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MBP recombinant protein and application thereof

A technology of recombinant protein and recombinant bacteria, applied in MBP recombinant protein and its application field, can solve the problems of high false positive rate of ELISA detection method, long time-consuming detection process, high cost, etc., to improve effectiveness and sensitivity, improve sensitivity and detection speed, and stability-enhancing effects

Pending Publication Date: 2021-02-12
SIMCERE DIAGNOSTICS CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex components of the blood sample, some components have non-specific binding with the ELISA plate, resulting in a high false positive rate of the ELISA detection method; the membrane strip method can only detect antibodies that recognize linear regions, resulting in a low detection rate of the membrane strip method
Therefore, currently commonly used detection methods, such as immunofluorescence, ELISA, western blotting, immunohistochemistry, etc., have the disadvantages of time-consuming detection process, cumbersome steps, large demand for antibodies, high cost, and low sensitivity. It is difficult to test a large number of samples at the same time or repeatedly test a certain sample
[0007] In summary, multiple sclerosis detection kits are currently scarce on the market, and conventional detection methods are limited

Method used

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  • MBP recombinant protein and application thereof
  • MBP recombinant protein and application thereof
  • MBP recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 MBP recombinant protein and plasmid construction

[0043] A signal peptide sequence and a transmembrane domain sequence are added to the MBP gene sequence to construct a MBP recombinant protein, wherein the amino acid sequence of the MBP recombinant protein is shown in SEQ ID No.1, and the nucleic acid sequence is shown in SEQ ID No.2. After the fusion protein was expressed, it was localized on the cell membrane, and the protein structure was proved to be normal by immunofluorescence experiments, and the structure of the chimeric gene was as follows: figure 1 shown.

[0044]

[0045]

[0046]

[0047]

Embodiment 2

[0048] Embodiment 2 Construction of the AD293 cell carrying the recombinant plasmid

[0049] 1) Construction of MBP recombinant plasmid: pcDNA3.1 plasmid was selected to construct the eukaryotic expression vector of enhanced green fluorescent protein (monomericEnhanced green fluorescent protein, mEGFP), and mEGFP was used to mark the complete open reading frame (Open Reading frame) of MBP recombinant protein. ORF), and at the same time allow the target protein to be fully expressed in AD293 cells as an antigen for subsequent detection and research.

[0050] 2) Cultivation of AD293 cells: AD293 cells were cultured by conventional experimental methods.

[0051] 3) Plasmid transfection: Firstly, according to the instructions of lipofectamine 3000 transfection reagent, the optimal concentration ratio of DNA to lipofectamine 3000 was explored to obtain the best transfection effect. The present invention explores the concentration through a 24-well cell culture plate, and finally f...

Embodiment 3

[0052] Embodiment 3 cell immunofluorescence method detects MBP antibody

[0053] Through repeated system optimization, the present invention finally establishes the following method system:

[0054] 1. Culture: Place the polylysine-treated cell slides in a 24-well cell culture plate, and then add AD293 cells carrying the recombinant plasmid for culture.

[0055] 2. Fixation: when the density of AD293 cells carrying the recombinant plasmid is 75%, fix with 2% paraformaldehyde fixative solution at room temperature for 10 min.

[0056] 3. Blocking: After the cells are fixed, block with a blocking solution at room temperature for 1 hour; wherein, the blocking solution is a PBS solution containing 5% goat serum albumin by mass fraction.

[0057] 4. Primary antibody incubation: After blocking, add the serum to be tested (1:20) diluted by the blocking solution doubling dilution method, and then incubate overnight at 4°C.

[0058] 5. Fluorescent secondary antibody incubation: After ...

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Abstract

The invention provides a MBP recombinant protein and an application thereof. Through antigen structure optimization and cell sublocalization transformation, the stability of the MBP recombinant protein in a CBA kit product is improved, sensitivity and a detection rate to MBP antibody detection are increased, and the specificity reaches 100%.

Description

technical field [0001] This application relates to the field of biotechnology, in particular to a MBP recombinant protein and its application. Background technique [0002] Multiple sclerosis (Multiple sclerosis, MS) is an acquired demyelinating disease of the human central nervous system (Central Nervous System, CNS). Genetic susceptibility or resistance to the disease is thought to be associated with genes located in or near the HLA-DR-DQ region on the short arm of the sixth chromosome. The autoimmune mechanism plays an important role in the process of MS-related demyelinating disease, which belongs to the inflammatory demyelinating disease in which specific antigen-mediated cellular immunity and humoral immunity participate together. At present, there is no evidence to explain which antigen is the most important in the etiology of MS, but there is strong evidence that myelin basic protein (Myelin Basic Protein, MBP) is more important. Due to the abnormal immune function ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
CPCG01N33/6857G01N21/6486
Inventor 张洋张超王春霞李诗濛任用
Owner SIMCERE DIAGNOSTICS CO LTD