Method and system for screening nanobody

Patent metadata

Field	Value
Publication number	US-10954550-B2
Application number	US-201515502438-A
Country	US
Kind code	B2
Filing date	Aug 6, 2015
Priority date	Aug 7, 2014
Publication date	Mar 23, 2021
Grant date	Mar 23, 2021

Abstract

Provided are a method for screening for nanobodies and a corresponding system. The method uses polymerase chain reactions and cDNA 5′ end rapid amplification technology to screen for and obtain nanobodies. The experiment cycle requires only approximately 21 days.

First claim

What is claimed is:

1. A method for screening a nanobody, comprising the following steps:
(1) extracting a nucleic acid sample from tissue or peripheral blood obtained from an animal after immunized;
(2) obtaining a sequencing result containing an antibody sequence based on the nucleic acid sample;
(3) constructing an antibody database based on the sequencing result containing the antibody sequence, wherein the step (3) comprises sub-steps of:
(3a) aligning the sequencing result containing the antibody sequence to a reference sequence to determine an immune-related gene sequence,
(3b) determining a nucleotide sequence of the antibody based on the immune-related gene sequence,
(3c) translating the nucleotide sequence of the antibody into an amino acid sequence, and
(3d) screening to obtain a VHH sequence based on the amino acid sequence, and constructing the antibody database;

(4) subjecting the antibody database to information analysis, to obtain sequence of the nanobody;
(5) subjecting serum obtained from the animal after immunized to protein mass spectrometry analysis to obtain a result of protein mass spectrometry;
(6) integrating information of the antibody database with the result of protein mass spectrometry for analysis to obtain the sequence of the nanobody; and
(7) expressing the nanobody via a protein expression system based on the sequence of the nanobody, to identify the nanobody.

2. The method according to claim 1 , wherein the animal is a Camelidae family animal.

3. The method according to claim 2 , wherein the Camelidae family animal is at least one selected from Camelus dromedarius, Camelus bactrianus, Lama guanicoe, Lama glama, Vicugna vicugna , and Vicugna pacos.

4. The method according to claim 1 , wherein the step (5) further comprises:
(5a) enriching IgG from the serum of the animal after immunized with Protein A/G to obtain an enriched product;
(5b) affinity purifying, by a chromatographic column conjugated with the antigen, the antibody from the enriched product to obtain a purified product;
(5c) subjecting the purified product to denaturation and reductive alkylation, and then lysing with protease to obtain an enzyme-digested peptide; and
(5d) subjecting the enzyme-digested peptide to protein mass spectrometry analysis on mass spectrometer to obtain a mass spectrometry result of the enzyme-digested peptide.

5. The method according to claim 4 , wherein the protease is at least one of pepsin, chymotrypsin, elastinase, trypsin, endoproteinase Lys-C, metalloendopeptidase Lys-N, endoproteinase Glu-C, aspartate endopeptidase Asp-N, and clostripain Arg-C.

6. The method according to claim 1 , wherein the antibody sequence includes a hypervariable region and a framework region.

7. The method according to claim 1 , wherein the nucleic acid sample is DNA or RNA.

8. The method according to claim 1 , wherein in the case that the nucleic acid sample is DNA, the antibody sequence in the nucleic acid sample is amplified by polymerase chain reaction (PCR); in the case that the nucleic acid sample is RNA, the antibody sequence in the nucleic acid sample is amplified by 5′-rapid amplification cDNA ends (5′-RACE) or PCR.

9. The method according to claim 8 , wherein the PCR is at least one of multiplex PCR, linear amplification mediated PCR, and nested PCR.

10. The method according to claim 1 , wherein the amplification product is sequenced on a high-throughput sequencing device.

11. The method according to claim 1 , wherein the immune-related gene sequence is at least one selected from a V gene, a D gene, a J gene and a C gene.

12. The method according to claim 1 , wherein the reference sequence is a known germline sequence in the absence of rearranging at least one of the V gene, the D gene, the J gene and the C gene.

13. The method according to claim 1 , wherein in the sub-step (3d), an indicator of determining the amino acid sequence to be the VHH sequence comprises at least one of:
A: a presence of any one of four conserved amino acids: 37F, 44E, 45R and 47G,
B: a presence of sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2 in a hinge region, and
C. absence of at least one portion of CH1.

14. The method according to claim 1 , wherein the step (2) further comprises the following sub-steps:
(2a) amplifying the antibody sequence in the nucleic acid sample to obtain an amplification product; and
(2b) subjecting the amplification product to sequencing to obtain the sequencing result containing the antibody sequence.

Assignees

• Bgi Shenzhen Co Ltd
• Bgi Shenzhen

Inventors

• Liu Xiao
• Nie Chao
• Zhang Wei
• Ren Zhe
• Zhang Ruifang
• Zeng Xiaojing
• Li Xinyang
• Yang Naibo

Classifications

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title

• C12Q1/6811 Primary

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C07K2317/22

  from camelids, e.g. camel, llama or dromedary · CPC title