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Anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and application thereof in immune diagnosis

A monoclonal antibody, schistosomiasis technology, applied in the direction of anti-animal/human immunoglobulin, application, introduction of foreign genetic material using vectors, etc., can solve the problems of difficult to meet on-site application, insufficient sensitivity of ELISA, and specificity of test results and other problems, to achieve a good application prospect, improve the accuracy and the effect of positive rate

Inactive Publication Date: 2011-09-14
JIANGSU INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a common disadvantage of this type of method is that the detected circulating antigen target is not clear, and the specificity of the detection results is problematic. In addition, the sensitivity of conventional ELISA is insufficient, and it is difficult to meet the needs of field application

Method used

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  • Anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and application thereof in immune diagnosis
  • Anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and application thereof in immune diagnosis
  • Anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and application thereof in immune diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1: Schistosoma japonicum signal transduction protein Sj14-3-3 gene cloning:

[0078] The signal transduction protein Sj14-3-3 gene of Schistosoma japonicum is synthesized by reverse transcription from the mRNA of Schistosoma japonicum adult worm tissue by RT-PCR technology, and the specific preparation method is as follows:

[0079] 1. Preparation of Schistosoma adult worm mRNA

[0080] Take 0.5 g of freshly isolated adults of Schistosoma japonicum, freeze in liquid nitrogen, pulverize in a ceramic mortar, and then use the mRNA purification kit (Illustra QuickPrep TM mRNA purification kit) was used to prepare and purify mRNA, and the operation method was strictly in accordance with the operating instructions of the kit. The purity and content of mRNA were measured by UV spectrophotometer.

[0081] 2. Sj14-3-3 gene amplification

[0082] 2.1 Primer design:

[0083] Sj14-3-3 1: tt ggatcc atgagggattcgttcgt, (the underline is Bam H 1 restriction site),

...

Embodiment 2

[0092] Embodiment 2: Construction of recombinant Sj14-3-3 protein expression plasmid

[0093] The present invention adopts pGEX-4T-3 as the expression carrier plasmid, and its preparation method is a common molecular biology method. That is, the plasmid is obtained by culturing a strain containing the expression vector and extracting it. After the plasmid was prepared, it was stored at -20°C until use.

[0094] Specifically, the expression vector plasmid pGEX-4T-3 and the recombinant plasmid Sj14-3-3 / pGEM-T were respectively Bam H 1, Sal 1 double digestion. The specific conditions are as follows. Plasmid (pGEX-4T-3 or Sj14-3-3 / pGEM-T) DNA 10 μL, Nco 1 1 μL, xho 1 1 μL, 5 μL of 10× Digestion Buffer; ddH 2 O 33 μL for a total volume of 50 μL. Warm in a constant temperature water bath at 37°C for 3 hours, and recover the linearized pGEX-4T-3 plasmid DNA and Sj14-3-3 DNA fragments by low agarose gel electrophoresis. The recovered Sj14-3-3 DNA fragment was connected wi...

Embodiment 3

[0095] Example 3: Construction of recombinant Sj14-3-3 protein expression engineering bacteria

[0096] The specific method is as follows.

[0097] Firstly, the bacterial clone containing the Sj14-3-3 / pGEX-4T-3 recombinant plasmid was selected, and the plasmid DNA was extracted. Then, the plasmid was transformed into Escherichia coli BL21(DE3) by electroporation.

[0098] The plasmid DNA extraction method was carried out according to the operating instructions of the plasmid DNA purification kit from Promega Company.

[0099] The electroporation method for transforming Escherichia coli is as follows.

[0100] Prepare competent cells first. Pick a fresh single colony of BL21 (DE3), inoculate it into a test tube containing 3 mL of LB liquid medium, and culture overnight at 37°C and 200 r / min. Then take 1 mL of the culture and inoculate it into a 2L Erlenmeyer shaker flask containing 500 mL of fresh LB medium, culture overnight at 37°C, 250-300 r / min, until OD 600 If it is l...

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Abstract

The invention discloses an anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and application thereof in immune diagnosis, and belongs to the technical field of immune diagnosis of parasitic diseases. The monoclonal antibody against recombinant Sj14-3-3 protein is generated by hybridoma JYQ5C6-1 which has the collection number of CCTCCC201118. The monoclonal antibody against recombinant Sj14-3-3 protein is applied to the detection of an antibody used by schistosoma infected patients or circulating antigen Sj14-3-3 protein in animal blood in schistosoma infected immune diagnosis. The prepared anti-Sj14-3-3 protein molecular specific monoclonal antibody can be used for establishing various immunological diagnosis methods for detecting anti-Sj14-3-3 protein circulating antigens. The time fluorescence resolution method for detecting circulating antigen Sj14-3-3 protein, established by using the anti-Sj14-3-3 protein specific monoclonal antibody, can further improve the accuracy and the positive rate of detecting the schistosoma circulating antigen Sj14-3-3 protein, and has good application prospect.

Description

technical field [0001] The invention relates to an anti-schistosoma japonicum Sj14-3-3 protein monoclonal antibody and its application in immunodiagnosis, belonging to the technical field of parasitic disease immunodiagnosis. Background technique [0002] In tropical and subtropical regions of the world, schistosomiasis is prevalent in 76 countries, nearly 600 million people are threatened by schistosomiasis infection, and more than 20 million people die from schistosomiasis every year. Although great achievements have been made in the prevention and control of schistosomiasis in China, schistosomiasis is still prevalent in 454 counties across the country, with nearly 420,000 patients (data from the national epidemiology survey in 2008). Health is an important public health issue. Establishing an effective diagnostic method for schistosomiasis, accurate diagnosis of schistosomiasis patients, livestock and intermediate hosts, and timely treatment are of great significance fo...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K14/435C12N15/12C12N15/70G01N33/577
Inventor 余传信钱春艳殷旭仁王玠宋丽君张伟梁幼生高琪
Owner JIANGSU INST OF PARASITIC DISEASES
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