Glutamine optical probe and preparation method and application thereof

An optical probe, glutamine technology, applied in the field of optical probes, can solve in situ, real-time, dynamic, high-throughput and high spatio-temporal resolution detection of non-viable cells and subcellular organelles, not suitable for living cell research, Separation, extraction, purification and other issues, to achieve the effect of eliminating sample processing steps, large dynamic changes in fluorescence, and easy maturation

Active Publication Date: 2019-04-23
EAST CHINA UNIV OF SCI & TECH
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, these detection methods in the prior art are not suitable for living cell research, and there are many defects: time-consuming sample processing proce...

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
  • Glutamine optical probe and preparation method and application thereof
  • Glutamine optical probe and preparation method and application thereof
  • Glutamine optical probe and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0072] The present invention also provides a preparation method for the above-mentioned glutamine optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the glutamine optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell 2) cultivating the host cell under conditions suitable for the expression of the expression vector, 3) isolating the glutamine optical probe.

[0073] The term "nucleic acid" or "nucleotide" as used in the present invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The term "variant" as used herein in reference to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion varia...

Embodiment 1

[0158] Example 1: Plasmids expressing glutamine-binding proteins

[0159] The glutamine-binding protein QBP (GlnH) gene in the Agrobacterium agrobacterium gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and EcoRI, and the pRSETb vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was used to transform MachI, and the transformed MachI was spread on LB plates (ampicillin 100ug / mL), and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

Embodiment 2

[0160] Example 2: Expression and detection of cpYFP optical probes at different fusion sites

[0161] In this example, the following sites were selected for fusion with cpYFP based on pRSETb-QBP to obtain the corresponding pRSETb-QBP-cpYFP plasmid: 177 / 178, 177 / 179, 177 / 180, 178 / 179, 178 / 180 or 179 / 180.

[0162] The DNA fragment of cpYFP was generated by PCR, and the DNA fragment was inactivated after adding phosphorous to the 5' end, and the pRSETb-glutamine binding protein linearized vector containing different break sites was amplified by reverse PCR at the same time. The linearized pRSETb-QBP and the phosphorylated cpYFP fragment at the 5' end were ligated under the action of PEG4000 and T4 DNA ligase to produce recombinant plasmids. These plates were picked on the Kodak multifunctional in vivo imaging system and picked up under the excitation of the FITC channel. Yellow The fluorescent clones were sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd. Shanghai Branc...

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 glutamine optical probe and a preparation method and application thereof. On one hand, the optical probe comprises glutamine sensitive polypeptide or a functional variant thereof and optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located in the sequence of the glutamine sensitivepolypeptide or functional variant thereof. The invention also relates to the preparation method of the probe and the application of the probe in the detection of amino acids. The glutamine optical probe has relatively smaller molecular weight and is easy to mature, has large fluorescence dynamic change and good specificity and can be expressed in different subcellular organelles of cells, and theamino acids can be quantitatively detected inside and outside the cells with high flux.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to a glutamine optical probe and its preparation method and application. Background technique [0002] Amino acid is a compound in which the hydrogen atom on the carbon atom of a carboxylic acid is replaced by an amino group, and is an organic compound containing a basic amino group and an acidic carboxyl group. The amino acids obtained after protein hydrolysis are all α-amino acids with amino groups connected to α-carbons, and there are only more than 20 kinds of them. They are the basic units of protein. Amino acids play a role in the human body through metabolism, such as synthesizing tissue proteins; synthesizing ammonia-containing substances such as acids, hormones, antibodies, and creatine; converting them into carbohydrates and fats; or oxidizing them into carbon dioxide, water, and urea to generate energy. [0003] Glutamine is one of the 20 essential amino acids. Gl...

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
IPC IPC(8): C07K19/00C12N15/62G01N21/64
CPCG01N21/6428C07K14/195C07K2319/60G01N2021/6439
Inventor 杨弋赵玉政李写顾燕芳胡晗阳陈念
Owner EAST CHINA UNIV OF SCI & TECH
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