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Staphylococcus aureus enterotoxin A nano antibody A13 and application and kit of nano antibody A13

A staphylococcal intestinal, nanobody technology, applied in applications, viruses/phages, vectors, etc., can solve the problems of high cost of detection methods, complicated operations, poor antibody specificity, etc. sexual effect

Inactive Publication Date: 2019-12-03
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the deficiencies and defects of the above-mentioned prior art, the object of the present invention is to provide a Staphylococcus aureus enterotoxin A nanobody A13, application and kit to solve the problem of poor antibody specificity in the prior art, high detection method cost and difficult operation. complex technical issues

Method used

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  • Staphylococcus aureus enterotoxin A nano antibody A13 and application and kit of nano antibody A13
  • Staphylococcus aureus enterotoxin A nano antibody A13 and application and kit of nano antibody A13
  • Staphylococcus aureus enterotoxin A nano antibody A13 and application and kit of nano antibody A13

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Construction of camel-derived nanobody phage display library

[0043] 1) Immunity of Bactrian camels

[0044] SEA was used as the immunogen to immunize adult male Alxa Bactrian camels by subcutaneous multipoint injection, and a total of five rounds of immunization were carried out. For the first immunization, Freund's complete adjuvant (Freund's complete adjuvant) was used to emulsify with an equal volume of immune antigen and then injected, and the immunization dose was 100 μg per mouse. Thereafter, booster immunization was performed every two weeks, and injected with Freund's incomplete adjuvant (Freund's incomplete adjuvant) and an equal volume of immunogen after emulsification. On the seventh day after the fifth booster immunization, the peripheral blood of Bactrian camels was collected to construct a nanobody phage display library.

[0045] 2) Separation of lymphocytes

[0046] Seven days after the last immunization, 200 mL of peripheral blood was col...

Embodiment 2

[0085] Example 2: Affinity panning of Nanobodies and their identification

[0086] 1) Affinity panning of Nanobodies: First, SEA was diluted with PBS (pH 7.4) to a final concentration of 50 μg / mL, and coated overnight at 4°C. The next day, after washing 5 times with PBST (10 mM PBS, 0.1% Tween-20 (v / v)), 5% BSA-PBS (or 5% OVA-PBS) was added to block for 1 hour at 37°C. Then wash 6 times with PBST, add 100 μL camel-derived single domain heavy chain antibody library (titer about 2.0×10 11 cfu), incubated at 37°C for 2 hours. Unbound phages were discarded, washed 10 times with PBST, added 100 μL of Glycine-HCl (0.2M, pH 2.2) to elute for 8 min, and immediately neutralized with 15 μL of Tris-HCl (1M, pH 9.1). Take 10 μL of the eluted phage to determine the titer, and the rest is used to infect 25 mL of the E.coli TG1 strain grown to the logarithmic phase for amplification. On the third day, the amplified phage was precipitated with PEG / NaCl, and the titer of the phage was deter...

Embodiment 3

[0089] Example 3: Sequencing of Nanobody Encoding Gene and Determination of its Amino Acid Sequence

[0090] The A13 clone was subjected to DNA sequencing, and the amino acid sequence of the nanobody could be obtained according to the DNA sequencing results and the codon table.

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Abstract

The invention discloses a staphylococcus aureus enterotoxin A (SEA) nano antibody A13 and application and a kit of the nano antibody A13. The nano antibody has small relative molecular mass, strong stability and high yield, can specifically recognize SEA, and has wider application and stronger specificity than conventional monoclonal antibodies. The invention discloses the nano antibody, a gene sequence encoding the nano antibody, a method for producing the nano antibody and a kit using the nano antibody. The nano antibody can avoid binding with staphylococcus aureus surface protein A, shows high specificity, has good stability and small molecular weight, and can be produced on a large scale.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Staphylococcus aureus enterotoxin A nanobody A13, its application and reagents. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, SA) is a common food-borne pathogen, and the food poisoning caused by it ranks first among Gram-positive bacteria. Toxins (Staphylococcal enterotoxins, SEs). SEs is a soluble extracellular toxin protein secreted by Staphylococcus aureus, with a similar structure, a molecular weight of 27.5-30kDa, good thermal stability, and is not destroyed by boiling at 100°C for 30 minutes. Generally, after heat treatment, although the bacterial cells can be killed, the enterotoxin produced by them is still active and pathogenic. According to serological classification, there are mainly serotypes such as A, B, Cs, D, E, etc. Among them, SEA has the highest incidence of food poisoning, and it mainly exists in animal foods with hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13G01N33/569
CPCC07K16/1271C07K2317/22C07K2317/33C07K2317/569C07K2317/94C12N2800/22G01N33/56938G01N2333/31
Inventor 季艳伟王建龙王妍入张道宏路云龙郭鹏利李想
Owner NORTHWEST A & F UNIV
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