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Anti-salmonella typhimurium nano antibody and application thereof

A Salmonella and nano-antibody technology, applied in the field of molecular biology, can solve the problems of difficult expression, low expression efficiency, weak recognition and binding ability, etc., and achieve the effect of high affinity, strong specificity and strong stability

Active Publication Date: 2021-03-30
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the deficiencies and defects of the above-mentioned prior art, the object of the present invention is to provide an anti-Salmonella typhimurium nanobody and its application, which solves the problem of weak recognition and binding ability of the antibody in the prior art to Salmonella enteritidis, difficulty in expressing and The problem of low expression efficiency

Method used

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  • Anti-salmonella typhimurium nano antibody and application thereof
  • Anti-salmonella typhimurium nano antibody and application thereof
  • Anti-salmonella typhimurium nano antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Construction of phage display nanobody immune library

[0034] 1) Camel immunization: take 5mL and dilute to 10 8 After the CFU / mL inactivated Salmonella typhimurium liquid was emulsified with an equal volume of Freund's complete adjuvant, a healthy Bactrian camel was injected subcutaneously at multiple points, and then immunized every two weeks for a total of five times. For the second to fifth immunizations, incomplete Freund's adjuvant was used for antigen emulsification, and blood sampling began after the fourth immunization. Boost the immunization 7-10 days after the fourth immunization, and then collect 20 mL of blood from the jugular vein of the blood collection tube one week after the booster immunization. At the same time, gently invert the blood collection tube to avoid blood coagulation, and then process the blood with the filter in the LeukoLOCK RNA (Invitrogen) kit , and then rinse the filter with 3mL phosphate buffer and 3mL RNAlater buffer in sequence, t...

Embodiment 2

[0061] Example 2: Affinity panning and identification of Nanobodies

[0062] 1) Affinity panning of nanobodies: first, inactivated Salmonella typhimurium was diluted to 10 with PBS (pH 7.4). 8 CFU / mL, coated overnight at 4°C. The next day, after washing 3 times with 0.05% PBST, 3% skimmed milk powder was added to block for 1 hour at 37°C. Then wash 3 times with PBST, add 150 μL camel-derived single domain heavy chain antibody library to each well, and incubate at 37°C for 1 hour. Unbound phages were discarded, the plate was manually washed 10 times with PBST, 100 μL of Glycine-HCl (0.2 M, pH 2.2) was added to elute for 8 min, and immediately neutralized with 4 μL of Tris-HCl (1 M, pH 9.1). Take 10 μL of the eluted phage to determine the titer, and the rest is used to infect and amplify the E.coli ER2738 strain cultured to the logarithmic phase. On the third day, the amplified phage was precipitated with PEG / NaCl, and the titer of the phage was determined.

[0063] During t...

Embodiment 3

[0066] Example 3: Sequencing of Nanobody Encoding Gene and Determination of its Amino Acid Sequence

[0067] The 10 positive clones were subjected to DNA sequencing, and the DNA sequencing results were analyzed with Bioedit software to obtain an anti-Salmonella typhimurium nanobody ST-Nb9, and the framework region and complementarity determining region of the antibody sequence were determined.

[0068] The amino acid sequence of the frame region FR1 of the anti-Salmonella typhimurium nanobody ST-Nb9 is shown in SEQ NO:1;

[0069] The amino acid sequence of the framework region FR2 of the anti-Salmonella typhimurium nanobody ST-Nb9 is shown in SEQ NO:2;

[0070] The amino acid sequence of the framework region FR3 of the anti-Salmonella typhimurium nanobody ST-Nb9 is shown in SEQ NO:3;

[0071] The amino acid sequence of the frame region FR4 of the anti-Salmonella typhimurium nanobody ST-Nb9 is shown in SEQ NO:4;

[0072] The amino acid sequence of the complementarity determin...

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Abstract

The invention provides an anti-salmonella typhimurium nano antibody ST-Nb9, the amino acid sequence of which comprises four framework regions FR1, FR2, FR3 and FR4, and also comprises three complementary determining regions CDR1, CDR2 and CDR3; wherein the sequence of the FR1 is shown as SEQ NO: 1, the sequence of the FR2 is shown as SEQ NO: 2, the sequence of the FR3 is shown as SEQ NO: 3, and the sequence of the FR4 is shown as SEQ NO: 4; the sequence of the CDR1 is shown as SEQ NO: 5, the sequence of the CDR2 is shown as SEQ NO: 6, and the sequence of the CDR3 is shown as SEQ NO: 7. The anti-salmonella typhimurium nano antibody provided by the invention has a unique variable region sequence, so that the antibody has specific recognition and binding capacity on salmonella enteritidis. The anti-salmonella typhimurium nano antibody provided by the invention has the advantages of easiness in expression and high expression efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a nanobody against Salmonella typhimurium and its application. Background technique [0002] Salmonella is a global foodborne disease. Diarrhea, vomiting, and fever will occur after the human body ingests bacteria-containing food. Children with typhoid fever and low immunity will develop symptoms such as sepsis, dehydration, acidosis, anuria, and heart failure. If first aid is not timely, it will be life-threatening. There are more than 2,000 known serotypes of Salmonella, and the common serotypes in clinical practice are mainly Salmonella typhimurium and Salmonella enteritidis. The application of well-regulated production operations and management systems such as hazard analysis and critical point control (HACCP) can largely reduce the occurrence of foodborne pathogens. [0003] At present, the methods for detecting Salmonella mainly include biochemical culture methods...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13G01N33/569
CPCC07K16/1235G01N33/56916C07K2317/569C07K2317/565C07K2317/567C07K2317/94G01N2333/255G01N2469/10
Inventor 王妍入张翠任亚荣王建龙季艳伟
Owner NORTHWEST A & F UNIV
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