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Nano antibody for resisting toxoplasma gondii SAG1 as well as coding gene and application thereof

A nano-antibody and Toxoplasma gondii technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of low detection rate and unsatisfactory demand, and achieve high affinity, excellent tissue penetration ability, and high water solubility sexual effect

Active Publication Date: 2018-09-07
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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  • Nano antibody for resisting toxoplasma gondii SAG1 as well as coding gene and application thereof
  • Nano antibody for resisting toxoplasma gondii SAG1 as well as coding gene and application thereof
  • Nano antibody for resisting toxoplasma gondii SAG1 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Recombinant SAG1 protein, the steps are as follows:

[0024] (1) Toxoplasma gondii cDNA was extracted by Trizol method, and PCR amplification primers were designed according to the Toxoplasma gondii SAG1 gene sequence on NCBI (GenBank number: HM776940.1);

[0025] (2) PCR amplification to obtain the SAG1 target gene fragment with a fragment size of 780bp, and construct the SAG1-pET28a recombinant vector;

[0026] (3) The SAG1-pET28a recombinant vector was transformed into competent cells BL21 by heat shock method, induced by IPTG at 37°C, expressed the recombinant protein, collected the bacteria, after ultrasonic disruption, purified by Ni-NTA nickel strain, and verified the recombinant SAG1 protein by SDS-PAGE get the expression ( figure 1 ).

Embodiment 2

[0028] To construct the Toxoplasma gondii-specific nanobody library, the steps are as follows:

[0029] (1) Purify the recombinant SAG1 protein (surface membrane antigen) of Toxoplasma gondii, then mix 1 mg with Freund's adjuvant in an equal volume, and immunize a camel (Camelus bactrianus). The experiment was carried out in Chifeng, Inner Mongolia, in 2015 May), once a week, a total of 4 times of immunization, to stimulate B cells to express antigen-specific nanobodies;

[0030] (2) After the 4 times of immunization, extract 100ml camel peripheral blood lymphocytes and extract total RNA;

[0031] (3) Synthesize cDNA by reverse transcription and use nested PCR (Nest PCR) to amplify the heavy chain variable region gene VHH;

[0032] (4) Use restriction endonucleases to perform double enzyme digestion on the amplified product of Nest PCR and the phagemid carrier pHEN 4, and purify the digested product according to the molar ratio of the optimized VHH insert fragment to the carr...

Embodiment 3

[0037] Panning of specific Nanobodies. Proceed as follows:

[0038] (1) Dissolve in 100mM NaHCO 3 , 30 μg recombinant SAG1 protein of Toxoplasma gondii at pH 8.2 was coated on NUNC microtiter plates, and placed overnight at 4°C;

[0039] (2) Add 100 μl of milk with a mass concentration of 3% the next day, and block at room temperature for 2 hours;

[0040] (3) After 2h, add 100μl 2×10 11 pfu contains the helper phage of the above-mentioned nanobody library, and acts at room temperature for 1 hour;

[0041] (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;

[0042] (5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to the recombinant SAG1 protein 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 res...

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Abstract

The invention discloses a nano antibody for resisting toxoplasma gondii SAG1 as well as a coding gene and application thereof. A VHH chain amino acid sequence of the nano antibody is as shown by SEQ ID NO. 4. The nano antibody comprises two VHH chains. The nano antibody for resisting the toxoplasma gondii SAG1 is obtained by immunizing a camel by virtue of toxoplasma gondii SAG1 antigen, obtainingan anti-SAG1 nano antibody library, and selecting a nano antibody with good performance from the nano antibody library. The nano antibody has high solubility and conformation stability and high antigen affinity and excellent tissue penetration capability, and has high affinity with SAG1 antigen, the affinity constant KD value is 1.66nM, the toxoplasma gondii can be efficiently detected, and the nano antibody can be used for preparing 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 the surface antigen SAG1 of toxoplasma gondii, its coding gene and its 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, convulsions...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/20C12N15/13G01N33/569
CPCG01N33/56905C07K16/20C07K2317/92C07K2317/565C07K2317/569G01N2333/45G01N2469/10
Inventor 孔庆明丁豪杰郑斌卓洵辉丁建祖楼涤陆绍红
Owner ZHEJIANG ACAD OF MEDICAL SCI
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