Staphylococcal enterotoxin B nano antibody B1, application thereof and kit

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

Inactive Publication Date: 2019-12-13
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 nanobody B1, 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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  • Staphylococcal enterotoxin B nano antibody B1, application thereof and kit
  • Staphylococcal enterotoxin B nano antibody B1, application thereof and kit
  • Staphylococcal enterotoxin B nano antibody B1, application thereof and kit

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] 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.

[0045] 2) Separation of lymphocytes

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

Embodiment 2

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

[0086] 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

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

[0090] The B1 clone is subjected to DNA sequencing, and the amino acid sequence of the nanobody can be obtained according to the DNA sequencing result and the codon table.

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PUM

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Abstract

The invention discloses a staphylococcal enterotoxin B nano antibody B1, an application thereof and a kit. The nano antibody obtained by the invention has the advantages of small relative molecular mass, strong stability and high yield, can specifically recognize SEB, and has wider application and stronger specificity than the conventional monoclonal antibody. The invention discloses the nano-antibody, a gene sequence for encoding the nano-antibody, a method for producing the nano-antibody and a kit applying the antibody. The nano antibody obtained by the invention can be prevented from beingcombined with staphylococcus aureus surface protein A, shows higher 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 B nanobody B1, 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): C07K16/12C12N15/13G01N33/569G01N33/68
CPCC07K16/1271C07K2317/22C07K2317/33C07K2317/565C07K2317/567C07K2317/569G01N33/56938G01N33/68
Inventor 王建龙季艳伟王妍入李想路云龙张敢为陈利莉
Owner NORTHWEST A & F UNIV
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