and so on. However, the traditional method of separating and extracting from breast milk, placenta or urine is difficult to meet the requirements of industrial production. Compared wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/62C12N15/85
CPCC07K2319/61C12N9/2462C12N15/85C12N2800/22C12Y302/01017
Inventor 成功昝林森王力
Owner NORTHWEST A & F UNIV
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