Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of anti-her-2 heavy chain antibody and its application

A HER-2 and heavy chain antibody technology, applied in the field of molecular immunology, can solve the problems of cumbersome monoclonal antibody development and production process, low specificity and instability of polyclonal antibody, and achieve excellent genetic resources and antibody resources. , strong affinity, reliable source effect

Active Publication Date: 2022-06-21
BEIJING INST OF COLLABORATIVE INNOVATION
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are monoclonal or polyclonal antibodies against HER-2, but the development and production process of monoclonal antibodies is cumbersome and complicated, and the specificity of polyclonal antibodies is not high and unstable. In contrast, heavy chain antibodies It has the advantages of high stability, high specificity, small molecular weight and large-scale production, and has broad application prospects

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of anti-her-2 heavy chain antibody and its application
  • A kind of anti-her-2 heavy chain antibody and its application
  • A kind of anti-her-2 heavy chain antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Panning of anti-HER-2 Nanobodies

[0028] Single-domain heavy-chain antibodies against HER-2 were panned from a single-domain heavy-chain antibody phage display library by solid-phase affinity panning. Dilute HER-2 protein with 1×PBS solution to 30-100μg / μL, coat on ELISA plate, add 100μL to each well, and coat overnight at 4°C; aspirate the coating solution, wash the plate 3 times with PBS, add to each well 300 μL of 4% skim milk, blocked at 37°C for 2 h; after washing the plate 3 times with PBS, add the phage display library (about 1×10 12 CFU), incubate at 37°C for 1 h; aspirate the unbound phage, wash the plate 5 times with PBS (increase the number of washes round by round, and the number of washes in each round is shown in Table 1), and then wash the plate with PBST for 3 times and then add 100 μL to wash the plate Desaturate (glycine-hydrochloric acid solution, pH 2.2) to elute the phages adsorbed in the plate wells, incubate at 37°C for 5 min, gently p...

Embodiment 2

[0036] Example 2 Expression and purification of anti-HER-2 nanobodies

[0037] The gene of the nanobody obtained in Example 1 was cloned into the expression vector pET-25b (cloned by Qingke Biotechnology Co., Ltd.), and the anti-HER-2 nanobody expression plasmid was constructed. The constructed expression plasmid was transformed into Escherichia coli BL21, and single clone was picked for induced expression. The single clone was inoculated into 1L LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C, 220rmp / min shaking until the bacterial liquid OD600 reached 0.5, adding IPTG with a final concentration of 0.1mM, 16°C, 80rmp / min min overnight induction culture. After the cultivation, the cells were collected by centrifugation, resuspended in 50 mL of PBS solution, and then sonicated on ice. The conditions were 200 w, crushed for 3 s, intermittent for 4 s, for a total of 30 min, and the supernatant was collected by centrifugation at 8000 g at 4 °C.

[0038] T...

Embodiment 3

[0039] Example 3 Affinity determination of anti-HER-2 nanobodies

[0040] The nanobody prepared in Example 2 was tested for affinity using the Octet@RED96 intermolecular interaction detection system (ForteBio Company). Octet@RED96 Intermolecular Interaction Detection System is based on biofilm layer interferometry (BLI) technology, which can measure the interaction between proteins and biomolecules without labeling with only a small amount of sample.

[0041] The human HER-2 protein was diluted to 10 μg / mL, and the Nanobody obtained in Example 2 was diluted to 50 μg / mL. The dilution used was PBS+0.1% Tween 20+0.1% BSA, and the samples were added according to Table 3. Affinity test chart see Figure 4 .

[0042] Table 3 Affinity detection sample loading table

[0043]

[0044] Equilibrium dissociation constant (affinity) K D (M)=k dis (1 / s) / k on (1 / Ms)

[0045] It has been detected:

[0046] k dis (1 / s)=0.0004768;

[0047] k on (1 / Ms)=567.7;

[0048] K D (M)=k ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to the field of molecular immunology, and specifically provides a HER-2 capture or detection method, which is characterized in that an anti-HER-2 heavy chain antibody is used as a capture or detection agent. The present invention also provides the preparation method and application of the anti-HER-2 heavy chain antibody. The affinity between the heavy chain antibody and HER‑2 protein reaches 8.4×10 ‑7 M, and has the advantages of small molecular weight, easy expression, low cost, and good stability.

Description

technical field [0001] The invention belongs to the field of molecular immunology, in particular to a phage display library and a nanobody recombinant expression technology, in particular to a heavy chain antibody that specifically recognizes HER-2 protein and its application. Background technique [0002] Heavy chain antibody (HcAb) is found in camels and sharks. It is an antibody that naturally lacks light chains and only consists of heavy chains. Clone its variable region to obtain a single-domain antibody composed of only the variable region of the heavy chain, called VHH (Variable domain of heavy chain of heavy chain antibody), also known as nanobody (nanobody), which is the smallest functional antigen Combine fragments. Different from ordinary antibodies, nanobodies are a peptide chain containing about 110 amino acids, and its molecular weight is about 1 / 10 of ordinary antibodies. Compared with ordinary antibodies and recombinant single-chain antibodies (single chain ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/71C07K16/32C12N15/13G01N33/68
CPCG01N33/68C07K14/71C07K16/32C07K2317/569C07K2317/565C07K2317/56C07K2317/92G01N2333/71
Inventor 徐良林坚周斌周鹏
Owner BEIJING INST OF COLLABORATIVE INNOVATION
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com