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Hybridoma for secreting anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as preparation method and application of hybridoma

A monoclonal antibody and hybridoma cell technology, applied in the fields of genetic engineering, monoclonal antibody preparation and immunodiagnosis, can solve the problems of unclear detection targets and insufficient sensitivity

Inactive Publication Date: 2015-05-13
JIANGSU INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have good specificity for the diagnosis of schistosomiasis, but the disadvantage is that the detection target is not clear and the sensitivity is generally insufficient

Method used

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  • Hybridoma for secreting anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as preparation method and application of hybridoma
  • Hybridoma for secreting anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as preparation method and application of hybridoma
  • Hybridoma for secreting anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as preparation method and application of hybridoma

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Schistosoma japonicum Sj Enolase gene cloning:

[0058] The Sj Enolase gene of Schistosoma japonicum is reverse-transcribed and synthesized from the mRNA of Schistosoma japonicum adult worm tissue by RT-PCR technology. The specific preparation method is as follows:

[0059] 1. Preparation of Schistosoma adult worm mRNA

[0060] Take 0.5 g of freshly isolated adults of Schistosoma japonicum, freeze them in liquid nitrogen, crush them in a ceramic mortar, and then use the mRNA purification kit (Illustra QuickPrepTM mRNA purification kit) from GE Healthcare to prepare and purify mRNA. The operation method is strictly according to the kit operating instructions. The purity and content of mRNA were measured by UV spectrophotometer.

[0061] 2. Sj Enolase gene amplification

[0062] 2.1 Primer design:

[0063] Sj Enolase 1: tt ggatcc at gaaaatggca attatagcga t, (the underline is the restriction site of BamH1),

[0064] Sj Enolase 2: tt gtcgac tc aaatttgag...

Embodiment 2

[0072] Embodiment 2: Construction of recombinant Sj Enolase protein expression plasmid

[0073] The present invention adopts pET28a 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 plasmid pET28a and extracting it. After the plasmid was prepared, it was stored at -20°C until use.

[0074] Specifically, the expression vector plasmid pET28a and the recombinant plasmid Sj Enolase / pGEM-T were cut with BamH1 and Sal1 respectively. The specific conditions are as follows. Plasmid (pET28a and Sj Enolase / pGEM-T) DNA 10 μL, BamH1 1 μL, Sal1 1 μL, 10× digestion buffer 5 μL; ddH 2 O 33 μL for a total volume of 50 μL. Incubate in a constant temperature water bath at 37°C for 3 hours, and recover linearized pET28a plasmid DNA and Sj Enolase DNA fragments by low agarose gel electrophoresis. The recovered Sj Enolase DNA fragment was connected with the expression plasmi...

Embodiment 3

[0075] Embodiment 3: Construction of recombinant Sj Enolase protein expression engineering bacteria

[0076] The specific method is as follows.

[0077] First, the bacterial clone containing the Sj Enolase / pET28a recombinant plasmid was selected, and the plasmid DNA was extracted. Then, the plasmid was transformed into Escherichia coli BL21(DE3) by electroporation.

[0078] The plasmid DNA extraction method was carried out according to the operating instructions of the plasmid DNA purification kit from Promega Company.

[0079] The electroporation method for transforming Escherichia coli is as follows.

[0080] Prepare competent cells first. Pick a fresh BL21 (DE3) single colony, inoculate it into a test tube containing 3 mL of LB liquid medium, and culture overnight at 37°C and 200 rpm. Then take 1 mL of the culture and inoculate it into a 2L Erlenmeyer shaker flask containing 500 mL of fresh LB medium, cultivate overnight at 37°C, 250-300 rpm, until OD 600 nm If it is l...

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Abstract

The invention relates to a hybridoma for secreting an anti-recombinant schistosoma japonica enolase specific monoclonal antibody as well as a preparation method and application of the hybridoma, and belongs to the technical fields of gene engineering, preparation of monoclonal antibodies and immunologic diagnosis. The hybridoma JYG-1 capable of secreting the anti-recombinant schistosoma japonica enolase (Sj Enolase protein) specific monoclonal antibody is preserved in the Common Microbiology Center of CCCCM (China Committee for Culture Collection of Microorganisms) and has the preservation number of CGMCC NO. 9910. The hybridoma JYG-1 can secrete the anti-recombinant schistosoma japonica enolase (Sj Enolase) specific monoclonal antibody JYGENmb-1. The monoclonal antibody JYGENmb-1 can be combined with Sj Enolase protein specificity of schistosoma japonica, a sandwich ELISA method for detecting Sj Enolase protein is provided, and the monoclonal antibody can be applied to establishing various immunological methods for detecting Sj Enolase protein in schistosoma japonica infected human and animal blood and excreting secretion.

Description

technical field [0001] The invention provides a hybridoma cell that secretes a specific monoclonal antibody against recombinant Schistosoma japonicum enolase (Sj Enolase protein), an anti-Sj Enolase specific monoclonal antibody and a preparation method thereof; The Enolase-specific monoclonal antibody is a capture antibody, and the anti-Sj Enolase-specific polyclonal antibody is used as a sandwich method to detect Sj Enolase protein. It belongs to the technical fields of genetic engineering, monoclonal antibody preparation and immunodiagnosis. Background technique [0002] Schistosomiasis is an important parasitic disease that seriously threatens human health in tropical and subtropical regions. It has been listed as the second most important public health problem under the government's key control in China. After more than half a century of hard work, the epidemic of schistosomiasis in my country has been effectively controlled, and the infection rate of schistosomiasis i...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573C12N9/88C12N15/70
Inventor 余传信高玒宋丽君沈双殷旭仁王玠柯雪丹
Owner JIANGSU INST OF PARASITIC DISEASES
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