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

Single-domain heavy-chain antibody aiming at adenovirus vector and application of single-domain heavy-chain antibody

A single-domain heavy chain antibody and adenovirus technology, which is applied in the direction of virus/phage, virus, vector, etc., to achieve the effect of broad application space, simple operation, and strong tissue penetration ability

Active Publication Date: 2022-06-10
INNER MONGOLIA AGRICULTURAL UNIVERSITY
View PDF14 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009]Currently, there is no report of single domain antibody against adenovirus vector

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
  • Single-domain heavy-chain antibody aiming at adenovirus vector and application of single-domain heavy-chain antibody
  • Single-domain heavy-chain antibody aiming at adenovirus vector and application of single-domain heavy-chain antibody
  • Single-domain heavy-chain antibody aiming at adenovirus vector and application of single-domain heavy-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Construction of a single-domain antibody library specific to adenovirus vector rAd26 and ChAd63

[0057] (1) Adenoviral vector from the original concentration of 1 × 10 13 vp / mL diluted to 2×10 11 vp / mL is used for immunization, 1 mL each time, to immunize a healthy female Bactrian camel;

[0058] Use 1 mL for the first and second immunization, 2×10 11 The vp / mL rAd26 adenovirus was used as an antigen to immunize Bactrian camels, and the third and fourth immunizations used 1 mL, 2×10 11 Bactrian camels were immunized with vp / mL ChAd63 adenovirus.

[0059] Immunize once every two weeks, collect basic serum before the first immunization, and collect serum one week after the last immunization to determine the antibody titer;

[0060] (2) After all 4 times of immunization, the camel peripheral blood lymphocytes were extracted and total RNA was extracted;

[0061] (3) reverse transcription of total RNA into cDNA;

[0062] (4) Utilize nested PCR to amplify th...

Embodiment 2

[0066] Example 2. Screening for specific single domain antibodies against adenovirus vector rAd26 and ChAd63

[0067] (1) Add the adenovirus vector-specific single-domain antibody phage display library constructed in Example 1 to 200ml of 2×YT medium, the OD 600nm value of the bacterial solution is not more than 0.3, and then at 37°C, 250r / min Incubate the library in a shaker for 2 hours;

[0068] (2) Add M13K07 helper phage, the infection ratio (quantity ratio) is M13K07:TG1=20:1, and infect at 37°C for 1 hour;

[0069] (3) Take the cultured bacterial solution, centrifuge at 4°C and 2 000×g for 10 minutes; discard the supernatant, resuspend, and inoculate the resuspended solution into 2×YT containing ampicillin and kanamycin ( 2×YT-AK) medium, 37°C, 220r / min constant temperature shaking overnight;

[0070] (4) The next day, centrifuge at 4°C and 7197×g for 15 minutes; take the supernatant, add 1% BSA, let stand at 4°C for 10 minutes, then add 20% PEG 8000 to precipitate pha...

Embodiment 3

[0074] Example 3. Screening of specific monoclones by phage-ELISA

[0075] (1) Randomly pick clones from the cell culture dishes containing phage after the above two rounds of selection and add them to 2×YT-AG (2×YT contains 100 μg / mL ampicillin and 2% glucose) liquid medium, 37°C , 250r / min constant temperature shaker overnight culture;

[0076] (2) The next day, add helper phage M13K07, infect for 2 hours, centrifuge at 14,000r / min for 5min, resuspend the bacteria in 2×YT-AK medium, culture overnight at 37°C, 250r / min constant temperature shaker;

[0077] (3) The next day, after centrifugation at 14000r / min for 5min, collect the supernatant, add 1% BSA, let stand at 4°C for 10min, then add 20% PEG8000, mix well, centrifuge at 7197×g for 20min, and resuspend in PBS recombinant phage;

[0078] (4) Add the recombinant phage to the pre-coated concentration of 2×10 7 vp / mL adenovirus vector rAd26 microtiter plate, with helper phage M13K07 as negative control, PBS buffer as bla...

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 invention relates to a single-domain heavy-chain antibody aiming at an adenovirus vector and application of the single-domain heavy-chain antibody. The VHH of the single-domain heavy-chain antibody comprises a framework region FR and a complementary determining region CDR, the amino acid sequence of the VHH of the single-domain heavy-chain antibody comprises an amino acid sequence shown in SEQ ID NO.8, and the nucleotide sequence of the VHH of the single-domain heavy-chain antibody comprises a nucleotide sequence shown in SEQ ID NO.9. The single-domain heavy-chain antibody as well as the nucleic acid, the vector and the host cell thereof can be used for detecting adenovirus vector reagents, adenovirus therapeutic antibody drugs, purification and preparation of adenovirus vector vaccines and the like. The single-domain heavy-chain antibody can be efficiently expressed in escherichia coli, can specifically recognize an adenovirus vector, has the advantages of good specificity and the like, and provides a wide application prospect for research and development of adenovirus therapeutic antibody disease drugs and detection reagents.

Description

technical field [0001] The invention belongs to the fields of biomedicine and molecular biology, and in particular relates to a single-domain heavy chain antibody against an adenovirus vector and its application. Background technique [0002] Adenovirus (adenovirus, Ad) belongs to the family Adenoviridae and is mainly extracted from five types of vertebrates, including birds, mammals, reptiles, amphibians and fish. The diameter of the adenovirus shell is 70-90nm. Non-enveloped, linear, double-stranded DNA viruses consisting of an outer capsid protein and inner genetic material. Viral DNA is wrapped in a protein shell, which contains three main proteins: hexon, penton, base, and fiber, as well as four minor capsid proteins IIIa, VI, VIII and IX. Adenoviruses can carry foreign genes and are used as gene delivery vehicles for vaccine development. Its advantages include: there are a variety of serotypes to choose from, and there are more than 100 types of adenoviruses that ha...

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 Applications(China)
IPC IPC(8): C07K16/08C12N15/13C12N15/70C12N7/00C12N7/02G01N33/58G01N33/569G01N33/543A61K39/42A61P31/20
CPCC07K16/081C12N15/70C12N7/00G01N33/581G01N33/56983G01N33/54306A61P31/20C07K2317/565C07K2317/567C07K2317/569C07K2317/22C12N2710/10051G01N2333/075G01N2469/10C12N2800/101A61K2039/505Y02A50/30
Inventor 包福祥郝彦霞程艺付山刘艳龙张慧敏敖可心吴玘瑶
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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