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Mcherry or mEOS nano antibody as well as preparation method and application of mcherry or mEOS nano antibody

A nano-antibody and antibody technology, applied in the field of bioengineering, can solve problems such as poor batch stability, poor acid-base stability, and variability, and achieve the effects of low cost, good stability, and strong affinity

Active Publication Date: 2021-08-03
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Both traditional mcherry antibody and mEOS antibody are obtained by immunizing animals, which have high cost and low yield, and the stability between batches is poor. The traditional antibody structure is a tetramer composed of two heavy chains and two light chains. Its acid-base stability is poor, and its variability

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  • Mcherry or mEOS nano antibody as well as preparation method and application of mcherry or mEOS nano antibody
  • Mcherry or mEOS nano antibody as well as preparation method and application of mcherry or mEOS nano antibody
  • Mcherry or mEOS nano antibody as well as preparation method and application of mcherry or mEOS nano antibody

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Embodiment 1

[0052] The preparation method of mcherry nanobody comprises the following steps:

[0053] (1) Construction of mcherry nanobody library:

[0054] (1.1) The protein used for immunization is the mcherry protein expressed by Escherichia coli. For the first immunization, take 1 mg of antigenic protein (including 0.25 mg of mcherry protein) and an equal volume of complete Freund's adjuvant, shake, mix and emulsify, and place on the neck of the alpaca Partial point injection, a total of four immunizations, once every two weeks, the amount of each immunization protein is 0.5mg (including 0.125mgmcherry protein), and the adjuvant used for booster immunization is Freund's incomplete adjuvant. About four days after the fourth immunization, collect at least 20mL alpaca whole blood, and use lymphocyte separation medium to separate PBMC.

[0055] (1.2) Extract the total RNA of PBMC (using the trizol method).

[0056] (1.3) RNA was reverse-transcribed into cDNA, and the operation steps wer...

Embodiment 2

[0093] The preparation method of mEOS nanobody comprises the following steps:

[0094] (1) Construction of mEOS nanobody library:

[0095] (1.1) The protein used for immunization is the mEOS protein expressed by Escherichia coli. For the first immunization, take 0.5mg of mEOS protein and an equal volume of Freund's complete adjuvant, shake, mix and emulsify, and inject it at the neck of the alpaca. Four times, one immunization every two weeks, each immunization protein dosage is 0.5 mg, and the adjuvant used for booster immunization is Freund's incomplete adjuvant. About four days after the fourth immunization, collect at least 20mL alpaca whole blood, and use lymphocyte separation medium to separate PBMC.

[0096] (1.2) Extract the total RNA of PBMC (using the trizol method).

[0097] (1.3) RNA was reverse-transcribed into cDNA, and the operation steps were carried out according to the instructions of PrimeScript™ II 1st Strand cDNASynthesis Kit (purchased from takara).

...

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Abstract

The invention relates to the technical field of bioengineering, in particular to an mcherry or mEOS nano antibody and a preparation method and application thereof, and relates to the technical field of bioengineering. By constructing an mcherry or mEOS nano antibody library, a specific positive monoclonal nano antibody is screened out, two mcherry nano antibodies capable of being combined with mcherry protein are detected, nucleotide sequences of the two mcherry nano antibodies are shown as SEQ ID NO.1 and SEQ ID NO.3, and seven mEOS nano antibodies capable of being combined with mEOS protein are detected. The nucleotide sequences of the primers are as shown in SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15 and SEQ ID NO. 17. Compared with a traditional antibody, the mcherry or mEOS nano antibody constructed by the invention has the advantages that the yield can be increased, the cost can be reduced, and better stability can be obtained.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an mcherry or mEOS nanobody and its preparation method and application. Background technique [0002] mcherry is a monomeric red fluorescent protein molecule widely used in biotechnology as a tracer, including molecular labeling and localization of cellular components. Because of its color and the photostability of the monomeric molecule, it is superior to other fluorescent protein tags for use as protein tags in the field of biomedical research. [0003] mEOS is a light-activated green-to-red fluorescent protein molecule. mEOS can emit strong green fluorescence, which irreversibly changes to red under 390 nm UV irradiation. In the field of biomedical research, it is usually used as a protein label for multicolor labeling of proteins, positioning and labeling of proteins, and tracking protein movement in living cells. [0004] Both the traditional mcherry antibody and m...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/70C07K16/46G01N33/68
CPCC07K16/18C12N15/70C07K2317/569C07K2317/92C07K2317/94C07K2317/22
Inventor 容益康吴靖李凯丽
Owner NANHUA UNIV
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