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Nano antibody for resisting methicillin-resistant staphylococcus as well as preparation method and application of nano antibody

A nanobody, construct technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the consumption of essential lipoproteins and toxicity-related lipoproteins, bacterial cell death, mislocalization of unprocessed lipoproteins and other problems, to achieve the effect of easy separation and purification, not easy to aggregate, and good stability of structure and physicochemical properties

Inactive Publication Date: 2022-01-28
南京中爱人工智能与生命科学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, blocking lipoprotein synthesis via inhibition of LspA function not only depletes essential lipoproteins as well as toxicity-associated lipoproteins, but can also lead to accumulation of mislocalized unprocessed lipoproteins, both of which lead to bacterial cell death

Method used

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  • Nano antibody for resisting methicillin-resistant staphylococcus as well as preparation method and application of nano antibody
  • Nano antibody for resisting methicillin-resistant staphylococcus as well as preparation method and application of nano antibody
  • Nano antibody for resisting methicillin-resistant staphylococcus as well as preparation method and application of nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Expression and purification of embodiment 1.LspA-MRSA protein

[0065] LspA-MRSA (uniport: PODTC2, 330-541 amino acids) is constructed on the pET28a vector, and the reading frame coding sequence is respectively Honeybee Melittin secretion signal peptide (KFLVNVALVFMVVYISYIYAA) from N-terminal to C-terminal, Gly-Ser junction sequence, RBD object Protein sequence, Gly-Ser linker, 3C protease cleavage site (LEVLFQGP), Gly-Ser linker, Avi tag (GLNDIFEAQKIEWHE), Gly-Ser linker, and 10×His tag.

[0066] The expression of RBD was secreted and expressed in Trichoplusia ni High Five suspension cells. The supernatant after expression was collected, filtered through a 0.22um filter, added 20mM imidazole, mixed with 3mL Ni-Smart beads filler (SA035100), and incubated at 4°C Incubate for 3 hours under ambient agitation for binding. Then add the supernatant-resin mixture to the gravity column, collect the resin, wash with 10 column volumes of buffer A (150mM NaCl, 20mM Tris HCl pH 8...

Embodiment 2

[0069] Example 2. Nanobody Screening

[0070] 2.1 Alpaca immunity

[0071] Before immunization, mix the immune antigen (2mg / mL, 500uL) prepared in Example 1 with the GERBU adjuvant (GERBUAJUVANT P#3111) at a volume ratio of 1:1 to form an emulsion, and then apply it to the neck of the alpaca The antigen-adjuvant emulsion was injected subcutaneously at 10 points close to the lymph nodes of the arch, and a booster immunization was carried out every 2 weeks. A total of 4 immunization injection experiments were carried out. Before the first immunization, 3 mL of blood was collected and coagulated at room temperature for 2 hours. Centrifuge at 3000 g for 5 min at room temperature, and collect the supernatant as pre-immune serum; the sera after the 1st to 4th immunizations were also collected according to this method, and then the serum antibody titers were determined by ELISA. After the 4th immunization, the titer of serum antibody is higher than 10 6 , collect 80 ml of blood in...

Embodiment 3

[0097] Example 3 Nanobody Purification

[0098] The gene encoding the nanobody was constructed into the pSb-init vector, and its C-terminus also had Myc tags and 6xHis tags. The constructed plasmid was transformed into Escherichia coli MC1061 for expression. The general process is as follows: the cells are treated with TB medium (0.17M KH) containing 25mg / L chloramphenicol 2 PO 4 and 0.72M K 2 HPO 4, 1.2% (w / v) peptone, 2.4% (w / v) yeast extract (yeast extract), 0.5% (v / v) glycerol (glycerol)), 37°C, 220rpm culture. When the bacteria grew to an OD of about 0.5, the temperature was lowered to 22° C., and the cells were continued to be cultured. When the OD reached about 1.5, 0.02% (w / v) arabinose was added to induce expression for 16 hours. Collect the cultured cells by centrifugation, resuspend each liter of cells with 20 mL of TES-high Buffer (0.5M sucrose, 0.5mM EDTA, and 0.2M Tris Tris-HCl pH 8.0), and incubate at 4°C for 30min. Then 40 mL of ice water was added and st...

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Abstract

The invention provides a nano antibody targeting a methicillin-resistant staphylococcus LspA protein as well as a preparation method and application of the nano antibody. The provided nano antibody mainly recognizes a binding motif region combined with the methicillin-resistant staphylococcus LspA, and comprises a framework region FR and complementary determining regions CDR1, CDR2 and CDR3. One specific amino acid sequence of the nano antibody is SEQ ID NO: 1. The nano antibody disclosed by the invention can be efficiently and specifically combined with methicillin-resistant staphylococcus LspA protein, and the affinity can reach a nanomole level; and meanwhile, the nano antibody provided by the invention can obtain an excellent effect in methicillin-resistant staphylococcus detection or diseases caused by methicillin-resistant staphylococcus, and can be applied to the fields of biology and medicine.

Description

technical field [0001] The invention belongs to the technical field of nanobody screening and preparation, and in particular relates to a nanobody against methicillin-resistant staphylococcus and its preparation method and application. Background technique [0002] Methicillin-resistant Staphylococcus aureus is a clinically common and highly toxic bacterium. Since the advent of penicillin in the 1940s, infectious diseases caused by Staphylococcus aureus have been largely controlled. However, with the widespread use of penicillin, some Staphylococcus aureus produce penicillinase, which can hydrolyze the β-lactam ring, showing resistance to penicillin. In the process of solving this problem, scientists have developed a new semi-synthetic penicillin that is resistant to penicillinase, namely methicillin. [0003] Methicillin has effectively controlled the enzyme-producing strains of Staphylococcus aureus after its clinical application, but Jevons screening in the UK found meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/70G01N33/569C12N9/48A61K39/40A61P31/04
CPCC07K16/1271C12N15/70G01N33/56938C12N9/52C12Y304/23036A61K39/40A61P31/04C07K2317/565C07K2317/567C07K2317/569C07K2317/92G01N2333/31
Inventor 马丁.卡弗瑞谈静泉
Owner 南京中爱人工智能与生命科学研究院有限公司
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