Immunolatex microsphere for detecting CpTI and preparation method thereof

A trypsin inhibition, microsphere technology, applied in chemical instruments and methods, measuring devices, instruments, etc., to achieve the effects of convenient operation, meticulous and rigorous preparation method, and simple material sampling

Inactive Publication Date: 2011-06-29
CHINESE RES ACAD OF ENVIRONMENTAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few research reports and related patented technologies at home and abroad on CpTI inhibitor immunoassay technology, and there is an urgent need to develop efficient, accurate and rapid new immunoassay technology and products

Method used

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  • Immunolatex microsphere for detecting CpTI and preparation method thereof
  • Immunolatex microsphere for detecting CpTI and preparation method thereof
  • Immunolatex microsphere for detecting CpTI and preparation method thereof

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preparation example Construction

[0032] The preparation method of the immune latex microspheres of detecting cowpea trypsin inhibitor described in the embodiment of the present invention, comprises the following steps:

[0033] 1) Design the full-length CpTI gene sequence, re-modify according to the preferential codons expressed by prokaryotic cells, and use the whole gene synthesis technology to construct the full-length CpTI gene expression sequence;

[0034] 2) Insert the CpTI gene fragment obtained in step 1) into the glutathione-S-transferase (GST) gene fusion expression vector by cutting with restriction enzymes to construct a bacterial fusion expression vector;

[0035] 3) After bacterial expression of the expression vector in step 2), the GST-CpTI fusion protein is obtained by purification with an affinity chromatography column;

[0036] 4) The GST-CpTI fusion protein antigen obtained in step 3) was dissolved in Freund's adjuvant to immunize mice, and the mouse anti-CpTI polyclonal antibody was obtain...

example

[0042] When the present invention is concretely implemented, comprise the following steps:

[0043] 1) Construct the full-length CpTI gene expression sequence:

[0044] (a) Design the full-length CpTI gene sequence, the result is as follows figure 2 shown;

[0045] (b) According to the re-modification of the preferential codons expressed by prokaryotic cells, the full-length CpTI gene was constructed using the whole gene synthesis technology, with a total of 333 base pairs, and cloned into the vector pGEM-T Easy. The results are as follows image 3 shown;

[0046] (c) Verify the correctness of the insertion of the CpTI gene fragment by colony PCR, the results are as follows Figure 4 shown;

[0047] (d) The cloned CpTI gene bacteria were amplified and cultured, and the plasmid was extracted and subjected to enzyme digestion and agarose electrophoresis analysis. The results are as follows: Figure 5 shown;

[0048] (e) DNA sequencing to ensure that the entire sequence of t...

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Abstract

The invention relates to an immunolatex microsphere for detecting a cowpea trypsin inhibitor (CpTI) and a preparation method thereof. The immunolatex microsphere is coated with a CpTI-resistant protein polyclonal antibody and has the particle size of 200nm. The preparation method of the immunolatex microsphere comprises the following steps of: designing a full-length CpTI gene sequence; inserting a CpTI gene fragment into a glutathione-S-transferase (GST) gene fusion expression vector through restriction enzyme digestion to construct a bacteria fusion expression vector; purifying by using an affinity chromatographic column to obtain GST-CpTI fusion protein; fusing a GST-CpTI fusion protein antigen into a Freund adjuvant to immunize a mouse, and separating and purifying to obtain an anti-mouse CpTI polyclonal antibody; and coupling the antibody with the activated microsphere by a chemical coupling method to obtain the immunolatex microsphere for detecting the CpTI protein. The invention has the advantages that: the preparation method is high in sensitivity and repeatability and easy to operate; and the prepared immunolatex microsphere has small and uniform particle size, the lowest detectable limit of 0.2mu g/ml of the CpTI fusion protein, and the related coefficient R2 of 0.67.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, in particular to an immune latex microsphere for detecting cowpea trypsin inhibitor and a preparation method thereof. Background technique [0003] Protease inhibitors are widely present in plants, especially in the storage organs of plants. The biological functions of protease inhibitors in plants mainly include two aspects: ① regulate the relevant physiological and biochemical processes in tissue cells by interacting with endogenous proteases; ② prevent the protein components in cells and tissues from being degraded by exogenous proteases . Its more important role is as a chemical defense factor against the attack of external herbivorous insects and microorganisms. Studies have confirmed that most plant-derived protease inhibitors have no inhibitory effect on plant endogenous proteases, but have specific inhibitory activity on exogenous proteases, which provides a natural evolutionary ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/531C07K17/08
Inventor 肖能文潘卫东王学霞李俊生
Owner CHINESE RES ACAD OF ENVIRONMENTAL SCI
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