Novel anti-human serum albumin antibody fragment, preparation method and application

An anti-human serum and albumin technology, applied in the field of biomedicine, can solve the problems of low expression level, difficult purification work, large molecular weight of human serum albumin fusion protein, etc., and achieve high affinity, high stability, and good specificity sexual effect

Active Publication Date: 2020-05-12
REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
View PDF4 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the covalent form of human serum albumin fusion protein has limited its wide application due to its large molecular weight, low expression level, and difficulty in purification.

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
  • Novel anti-human serum albumin antibody fragment, preparation method and application
  • Novel anti-human serum albumin antibody fragment, preparation method and application
  • Novel anti-human serum albumin antibody fragment, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of anti-human serum albumin single domain antibody library

[0028] Two Xinjiang Bactrian camels were immunized with human serum albumin mixed with Freund's adjuvant, once a week for a total of seven times. After the immunization, the peripheral blood cells of camels were extracted, and the lymphocytes were frozen in dry ice and sent to Nanjing Jaxes Biotechnology Co., Ltd. for the extraction and separation of single domain antibody fragments. The steps of extraction and isolation of single-domain antibody fragments are as follows: extract total RNA, synthesize cDNA by reverse transcription, amplify single-domain antibody fragments by nested PCR, digest with restriction endonucleases, ligate into phage display vectors, and electroporate into competent cells. Two independent single-domain antibody phage display libraries against human serum albumin were successfully constructed. The library capacity was determined by counting the number of single...

Embodiment 2

[0029] Example 2: Screening process of single domain antibody against human serum albumin

[0030] Coupling human serum albumin on the ELISA plate overnight, adding the phage library after blocking with blocking solution, washing with PBST for several times, using TEA eluent to dissociate the phage that specifically binds to human serum albumin, and use it for infection Escherichia coli cells in the logarithmic growth phase, and the phages were expanded for the next round of screening. Each library was enriched by more than 500 times after three rounds of Bio-panning, achieving the purpose of using phage display technology to screen the specific antibody binding to human serum albumin in the antibody library.

Embodiment 3

[0031] Embodiment 3: use the enzyme-linked immunoassay method (ELISA) of phage to screen specific single positive clone

[0032]Randomly select 600 single colonies from the two libraries to inoculate and culture. After growing to the logarithmic phase, IPTG induces expression. After the bacteria are collected by centrifugation, the crude antibody is obtained from the periplasm by osmotic shock method, and the enzyme coated with human serum albumin is added. Incubate in the standard plate, wash the plate with PBST, use mouse Anti-HA tag antibody as primary antibody, goat anti-mouse alkaline phosphatase conjugate antibody (goat anti-mouse alkaline phosphatase conjugate) as secondary antibody, add alkaline phosphatase Develop color and read the absorbance value, and judge the sample wells with OD value more than 3 times higher than the control wells as positive control wells. All positive clones were cultured, plasmids were extracted and sequenced to obtain multiple single-domain...

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 belongs to the field of biomedicine, and in particular relates to an anti-human serum albumin single-domain antibody and a preparation and application thereof. A single-domain antibody with good specificity, relatively high affinity and stability is finally obtained, and the single-domain antibody can specifically recognize human serum albumin, prolongs the half-life period of a biological drug, and is beneficial to beneficial effects of the biological drug in treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the preparation and application of an anti-human serum albumin single domain antibody. Background technique [0002] Pharmacokinetics is a key parameter for protein-based drugs. Typically, longer serum half-lives mean that peptide and protein drugs can use lower doses and achieve better therapeutic effects. At present, a variety of technical methods have been used to prolong the half-life of such drugs, including PEGylation of proteins, fusion with IgG-Fc fragments, and fusion expression with human serum albumin. Human serum albumin (HSA) has been successfully used in various fusion constructions, including Factor VIII, Factor IX, etc., due to its long half-life (19-day serum half-life), good stability, and non-immunogenicity. However, the covalent form of human serum albumin fusion protein has limited its wide application to some extent due to its large molecular weight,...

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
IPC IPC(8): C07K16/18C07K19/00C12N15/13A61K47/68
CPCA61K47/6803C07K16/18C07K2317/569C07K2317/92C07K2317/94C07K2319/31
Inventor 郜鹏何志娟仲夏马琳郭树华
Owner REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap