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Method for qualitatively and quantitatively determining protein and application

A quantitative determination and protein technology, applied in the field of protein detection, can solve the problems of poor detection accuracy and specificity, affecting detection accuracy, etc.

Pending Publication Date: 2021-07-09
广州辉骏生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the above-mentioned method also has the following disadvantages: for example, the Coomassie brilliant blue method has more restrictions on the protein molecular size in the test sample; the ultraviolet absorption method is based on the light absorption value of the aromatic amino acid residues in the protein at 280nm. The content of aromatic amino acids in different proteins is different, and the detection accuracy and specificity of this method are not good; at the same time, for the traditional biuret method, BCA method and Lowry method, it is not suitable for those containing poorly soluble Determination of protein content in grain samples of gliadin and gluten, as well as most animal and plant industrial derivatives
Moreover, the components such as starch, lipid and fiber in the sample system to be tested will also affect the detection accuracy of the above laboratory methods

Method used

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  • Method for qualitatively and quantitatively determining protein and application
  • Method for qualitatively and quantitatively determining protein and application
  • Method for qualitatively and quantitatively determining protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Using luciferase NanoLuc@ as a reporter enzyme, construct vectors containing Protein G-LgBit gene and ProteinL-SmBit gene respectively, purify and obtain the above two fusion proteins, detect the luminescence value of the analyte with known concentration, and use the analyte The concentration of the substance is the abscissa, and the luminescence value is the ordinate to draw a standard curve.

[0034] 1. Construct the ProteinG-LgBit and ProteinL-Smbit genes into the pET-28a(+) prokaryotic expression vector, and perform sequencing verification

[0035] (1) Artificially synthesize the ProteinG-LgBit sequence (SEQ ID NO.9), and connect it into the HindIII and NotI restriction sites of the pET28a(+) vector, transform Stbl3 competent cells, screen positive colonies for sequencing, and the sequence is correct Plasmids were extracted from colonies and verified by enzyme digestion.

[0036] (2) Artificially synthesize the ProteinL-Smbit sequence (SEQ ID NO.10), and connect it ...

Embodiment 2

[0058] Using split horseradish peroxidase as a reporter enzyme, construct vectors containing ProteinG-HRP-L gene and ProteinL-HRP-R gene respectively, purify and obtain the above two fusion proteins, and detect the luminescence value of the analyte with known concentration , and draw a standard curve with the concentration of the analyte as the abscissa and the fluorescence value as the ordinate.

[0059] 1. Construct ProteinG-HRP-L and ProteinL-HRP-R genes into pET-28a(+) prokaryotic expression vector, and perform sequencing verification

[0060] (1) Artificially synthesize the gene sequence of ProteinG-HRP-L as shown in SEQ ID NO.11, connect it into pET28a(+) between HindIII and XhoI restriction sites, transform the ligation product into Stbl3 competent cells, and screen positive colonies Sequencing was carried out, and plasmids were extracted from colonies with correct sequencing, and verified by enzyme digestion.

[0061] (2) Synthesize the gene sequence of ProteinL-HRP-R...

Embodiment 3

[0070] Using the split peroxidase APEX2 as the reporter enzyme, construct vectors containing the ProteinG-AP gene and the ProteinL-EX2 gene respectively, purify the above two fusion proteins, detect the luminescence value of the analyte with a known concentration, and use the analyte The concentration of the substance is the abscissa, and the luminescence value is the ordinate to draw a standard curve.

[0071] 1. Construct the ProteinG-AP (SEQ ID NO.13) and ProteinL-EX2 (SEQ ID NO.14) genes into the pET-28a(+) prokaryotic expression vector, and insert the correct sequences into the pET28a(+) vector respectively Between the HindIII / XhoI and NdeI / XhoI restriction sites, the ligation product was transformed into Stbl3 competent cells, the positive colonies were screened for sequencing, and the plasmids were extracted from the correctly sequenced colonies.

[0072] 2. Expression and purification of two fusion proteins

[0073] The extraction and purification of the fusion protei...

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Abstract

The invention provides a method for qualitatively and quantitatively determining protein and application, and relates to the technical field of protein detection. The method comprises the following steps that proteins are detected based on a ternary complex system, two proteins which are respectively and specifically combined with different epitopes in a solution to be detected and do not interact with each other are sequentially combined with different fragments of a reporter enzyme to form a fusion gene, and expressing and purifying to obtain two fusion proteins, protein fragments of reporter enzymes contained in the two fusion proteins can be recombined when approaching each other, when a reporter enzyme substrate is added, an optical signal can be released, and meanwhile, the luminous degree of a reference standard substance (or a reference material) of the detected protein is positively correlated with the concentration of the detected protein, and the high-expression cell strain can be screened in a high-throughput manner through detection data of light-emitting reading.

Description

technical field [0001] The invention belongs to the technical field of protein detection, and in particular relates to a method and application for qualitatively and quantitatively measuring protein. Background technique [0002] At present, the commonly used protein detection methods generally use radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) to quantitatively and qualitatively analyze the protein contained in the sample, but expensive detection equipment is required, and the operation process is complicated. Therefore, detection and analysis cannot be performed anytime and anywhere. [0003] In addition to the above detection methods, biochemical detection methods such as biuret method, BCA method, Coomassie brilliant blue method, Lowry method and ultraviolet absorption method are often used in biochemical laboratories to determine protein content. But above-mentioned method also has the following shortcoming: for example, Coomassie brilliant blue me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/569G01N33/58G01N21/64G01J1/00
CPCG01N33/535G01N33/581G01N33/569G01J1/00G01N21/6486
Inventor 刘郁林包煜贤屈义
Owner 广州辉骏生物科技股份有限公司