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Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof

A nanobody, sequence technology, applied in the field of biotechnology or biomedicine

Active Publication Date: 2015-05-27
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Belgian scientist Hamers.R first discovered in camel blood that ordinary antibody proteins consist of two heavy chains and two light chains, while the new antibodies found in camel blood have only two heavy chains and no light chains. Antibodies can tightly bind to antigens like normal antibodies, but they don't stick to each other like single-chain antibodies and aggregate into clumps

Method used

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  • Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof
  • Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof
  • Nano-antibody aiming at SEA (Soluble Egg Antigen), and coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] This embodiment constructs the Schistosoma japonicum SEA-specific nanobody library, the steps are as follows:

[0021] (1) First, purify Schistosoma japonicum SEA, then mix 1 mg of SEA antigen with Freund's adjuvant in equal volume, and immunize an alpaca (Alpa-Vet, www.alpa-vet.be) once a week, 7 times in total , to stimulate B cells to express antigen-specific nanobodies;

[0022] (2) After the 7 times of immunization, extract 100ml alpaca peripheral blood lymphocytes and extract total RNA;

[0023] (3) Synthesize cDNA and amplify VHH by nested PCR;

[0024] (4) Digest 20ug of phage display vector and 10ul of VHH with restriction enzymes pstI and NotI and connect the two fragments;

[0025] (5) The ligation product was transformed into electroporation-competent cells TG1, and the SEA nanobody library was constructed and the storage capacity was determined. The storage capacity was 8.9×10 9 .

Embodiment 2

[0027] This embodiment screens SEA-specific Nanobodies, the steps are as follows:

[0028] (1) Dissolve in 100mM NaHCO 3 , 20ug SEA of pH 8.2 was coated on the NUNC microtiter plate, and placed overnight at 4°C;

[0029] (2) Add 100ul 3% milk the next day, and block at room temperature for 2 hours;

[0030] (3) After 2 hours, add 100ul 2×10 11 tfu contains the helper phage of the SEA nanobody library, and acts at room temperature for 1 hour;

[0031] (4) Wash 10 times with 0.05% PBS+Tween-20 for the first round of panning / 20-25 times for the second round / 20 times for the third round to remove non-specifically bound phages;

[0032] (5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to SEA, and infect Escherichia coli TG1 in the logarithmic growth phase, culture at 37°C for 1 hour, produce and purify the phage for the next round of screening, the same The screening process was repeated for 3-4 rounds, and the enrichment was obtained step by st...

Embodiment 3

[0034] In this example, phage enzyme-linked immunosorbent assay (ELISA) was used to screen specific single positive clones, and the steps were as follows:

[0035] (1) From the cell culture dish containing phage after the above 3-4 rounds of screening, select 96 single colonies and inoculate them in TB medium containing 100ug / ml ampicillin, grow to the logarithmic phase, and add the final concentration 1 mM IPTG, culture overnight at 28°C.

[0036] (2) Use the infiltration method to obtain the crudely extracted antibody, transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour;

[0037] (3) Wash off unbound antibody with PBST, add mouse anti-His antibody (mouse anti-HIS antibody, R&D system), and place at room temperature for 1 hour;

[0038] (4) Wash off the unbound antibody with PBST, and add anti-mouse alkaline phosphatase conjugate (goat anti-mouse AP labeled antibody, sigma).

[0039] (5) Wash off the unbound antibody with PBST...

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Abstract

The invention discloses a nano-antibody aiming at the epi-position of an SEA (Soluble Egg Antigen), and simultaneously discloses a gene sequence for coding the nano-antibody and a host cell for expressing the nano-antibody. By means of the nano-antibody gene sequence and the host cell disclosed by the invention, high-efficiency expression of the nano-antibody can be carried out in escherichia coli; and the nano-antibody is applied to researching and developing schistosome detection reagents.

Description

technical field [0001] The present invention relates to the field of biotechnology or biomedicine, and relates to a nanobody against Schistosoma japonicum soluble egg antigen (Soluble Egg Antigen, SEA), its coding sequence and application. Background technique [0002] Schistosoma is widely distributed all over the world, especially in developing countries. There are five species of schistosomiasis that can infect and cause schistosomiasis in humans. In my country, Schistosoma japonicum is the main species. The deposition of Schistosoma japonicum eggs can cause blood vessel blockage, tissue necrosis, granulomatous reaction and tissue fibrosis. Therefore, the eggs play an important role in the pathogenic process of Schistosoma japonicum on human body. For the study of schistosomiasis, the development of ideal diagnostic reagents and vaccines are two main aspects. Initially, the diagnostic method of schistosomiasis was mainly based on routine stool examination, which could n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/68
Inventor 陈睿马格斯 斯蒂芬陆绍红孔庆明童群波郑斌楼涤丁建祖
Owner ZHEJIANG ACAD OF MEDICAL SCI
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