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Kit and method for detecting mu-conotoxin (CTX) by using single-chain antibody of gene engineering

A single-chain antibody, conotoxin technology, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of low sensitivity, high variability, high cost, etc., and achieves low cost, simple production process, and convenient and fast use. Effect

Inactive Publication Date: 2011-06-15
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

Commonly used detection methods include high-performance liquid chromatography / quadrupole-time-of-flight mass spectrometry (HPLC / Q-TQFMS) coupled technology, but it is expensive, time-consuming, high variability, low sensitivity, limited by the amount of samples to be tested, Unable to provide definitive evidence for specific toxins, a simple and effective method for the detection of μ-CTX is lacking at this stage

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  • Kit and method for detecting mu-conotoxin (CTX) by using single-chain antibody of gene engineering
  • Kit and method for detecting mu-conotoxin (CTX) by using single-chain antibody of gene engineering
  • Kit and method for detecting mu-conotoxin (CTX) by using single-chain antibody of gene engineering

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Embodiment 1

[0038] A kit for detecting μ-CTX using a genetically engineered single-chain antibody was made according to the following reagent composition:

[0039] (1) Sample extraction solution: deionized water;

[0040] (2) Standard reagents: TRX-CTX toxin samples containing 0.01, 0.1, 1.0, and 10 ng / mL respectively; the standard reagent preparation method is as follows:

[0041] A Construction of recombinant plasmid pET32a-CTX

[0042] double-stranded CTX gene inserted into the same Bam H I and EcoR In the double-digested pET32a vector, connect overnight with T4 DNA ligase, pET32a- CTX The recombinant plasmid was identified by sequencing.

[0043] Induced expression of B TRX-CTX fusion protein

[0044] Using CaCl 2 Competent state BL21 . pET32a- CTX Plasmid transformation into E. coli BL21 middle. The clones that were successfully transformed were picked and inoculated into LB medium containing ampicillin to BL21 Empty bacteria were negative controls, and pET32a-con...

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Abstract

The invention provides a kit and a method for detecting mu-conotoxin (CTX) by using a single-chain antibody of gene engineering. Reagents in the kit comprise sample extracting solution, a standard reagent, an enzyme-labeled antigen reagent, cleaning solution, a bovine serum albumin (BSA) reagent, a color development substrate and stop solution. The method comprises the following steps of: processing samples; balancing the reagents; washing; adding the samples; incubating; washing; developing; stopping; and calculating the content of the mu-CTX in the samples. By the kit and the method, the single-chain antibody of the gene engineering for resisting mu-CTX micromolecules can be effectively expressed in escherichia coli; and the kit can be produced on a large scale, has an extremely simple whole production process, saves time, labor and money, can be conveniently and fast used and has low cost.

Description

technical field [0001] The present invention relates to a detection kit for detecting marine toxins by using a genetically engineered single-chain antibody, more particularly to a kit for detecting μ-conotoxin (μ-Conotoxin, μ-CTX) by using a genetically engineered single-chain antibody, further It relates to a method for detecting μ-CTX using the kit. Background technique [0002] Conotoxin (CTX) is a small peptide toxin secreted by Conus, a carnivorous mollusc living in tropical oceans, used to anesthetize prey. Similar to tetrodotoxin, μ-CTX is a typical sodium ion channel inhibitor. The poisoned person will experience symptoms such as limb numbness and paralysis, and severe cases may lead to death. Commonly used detection methods include high-performance liquid chromatography / quadrupole-time-of-flight mass spectrometry (HPLC / Q-TQFMS) coupled technology, but it is expensive, time-consuming, high variability, low sensitivity, limited by the amount of samples to be tested, ...

Claims

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Application Information

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IPC IPC(8): G01N33/543
Inventor 余晟兴庄振宏汪世华李恒
Owner FUJIAN AGRI & FORESTRY UNIV
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