Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of codon-optimized recombinant human lysozyme gene and its application

A technology of codon optimization and human lysozyme, which is applied in the fields of molecular biology and cell engineering, can solve the problems of unsatisfactory expression, low expression of recombinant protein, and high cost of purification and production.

Active Publication Date: 2021-07-13
NORTHWEST A & F UNIV
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of codon-optimized recombinant human lysozyme gene and its application
  • A kind of codon-optimized recombinant human lysozyme gene and its application
  • A kind of codon-optimized recombinant human lysozyme gene and its application

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 Table 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 strains were picked, plasmids were extracted, HindIII and XhoI were digested and identified, 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 mammary epitheli...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a codon-optimized recombinant human lysozyme gene and application thereof. The codon-optimized recombinant human lysozyme gene is obtained by comparing the high-frequency codons and low-frequency codons of bovine mammary gland dominant milk protein to human lysozyme codons. Global optimization (Lyzop) and optimization at codon 23 after translation initiation (Lyzop23) were performed. The optimized codons significantly improved the stability of human lysozyme mRNA and its mRNA expression in bovine mammary gland cells. It was proved by western blotting that the expression level of the codon-optimized recombinant human lysozyme gene in bovine mammary gland cells was 2.18 times that of the wild type. It provides tool genes for the efficient production of recombinant human lysozyme in cell bioreactors and bovine mammary gland bioreactors in the future. At the same time, the codon-optimized recombinant human lysozyme gene was fused and expressed with luciferase, and by analyzing the expression of luciferase, the rapid and accurate evaluation of the codon optimization effect was realized, which provided a new reliable method for future gene codon optimization. Evaluation method.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com