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Anti-toxoplasma gondii TRX (Thioredoxin) nano-antibody, encoding gene and application thereof

A nanobody, Toxoplasma gondii technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of inability to meet demand, low detection rate, etc., and achieve excellent tissue penetration ability, efficient detection, and high conformational stability. Effect

Active Publication Date: 2018-09-04
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For mild and chronic patients, the detection rate of conventional CAg detection methods is low and cannot meet the needs

Method used

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  • Anti-toxoplasma gondii TRX (Thioredoxin) nano-antibody, encoding gene and application thereof
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  • Anti-toxoplasma gondii TRX (Thioredoxin) nano-antibody, encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Recombinant TRX protein, the steps are as follows:

[0025] (1) Trizol method was used to extract Toxoplasma gondii cDNA, and PCR amplification primers were designed according to the Toxoplasma gondii TRX gene sequence (GenBank number: XM_002370147) on NCBI, and the TRX gene fragment was obtained by PCR amplification;

[0026] (2) Construct the TRX gene fragment into the pET-28a expression vector, and transform the competent cell BL21;

[0027] (3) IPTG induces expression, collects bacteria, and after sonication, the protein is mainly expressed in the supernatant, and the recombinant protein is 15KDa, which is consistent with the expected size; the supernatant is purified, dialyzed and refolded using a nickel column, and detected by SDS-PAGE The result is as figure 1 shown.

Embodiment 2

[0029] To construct the Toxoplasma gondii-specific nanobody library, the steps are as follows:

[0030] (1) Purify the recombinant TRX protein of Toxoplasma gondii, then mix it with Freund's adjuvant in an equal volume of 1 mg, and immunize a camel (Camelus bactrianus, the experiment was carried out in Chifeng, Inner Mongolia, in May 2015), Once a week, a total of 3 times of immunization to stimulate B cells to express antigen-specific nanobodies;

[0031] (2) After the 3 times of immunization, extract 100ml camel peripheral blood lymphocytes and extract total RNA;

[0032] (3) Synthesize cDNA by reverse transcription and use nested PCR (Nest PCR) to amplify the heavy chain variable region gene VHH;

[0033] (4) Use restriction endonucleases to double-digest the amplified product of Nest PCR and the phagemid carrier pHEN 4, and divide the purified digested product according to the molar ratio of the optimized VHH insert fragment to the carrier molecule: 3: 1. Connecting and ...

Embodiment 3

[0038] Panning of specific Nanobodies. Proceed as follows:

[0039] (1) Dissolve in 100mM NaHCO 3 , 20 μg Toxoplasma gondii recombinant TRX antigen at pH 8.2 was coated on NUNC microtiter plates, and placed overnight at 4°C;

[0040] (2) Add 100 μl of milk with a mass concentration of 3% the next day, and block at room temperature for 2 hours;

[0041] (3) After 2h, add 100μl 2×10 11 tfu contains the helper phage of the above-mentioned nanobody library, and acts at room temperature for 1 hour;

[0042](4) Wash 10 times with 0.05% PBS+Tween-20 for the first round of panning / 20-25 times for the second round to remove non-specifically bound phages;

[0043] (5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to the recombinant TRX antigen of Toxoplasma gondii, and infect Escherichia coli TG1 in logarithmic growth phase, culture at 37°C for 1 hour, produce and purify the phage for the next round The panning was gradually enriched, and the results...

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Abstract

The invention discloses an anti-toxoplasma gondii TRX (Thioredoxin) nano-antibody, an encoding gene and the application thereof. VHH chain amino acid sequence of the antibody is as shown in SEQ ID No.4. The nano-antibody comprises two VHH chains. The anti-toxoplasma gondii TRX nano-antibody is obtained by immunizing a camel by a toxoplasma gondii TRX antigen to obtain an anti-TRX nano-antibody library, and then screening to obtain the anti-toxoplasma gondii TRX nano-antibody with good performance from the nano-antibody library. The anti-toxoplasma gondii TRX nano-antibody has high water solubility and conformational stability, strong antigen affinity, excellent tissue penetrating capacity, can detect toxoplasma gondii highly effectively, and can be used for preparing a toxoplasma gondii detection kit.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a nanobody against toxoplasma gondii thioredoxin (Thioredoxin, TRX) and its coding gene and application. Background technique [0002] Toxoplasmosis is a zoonotic protozoan disease caused by Toxoplasma gondii (T. gondii). The transmission of Toxoplasma gondii through the placenta to the fetus mainly occurs in the acute infection stage of the mother. Once it invades the placenta, 95% of the fetuses will not be spared, directly affecting fetal development, severe teratogenicity and even death, which can cause miscarriage, stillbirth and premature birth in pregnant women. Increased complications of pregnancy. The clinical manifestations of children with congenital toxoplasmosis are relatively typical, and generally have four major characteristics of hydrocephalus or cerebellar malformation, brain calcification, chorioretinitis, mental retardation, convulsio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/20C12N15/13G01N33/569
CPCG01N33/56905C07K16/20C07K2317/565C07K2317/569C07K2317/567
Inventor 陆绍红丁豪杰孔庆明郑斌卓洵辉丁建祖楼涤
Owner ZHEJIANG ACAD OF MEDICAL SCI
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