High-activity transposase and use thereof

A transposase, high activity technology, applied in the fields of molecular biology and biomedicine

Pending Publication Date: 2021-06-04
SHANGHAI CELL THERAPY RES INST +1
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the enzyme activity of the existing PiggyBac transferase mutants has been improved compared with the wild-type PiggyBac transferase, it still cannot meet the higher and stricter requirements for enzyme activity. Therefore, it is still necessary to study the PiggyBac transferase with high enzyme activity.

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
  • High-activity transposase and use thereof
  • High-activity transposase and use thereof
  • High-activity transposase and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The acquisition of embodiment 1 highly active bz-hyPBase mutant

[0053] Based on the original sequence (amino acid sequence shown in SEQ ID NO: 1) of the existing highly active piggybac transposase (hyPBase for short), we made the following changes to obtain the protected baize piggyBac transposase (bz-hyPBase for short) sequence information:

[0054] (1) Based on human codon usage preferences, we optimized the codons of the existing highly active piggybac transposase to obtain the nucleotide sequence shown in SEQ ID NO:4 to improve the expression level of the transposase;

[0055] (2) The human c-myc nuclear localization signal is added after the start codon to improve the integration efficiency of foreign genes in host cells;

[0056] (3) The following method was used to randomly mutate the nucleotide sequence shown in SEQ ID NO:4 to obtain a mutant whose transposition efficiency was significantly better than that of the existing highly active piggybac transposase, ...

Embodiment 2

[0084] Example 2 bz-hyPBase has higher transposition efficiency in yeast

[0085] We inserted the transposon with the G418 resistance gene into the URA3 gene in the yeast plasmid PRS316 to disrupt the expression of the URA gene, and cloned the transposase with an inducible promoter into PRS316 to generate the plasmid PRS316-URA- For Pbase, plasmids carrying different transposases WT PBase, hyPBase, optimized hyPBase, and bz-hyPBase were prepared in parallel. The plasmid was transformed into ura-deficient Saccharomyces cerevisiae BJ2168, which could not survive in ura-deficient medium. The expression of transposase is turned on under the regulation of the inducer galactose, which promotes the transposition of the transposon, the transposition of the transposon, the normal expression of the URA gene, and the normal growth of the transposed clone in the ura-deficient medium . The transposition efficiency of the transposase in Saccharomyces cerevisiae can be calculated by counti...

Embodiment 3

[0087] Example 3 bz-hyPBase has higher gene editing efficiency in CHO cells

[0088] We cloned optimized hyPBase and bz-hyPBase into mammalian cell expression vectors to generate plasmid ploxP-optimized hyPBase (structure the same as Figure 5 , only the Figure 5 The medium transposase was replaced by bz-hyPBase with optimized hyPBase) and ploxP-bz-HyPB( Figure 5 ) to express the transposase. The promoters of optimized hyPBase and bz-hyPBase are connected with human c-myc nuclear localization signal. The transposon with the EGFP gene was cloned into the vector pSAD-EGFP ( Image 6 ) to express green fluorescent protein. The two plasmids expressing transposase and transposon were co-electrotransformed into CHO cells, and the transposon with EGFP would be inserted into the genome under the action of transposase to stably express green fluorescent protein. After two subcultures Afterwards, on the 7th and 14th day, the cells expressing the green fluorescent protein were cou...

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 belongs to the field of molecular biology and biological medicine, and particularly relates to a high-activity transposase and use thereof. The high-activity transposase has an amino acid sequence as shown in SEQ ID NO: 2 and a coding nucleotide sequence of the amino acid sequence is shown as SEQ ID NO:3. When the high-activity transposase based on the amino acid sequence is applied to a transposon system, the gene transfer activity of transposons can be remarkably improved. The high-activity transposase and the nucleotide sequence for coding the high-activity transposase can be used for constructing a gene transfer system, and prepare or used as drugs, preparations or tools for genome research, gene therapy, cell therapy or multifunctional stem cell induction and / or differentiation.

Description

technical field [0001] The invention belongs to the field of molecular biology and biomedicine, and specifically relates to a high-activity transposase and its application. Background technique [0002] A DNA transposon is a mobile DNA sequence that can be transposed from one location in the genome to another through a series of processes such as cleavage and reintegration. PiggyBac (PB) transposon is a DNA transposon isolated from the Trichoplusiani TN368 cell line, which can be specifically inserted into the "TTAA" site, with the help of transposase, PiggyBac transposition The target gene can be precisely cut out from the host without remaking the host chromosome. PB transposons have no potential viral genotoxicity, can carry longer foreign gene fragments (up to 150kb), and have a strong remodeling type. The transgene mediated by PB transposase has the characteristics of high integration efficiency, stable integration, long-term expression, single-copy integration, posit...

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 Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/81C12N15/85C12N1/19C12N5/10C12R1/865
CPCC12N9/1241C12N15/81C12N15/85C12N2800/107C12N2800/22C07K14/435C07K19/00C12N5/10C12N9/10C12N9/12C12N9/22C12N15/62
Inventor 文雯宋姗姗刘韬刘祥箴金华君钱其军
Owner SHANGHAI CELL THERAPY RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap