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mcherry or meos nanobodies and their preparation methods and applications

A nanobody and expression carrier 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: 2022-07-22
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

Method used

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  • mcherry or meos nanobodies and their preparation methods and applications
  • mcherry or meos nanobodies and their preparation methods and applications
  • mcherry or meos nanobodies and their preparation methods and applications

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Experimental program
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Effect test

Embodiment 1

[0052] The preparation method of mcherry nanobody includes 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, 1 mg of antigenic protein (including 0.25 mg of mcherry protein) was mixed with an equal volume of Freund's complete adjuvant and emulsified. Partial point injection, a total of four immunizations, once every two weeks, each immunization protein dosage is 0.5mg (including 0.125mg mcherry protein), and the adjuvant used for booster immunization is incomplete Freund's adjuvant. About four days after the fourth vaccination, at least 20 mL of alpaca whole blood was collected, and PBMCs were isolated using lymphocyte separation medium.

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

[0056] (1.3) Reverse transcription of RNA into cDNA. The operation steps are carried out according to the instructio...

Embodiment 2

[0093] The preparation method of mEOS nanobody includes 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, 0.5 mg of mEOS protein was mixed with an equal volume of Freund's complete adjuvant and emulsified. Four times, once every two weeks, the dose of protein for each immunization was 0.5 mg, and the adjuvant used for boosting immunization was incomplete Freund's adjuvant. About four days after the fourth vaccination, at least 20 mL of alpaca whole blood was collected, and PBMCs were isolated using lymphocyte separation medium.

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

[0097] (1.3) Reverse transcription of RNA into cDNA. The operation steps are carried out according to the instructions of PrimeScript™ II 1st Strand cDNASynthesis Kit (purchased from takara).

[0098] (1.4) Amplify the VHH fragment b...

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Abstract

mcherry or mEOS nanobodies and preparation methods and applications thereof relate to the technical field of bioengineering. The present invention screens out specific positive monoclonal nanobodies by constructing mcherry or mEOS nanobody libraries, and detects the antibodies that can bind to mcherry proteins. Two mcherry nanobodies whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3, and seven mEOS nanobodies that can bind to mEOS protein, whose nucleotide sequences are 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, SEQ ID NO.17. Compared with traditional antibodies, the mcherry or mEOS nanobodies constructed in the present invention can improve yield, reduce costs, and obtain better stability.

Description

technical field [0001] The present invention relates to the technical field of bioengineering, in particular to an mcherry or mEOS nanobody and a preparation method and application thereof. Background technique [0002] mcherry is a monomeric red fluorescent protein molecule that is widely used in biotechnology as a tracer, including molecular labeling and localization of cellular components. Because of its color and photostability of monomeric molecules, its use as a protein tag in biomedical research is superior to other fluorescent protein tags. [0003] mEOS is a light-activated green to red fluorescent protein molecule. mEOS emits strong green fluorescence and irreversibly turns red upon irradiation with UV light at 390 nm. It is usually used as a protein label in the field of biomedical research, for multi-color labeling of proteins, positioning and labeling proteins, and tracking protein movement in living cells. [0004] Both traditional mcherry antibodies and mEO...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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