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A codon-optimized β-galactosidase gene and its application

A technology of galactosidase and codon optimization, applied in the field of β-galactosidase gene and its application

Active Publication Date: 2019-03-19
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no codon-optimized expression scheme for LacZ gene that can be highly expressed in bovine mammary glands

Method used

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  • A codon-optimized β-galactosidase gene and its application
  • A codon-optimized β-galactosidase gene and its application
  • A codon-optimized β-galactosidase gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The optimal design and synthesis of embodiment 1β-galactosidase gene codon

[0016] The proteins with the highest content in milk are αs1-casein (casein alpha s1, CSN1S1), αs2-casein (casein alpha s2, CSN1S2), β-casein (casein beta, CSN2), K-casein (casein kappa , CSN3), β-lactoglobulin (beta-lactoglobulin), α-lactalbumin (lactalbumin, alpha-LALBA), bovine milk serum albumin (albumin, ALB), etc. The contents of these proteins in milk are shown in Table 1. According to the weights of several proteins with the highest content in milk, and the codon usage frequency and frequency of the genes encoding these proteins in the bovine mammary gland, the optimal codons were selected (see Table 2), and 1025 LacZ genes were selected. Amino acid codons are replaced one by one. The optimized gene is called OLacZ (optimized LacZ). Blast compared the nucleotide sequence before and after codon optimization, the similarity was 77%, and the amino acid sequence before and after optimiza...

Embodiment 2

[0022] Example 2 pCMV-LacZ and pCMV-OLacZ plasmids were transfected into HC11 cells

[0023] HC11 cells were seeded into 24-well plates, and the cells grew to 70% density of the plate. Using LipofectamineTM2000, according to liposome: DNA=2.5:1 (μL / μg), the plasmid pCMV-LacZ or pCMV-OLacZ were respectively unsaturated transfected to HC11 cells in the same amount. At 37°C, 5% CO 2 After 6 hours of cultivation under the same conditions, the complete culture medium was replaced to continue the cultivation.

[0024] After 48 hours of transfection of cells with plasmids pCMV-LacZ and pCMV-OLacZ, the cells were stained according to the instructions of the β-galactosidase in situ staining kit. The plasmid is transfected into the cells, and the cells express the β-galactosidase gene (OLacZ). Using X-Gal as a substrate, a dark blue product will be generated under the catalysis of β-galactosidase, so that the blue cells can be easily observed under an optical microscope. Incubate at...

Embodiment 3

[0030] Example 3 shows that codon-optimized OLacZ exhibits mammary gland specificity to a certain extent at the mouse level

[0031] Several pCMV-LacZ and pCMV-OLacZ transgenic mouse families were obtained by pronuclear injection and transplantation of pCMV-LacZ and pCMV-OLacZ vector DNA respectively through mouse fertilized eggs. After mating, pregnancy, calving, and about 10 days of lactation, the female mice were killed, and the organs were collected after perfusion. Quantitative detection of β-galactosidase activity per unit weight organ of each transgenic mouse by ONPG method (Beiyuntian Company). The test results showed that the codon-optimized OLacZ transgenic mice had a certain mammary gland-specific optimization trend (pCMV-LacZ female mice=4, pCMV-OLacZ female mice=6) (see Table 5).

[0032] Table 5 Expression of β-galactosidase in various organ units of pCMV-LacZ and pCMV-OLacZ transgenic mice during lactation

[0033]

[0034] According to the codon preference...

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Abstract

The invention discloses a codon optimized beta-galactosidase gene and an application thereof and belongs to the technical field of biochemistry and molecular biology. The nucleotide sequence of beta-galactosidase is shown as a sequence table SEQ ID NO.1. According to the preference of relevant high-expression protein codons in cow milk, codons of the bacterium-derived beta-galactosidase gene are optimized. The optimized OLacZ gene is verified in mouse mammary epithelial cells, and it is proved that the expression quantity of the gene is remarkably increased by about 3.7 times compared with that of a wild LacZ gene. The codon optimized OLacZ gene can be better effectively expressed in mammary tissue of a mother mouse in a lactation period. Beta-galactosidase serves as a reporter gene and is efficiently expressed in cow mammary tissue, and the effective tool gene is provided for further developing a mammary gland bioreactor.

Description

technical field [0001] The invention belongs to the technical field of biochemistry and molecular biology, and in particular relates to a codon-optimized β-galactosidase gene and application thereof. Background technique [0002] The LacZ gene is a gene in the lactose operon of Escherichia coli, and the β-galactosidase gene is a tetramer composed of four subunits, which can catalyze the hydrolysis of lactose. β-galactosidase is relatively stable, and when stained with X-Gal as a substrate, it turns blue, which is convenient for detection and observation. LacZ gene is a widely used reporter gene in molecular biology and genetic engineering, especially for the in situ expression of proteins detected by histochemical staining, and its expression products have basically no toxic effect on the survival and growth of cells. However, the detection sensitivity of LacZ gene is not high, and its detection results are affected by various factors such as gene expression and staining. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N15/85C12N5/10
CPCC12N9/2471C12Y302/01023
Inventor 曾凡一张敬之高越何佳平张斯敏
Owner SHANGHAI CHILDRENS HOSPITAL
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