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

Human agkistrodon acutus venom immune ScFv antibody library and construction method thereof

A technology of a pit viper and a construction method, which is applied in the field of human-derived pit viper venom phage antibody display, and can solve the problems of reduced titer, increased dosage of antiserum, different toxicity and immunogenicity, etc.

Inactive Publication Date: 2013-10-23
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the quality of antivenom has been greatly improved after decades of preparation technology development, it is still mainly obtained by super-immunizing horses or sheep with whole snake venom.
Since snake venom is a complex protein mixture, containing more than 100 kinds of diverse venom proteins, its toxicity and immunogenicity are different, the current whole snake venom immunization method has the following problems: (1) potency will be affected by non-toxin components (2) Antivenom serum is an animal-derived antibody, which contains a large number of non-specific snake venom antibodies and many foreign proteins, and a considerable proportion of patients are due to Immediate hypersensitivity reaction due to allergy to animal protein; (3) exogenous protein can also initiate an anti-foreign protein response, causing delayed allergic reaction mediated by immune complexes, leading to glomerulonephritis and even renal failure , such as serum sickness
(4) Because Agkistrodon acutus venom is very toxic, animals can only tolerate small doses of venom each time they are immunized. In this state, some toxic components of snake venom may only have sub-immunogenic amounts, and immunization operations must use A method in which the dose of immunization is increased gradually, possibly over several months
(5) Animal-derived antivenom has high production and storage costs
At present, there are no related research reports

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
  • Human agkistrodon acutus venom immune ScFv antibody library and construction method thereof
  • Human agkistrodon acutus venom immune ScFv antibody library and construction method thereof
  • Human agkistrodon acutus venom immune ScFv antibody library and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 primer design

[0055] Amplification of the variable region gene of a Agkistrodon venom antibody

[0056] 1. Design of primers for human antibody V gene region amplification:

[0057] The reference material comes from "Daniele Sblattero, Andrew Bradbury, A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology3 (1998) 271–278."; V BASE database website: http: / / vbase.mrc-cpe.cam.ac.uk

[0058] The primer sequences involved are as follows:

[0059]

[0060] According to the above reference sequence and the planned T7 Phage Select System (Novagen) phage sequence, this experiment designed the antibody variable region gene V H , V L The primers introduce EcoRI and HindIII restriction endonuclease sites at the upstream and downstream respectively. Specifically designed primers are as follows:

[0061]

[0062]

[0063] where r=a / g, y=c / t, m=a / c, k=g / t, s=c / g, w=a / t, h=a / c / t, b=c / g / t, v=a / c / g, d=a / g / t, n=a / c / g / ...

Embodiment 2

[0064] Example two total cDNA extraction

[0065] 1. Extract total RNA from whole blood of positive patients

[0066] 1) Pre-experiment - small amount of total RNA extraction

[0067] Thaw the whole blood stored at -70°C on ice, take 3ml and put it into a 15ml centrifuge tube, add 6ml TRNzol-A+, 3ml chloroform, and mix thoroughly for 5 minutes. Centrifuge at 10,000g for 10 minutes, absorb about 2.5ml of the supernatant and add to a new 15ml centrifuge tube, add 2.5ml of isopropanol and 250μl of sodium acetate, mix by inverting and place on ice for 1 hour. Centrifuge at 10,000g for 20 minutes, wash the pellet twice with 70% ethanol, dry at 37°C, add 50μl TE to dissolve and transfer to a 1.5ml centrifuge tube. Determination of total RNA: OD260 / 280=1.94, 20ng / μl, 3ml of whole blood can get a total of about 1μg of RNA. According to the estimation of 1% as mRNA, the total mRNA is about 10-30ng, which is far from enough to meet the requirement of RT-PCR.

[0068] 2) Extract a lar...

Embodiment 3

[0078] Example 3 PCR Amplification of Total Antibody Gene V Segments

[0079] Main reagents: polymerase. (CatNo.: DR010A, TaKaRa Company), gel recovery DNA purification kit (CatNo.: 28706, Qiagen Company).

[0080] 1) to V L and V H Amplification of the area:

[0081] V H Region; upstream primers VL1-01-F, VL1-02-F and downstream primers VL1-01-R, VL1-02-R are paired to amplify V L 1 region; upstream primers VL2-01-F, VL2-02-F, VL2-03-F paired with downstream primers VL2-01-R, VL2-02-R to amplify the VL2 region.

[0082] The 20μl reaction system is as follows:

[0083]

[0084] PCR reaction program:

[0085]

[0086] The amplified product was subjected to 1.5% agarose gel electrophoresis, and the loading volume was 3 μl / well. The results were as follows: For V L Region amplification, the target fragment size is about 360bp, such as figure 2 shown. to V H For the amplification of the region, the target fragment is about 370bp, such as image 3 shown.

[0087]...

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

PropertyMeasurementUnit
Titeraaaaaaaaaa
Login to View More

Abstract

The invention relates to the antibody engineering medicine and particularly relates to the phage antibody display technology, namely a construction method of a human agkistrodon acutus venom immune ScFv antibody library. The method comprises the following steps of: performing PCR (polymerase chain reaction) amplification by use of human specific IgG light chain and heavy chain primers to obtain PCR products; mixing all PCR products to obtain a light chain PCR mixed product and a heavy chain PCR mixed product respectively; introducing specific primers by taking the light chain PCR mixed product and heavy chain PCR mixed product as templates, and performing PCR amplification to obtain a Linker-VL fragment and a VH-Linker fragment; connecting the obtained Linker-VL fragment and the obtained VH-Linker fragment to obtain a VH-Linker-VL fragment; and importing the obtained VH-Linker-VL fragment into a phage cloning vector to obtain an original library of the human agkistrodon acutus venom immune ScFv antibody library.

Description

technical field [0001] The invention relates to antibody engineering medicine, in particular to a phage antibody display technology, in particular to a human-derived Agkistrodon venom phage antibody display technology. Background technique [0002] Venomous snake bite is one of the common clinical emergencies. It has the characteristics of acute onset, rapid changes, many complications, high disability and fatality rate, etc. It is very difficult to treat, especially for patients with multiple organ failure, the treatment is even more difficult. Agkistrodon viper (pentapod viper) is one of the three most common venomous snakes in my country. After being bitten by Agkistrodon viper, the most acute reaction is tissue inflammation, edema, necrosis, bleeding without coagulation, etc. Among them, abnormal blood coagulation is the most common The main characteristic toxic reaction, and easily secondary systemic multiple organ failure. The pathological basis is that Agkistrodon ve...

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): C40B50/06C40B40/10C07K16/18
Inventor 刘明华张雷曹宇亮
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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