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

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

Active Publication Date: 2014-09-17
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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  • Schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody and preparation method thereof
  • Schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody and preparation method thereof
  • 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 Sj TGR 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 Sj TGR 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 Week, a total of 4 booster immunizations. 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 solution (10 μg / mL) of recombinant Sj TGR protein, overnight at 4°C, block with 5% skimmed milk powder PBS the next day, 37°C for 2h, and then mix with doubly diluted immune camel serum Then r...

Embodiment 2

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

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

[0077] Trizol was used to extract the total RNA of Sj TGR immunized camel peripheral blood leukocytes, and the method was as follows:

[0078] 1) Take about 1×10 7 One Sj TGR was used to immunize the peripheral ...

Embodiment 3

[0152] Example 3: Anti Sj Construction of phage display library of TGR single region antibody

[0153] The linearized phagemid pCANTab5E DNA and the DNA fragment encoding the nanobody were prepared by Sfi1 and Not1 double digestion method. The enzyme digestion reaction system is as follows:

[0154] pCANTab5E DNA fragment 30 μL

[0155] 10×Buffer4 5 μL

[0156] 100×BSA 0.5 μL

[0157] Not1-HF 1 μL

[0158] wxya 2 O 2.5 μL

[0159] 49 μL total

[0160] After mixing and centrifuging, place in a 37°C water bath to react for 4 h. Add 1 μL of Sfi1, mix well and centrifuge, then place in a water bath at 50 °C for 3 h.

[0161] Recovery of digested products: The digested products were subjected to 1% agarose gel electrophoresis, and after electrophoresis at 100 V for 60 min, the status of the digested products was observed. The gel block containing the target fragment was excised from the agaro...

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Abstract

The invention relates to a schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody and a preparation method thereof, and belongs to the technical field of molecular biology, immunology and pharmacy of a pharmaceutical technology. The invention provides a recombinant phage display library for displaying an Sj TGR protein single-domain antibody, the Sj TGR protein resistant single-domain antibody and a preparation method thereof. The Sj TGR resistant single-domain antibody disclosed by the invention can be specifically combined with an Sj TGR protein and / or has Sj TGR inhibition activity and can be used for preparing a new drug for curing schistosomiasis or a targeting vector of the drug for cure. The schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody disclosed by the invention lays a good foundation for developing the new drug for curing the schistosomiasis and a new curing method and has important significance on controlling schistosomiasis prevalence.

Description

technical field [0001] The invention relates to the construction of a recombinant phage display library capable of displaying a single-region antibody against Sj TGR protein by using immunological technology and phage display technology, and screening recombinant phage capable of displaying and combining with Sj TGR protein, and preparing and purifying the recombinant phage by using genetic engineering technology The single-region antibody against Sj TGR protein can bind to Sj TGR protein and / or inhibit the enzyme activity of Sj TGR, which can destroy the redox balance in the physiological metabolic process of Schistosoma japonicum, resulting in the death of Schistosoma japonicum. The antibody can also be used as a targeting carrier for chemical inhibitors of Sj TGR 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 pharmac...

Claims

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Application Information

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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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