Staphylococcus aureus enterotoxin B nanometer antibody B7, application and reagent kit

A staphylococcus enteric and nano-antibody technology, applied in the biological field, can solve the problems of high cost of detection methods, complicated operation, poor antibody specificity, etc., and achieve the effect of small relative molecular weight, wide application and high specificity

Active 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 B nanob

Method used

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  • Staphylococcus aureus enterotoxin B nanometer antibody B7, application and reagent kit
  • Staphylococcus aureus enterotoxin B nanometer antibody B7, application and reagent kit
  • Staphylococcus aureus enterotoxin B nanometer antibody B7, application and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, Construction of camel-derived nanobody phage display library

[0044] 1) Immunity of Bactrian camels

[0045] Adult male Alxa Bactrian camels were immunized by subcutaneous multipoint injection with SEB as the immunogen, and 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.

[0046] 2) Separation of lymphocytes

[0047] Seven days after the last immunization, 200 mL of peripheral blood was collected with ...

Embodiment 2

[0087] Example 2: Affinity panning of Nanobodies and their identification

[0088] 1) Affinity panning of Nanobodies: First, SEB 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

[0091] Example 3: Sequencing of Nanobody Encoding Gene and Determination of its Amino Acid Sequence

[0092] The B7 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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PUM

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Abstract

The invention discloses a staphylococcus aureus enterotoxin B nanometer antibody B7, an application and a reagent kit. The obtained nanometer antibody has small relative molecular mass, strong stability and high yield, can specially recognize SEB, and is broader in use and higher in specificity compared with a conventional monoclonal antibody. The invention discloses the nanometer antibody and a gene sequence for coding the nanometer antibody, a method for producing the nanometer antibody, and a reagent kit applying the antibody. The obtained nanometer antibody can prevent combination with surface protein A of the staphylococcus aureus, shows higher specificity, has good stability and small molecular weight, and can realize mass production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Staphylococcus aureus enterotoxin B nanobody B7, an application and a reagent. 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, SEB is the most virulent and most stable to heat in the SEs family, and mainly exists in meat, ...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/00C12N15/70G01N33/53
CPCC07K16/005C07K16/1271C07K2317/565C07K2317/567C12N15/70G01N33/53G01N2333/31
Inventor 季艳伟郭鹏利王建龙王妍入陈利莉路云龙李想
Owner NORTHWEST A & F UNIV
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