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Nano antibody of clostridium difficile glutamate dehydrogenase, and encoding sequence, screening method and application thereof

A technology of bacterial glutamate dehydrogenase and nanobody, applied in the field of immunology, to achieve the effect of low requirements, simple operation and good repeatability

Active Publication Date: 2019-02-15
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no related GDH nanobodies and related detection reagents at home and abroad.

Method used

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  • Nano antibody of clostridium difficile glutamate dehydrogenase, and encoding sequence, screening method and application thereof
  • Nano antibody of clostridium difficile glutamate dehydrogenase, and encoding sequence, screening method and application thereof
  • Nano antibody of clostridium difficile glutamate dehydrogenase, and encoding sequence, screening method and application thereof

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preparation example Construction

[0051] In the present invention, the preparation method of the nanobody is preferably obtained by recombinant expression of a prokaryotic expression system, and specifically preferably includes the following steps:

[0052] A. inserting the coding sequence of the Nanobody into a vector to obtain a recombinant vector;

[0053] B. Transform the recombinant vector into a competent state, and culture the transformed competent state on a flat plate to screen positive clones;

[0054] C. After expanding the culture of the positive clones, IPTG induced culture, and centrifuged the bacteria solution to resuspend the bacteria;

[0055] D. Breaking up the bacterium, purifying the obtained supernatant, and refolding the obtained purified protein solution through PBS dialysis to obtain recombinant nanobody.

[0056] In the present invention, the multiple cloning sites for inserting the coding sequence of the nanobody into the vector are Nde I and Xho I. The ligating enzyme is T4 DNA lig...

Embodiment 1

[0101] 1. Spleen blood RNA extraction and reverse transcription

[0102] After the camel spleen tissue was taken out from -80°C (taken from a Bactrian camel that was inspected and quarantined in a slaughterhouse in Alxa Left Banner, Inner Mongolia), liquid nitrogen was added to crush the specimen, Trizol reagent was added in a certain proportion, and chloroform was used to Total RNA was extracted with isopropanol. The extracted RNA was extracted with the mRNA purification kit, and the cDNA was obtained with Oligo dT through the cDNA reverse transcription kit.

[0103] 2. Amplification of VHH gene and construction of recombinant vector

[0104] Design two pairs of upstream and downstream primers for nested PCR of the target fragment, the primer sequences are:

[0105] CALL001: 5'-GTCCTGGCTGCTCTTCTCAAAAG-3' (SEQ ID NO: 10)

[0106] CALL002: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 11)

[0107] VHH-F: 5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3' (SEQ ID NO: 12);

[0108...

Embodiment 2

[0123] Preparation of GDH recombinant protein method

[0124] 1. According to the instructions of the Tiangen Bacteria Genomic DNA Extraction Kit, perform genome extraction on the Clostridium difficile CD630 strain provided by the Laboratory Department of the hospital.

[0125] 2. Design PCR primers to amplify the GDH protein gene, and use the whole genome as a template to amplify the target gene.

[0126] 3. The target gene of the PCR product was ligated with the expression vector PET30a, ligated with T4 DNA ligase at 16°C for 4 hours, and then ligated overnight at 4°C.

[0127] 4. Take 10 μl of the ligation product and add it to 100 μl of BL21 competent cells. After mixing, place it on ice for 30 minutes, then place it in a water bath at 42°C for 90 seconds, then place it on ice for 5 minutes, and add 900 μl of LB / kana medium , shake at 200 rpm at 37°C for 1 h, centrifuge at 5000 rpm for 10 min, take 100 μl of the supernatant to resuspend the bacteria, spread on LB / Kana sol...

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Abstract

The invention provides a nano antibody of clostridium difficile glutamate dehydrogenase, and an encoding sequence, a screening method and the application thereof and belongs to the technical field ofimmunology. The invention provides an amino sequence of the nano antibody of clostridium difficile glutamate dehydrogenase. By using a phage display technology, a sequence with a constant region specific gene is inserted into a vector of a phage encoding coat protein, after recombination expression, an exogenous gene expression product is fused with the phage encoding coat protein and demonstratedon the surface of phage to form a phage demonstration library, phage monocloning of the nano antibody is expressed through screening, and the nano antibody is prepared through sequencing. Phage-ELLSAidentification shows that the phage that the nano antibody is expressed on the surface can be specifically combined with clostridium difficile glutamate dehydrogenase antigen and has a good developing property, the result shows that the nano antibody has a good combination property with glutamate dehydrogenase, therefore, the nano antibody can be adopted to replace a common antibody applied to aclostridium difficile immunodetection kit.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to a nanobody of Clostridium difficile glutamic acid dehydrogenase, its coding sequence, its screening method and application. Background technique [0002] A heavy-chain antibody naturally deficient in light chains is present in the peripheral blood of camels, which contains only a heavy-chain variable domain and two conventional CH2 and CH3 domains. The single-domain antibody obtained by cloning the variable region of the heavy chain by PCR technology is called VHH antibody (variable domain of heavy-chain antibody, VHH). nm, 4nm long, so it is also called nanobody (nanobody, Nb). Nanobody is the smallest antibody with complete antigen-binding activity known so far. Due to its small size, it has strong antigen-antibody binding ability and can be highly expressed in prokaryotic and eukaryotic cells. In addition, it also has a stable structure and high hydrophilicity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N15/70C12N15/10G01N33/569
CPCC07K16/005C07K16/40C07K2317/565C07K2317/567C07K2317/569C12N15/1037C12N15/70G01N33/56911G01N2333/33G01N2469/10
Inventor 徐广贤方媛郭乐潘俊斐蒋丹屈昱良
Owner NINGXIA MEDICAL UNIV
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