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Anti-Schistosoma japonicum thioredoxin glutathione reductase sj TGR single-domain antibody and preparation method thereof

A technology of schistosome thioredoxin and single-region antibody, which is applied in the direction of anti-enzyme immunoglobulin, antibody, peptide library, etc., and can solve the problems that anti-SjTGR single-region antibody has not been reported.

Active Publication Date: 2016-03-23
JIANGSU INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no report on the research on anti-SjTGR single domain antibody at home and abroad

Method used

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  • Anti-Schistosoma japonicum thioredoxin glutathione reductase sj TGR single-domain antibody and preparation method thereof
  • Anti-Schistosoma japonicum thioredoxin glutathione reductase sj TGR single-domain antibody and preparation method thereof
  • Anti-Schistosoma japonicum thioredoxin glutathione reductase sj TGR single-domain antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Recombinant SjTGR protein immunization of Xinjiang Bactrian camels and preparation of serum antibodies

[0067] Before immunization, blood was collected from camel jugular vein, serum was separated, and stored at low temperature for later use. For the first immunization, the recombinant purified SjTGR protein (500 μg) complete Freund's adjuvant antigen was injected subcutaneously into the neck skin of Bactrian camels in Xinjiang, followed by booster immunization with the same dose of incomplete adjuvant antigen, and the immunization interval was 2 weeks. A total of 4 booster immunizations were given. Two weeks after the first immunization, 50 mL of blood was collected from the jugular vein of the camel, and the serum was separated. Coat the ELISA plate with carbonic acid buffer (10 μg / mL) of the recombinant SjTGR protein, overnight at 4°C, block with 5% skimmed milk powder PBS the next day, 37°C for 2 hours, and then react with the doubly diluted immune came...

Embodiment 2

[0075] Embodiment 2: Preparation of gene fragments encoding camel anti-SjTGR nanobody

[0076] SjTGR immunized camel anticoagulant blood was centrifuged at 1000rpm for 10min to separate plasma and blood cells. Use percoll (GEHealth product) to prepare camel white blood cell separation liquid with a specific gravity of 1.07, add anticoagulant blood cells to the upper layer of white blood cell separation liquid, centrifuge at 4°C for 30 min at a speed of 3000g, and collect the white cell layer at the interface in a new sterile Add sterile PBS to the centrifuge tube to suspend and wash the cells, centrifuge at 1000rpm for 10min, remove the supernatant, then add PBS, wash the cells 3 times repeatedly, collect the cell pellet, and count.

[0077] Trizol was used to extract the total RNA of SjTGR immune camel peripheral blood leukocytes, the method is as follows:

[0078] 1) Take about 1×10 7 One SjTGR was used to immunize the peripheral blood leukocytes of Bactrian camels in Xinj...

Embodiment 3

[0152] Embodiment 3: anti Sj Construction of phage display library of TGR single region antibody

[0153] The linearized phagemid pCANTab5EDNA and the DNA fragment encoding the nanobody were prepared by the Sfi1 and Not1 double enzyme digestion method. The enzyme digestion reaction system is as follows:

[0154] pCANTab5EDNA fragment 30 μL

[0155] 10×Buffer45μL

[0156] 100×BSA0.5μL

[0157] Not1-HF 1 μL

[0158] wxya 2 O2.5μL

[0159] 49 μL total

[0160] After mixing and centrifuging, put it in a water bath at 37°C for 4 hours. Add 11 μL of Sfi, mix well and centrifuge, then place in a 50°C water bath for 3 hours.

[0161] Recovery of enzyme-digested products: Perform 1% agarose gel electrophoresis on the above-mentioned enzyme-cleaved products, and observe the status of the enzyme-cleaved products after electrophoresis at 100V for 60 minutes. The gel block containing the target fragment was excised from the agarose gel, and the double-enzyme-digested linearized p...

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Abstract

Anti-Schistosoma japonicum thioredoxin glutathione reductase Sj ? The invention relates to a TGR single-region antibody and a preparation method thereof, which belong to the technical fields of molecular biology, immunology and pharmacy of biotechnology. The present invention provides an anti- Sj ? Recombinant phage display library of single-region antibody of TGR protein, anti- Sj ? Single region antibody of TGR protein and preparation method thereof, described anti Sj ? TGR single domain antibody can be combined with Sj ? TGR protein specifically binds, and / or has inhibitory Sj The TGR enzyme activity can be used to prepare new drugs for treating schistosomiasis or targeted carriers of therapeutic drugs. The invention lays a good foundation for the development of new schistosomiasis therapeutic drugs and new treatment methods, and has important significance for controlling the prevalence of schistosomiasis.

Description

technical field [0001] The invention relates to constructing a recombinant phage display library capable of displaying single-region antibodies against SjTGR protein by adopting immunological technology and phage display technology, and screening recombinant phage capable of displaying and combining with SjTGR protein, and preparing and purifying recombinant anti-SjTGR by using genetic engineering technology The single-region antibody of the protein can bind to the SjTGR protein and / or inhibit the enzyme activity of the SjTGR, which can destroy the redox balance in the physiological metabolic process of the schistosomiasis, resulting in the death of the schistosomiasis. The antibody can also be used as a targeting carrier for chemical inhibitors of SjTGR enzyme activity, is suitable for the development and research of new schistosomiasis therapeutic drugs and targeted treatment methods, and belongs to the technical fields of molecular biology, immunology and pharmacy of biotech...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/10C07K16/40A61K39/395A61P33/12G01N33/573
CPCY02A50/30
Inventor 宋丽君姚媛余传信殷旭仁沈双高玒
Owner JIANGSU INST OF PARASITIC DISEASES
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