Human Abzyme Production via Selective Vκ Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The development of human abzymes with enzyme activity has been hindered by the limited availability of fully human sequences from Bence Jones proteins, and existing methods struggle to efficiently obtain human abzymes with antiviral activity against pathogens like rabies and influenza viruses.
Innovation Solution
A two-stage PCR reaction using primers designed for the leader sequence of Vκ genes belonging to subgroup II allows for the selective amplification and production of human antibody κ light chains with high antiviral activity, specifically a polypeptide with a variable domain represented by the amino acid sequence shown in SEQ ID NO: 50, which exhibits nucleolytic and antiviral properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If human abzymes are obtained from Bence Jones proteins of multiple myeloma patients, then fully human sequences can be obtained, but the availability is limited and only a few contain enzyme activity
Solution Approach 1:
The patent uses PCR technology as an intermediary method to amplify and obtain human antibody light chain genes from human cDNA, bypassing the need to directly extract from Bence Jones proteins. This mediator approach allows selective amplification of Vκ genes belonging to subgroup II, which are more likely to produce abzymes with enzyme activity, thereby improving both availability and efficiency.
Solution Approach 2:
The patent creates copies of the desired human antibody light chain genes through PCR amplification from human cDNA library. By using specific primers that target Vκ genes of subgroup II, the method generates sufficient copies of candidate genes for further analysis and abzyme production, overcoming the scarcity of suitable human sequences from multiple myeloma patients.
2Ease of manufacture
If conventional PCR methods are used to amplify human antibody light chains, then amplification can be performed, but selectivity and effectiveness are insufficient
Solution Approach 1:
The patent applies local quality by designing primers with specific sequences that match the leader region of Vκ genes belonging to subgroup II. This localized specificity in the primer design allows selective amplification of the desired gene subset from the complex human cDNA library, achieving both ease of manufacture and high selectivity.
Solution Approach 2:
The patent optimizes PCR parameters including primer sequence, annealing temperature, and cycling conditions to enhance the selectivity and effectiveness of amplification. By adjusting these parameters, the method achieves preferential amplification of Vκ genes from subgroup II while minimizing non-specific amplification of other antibody genes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the production of a novel human antibody κ light chain with high antiviral activity against rabies and influenza viruses, offering a potential treatment with minimal side effects, and provides a primer set and vector for efficient expression and purification.
Implementation Method 1
two-stage PCR reaction using a primer designed for a first primer on the basis of leader sequence which is characteristic of a Vκ gene belonging to a subgroup II
Implementation Method 2
a first primer for first-stage PCR reaction, the first primer being a polynucleotide having a domain hybridizable with the template in the first stage PCR reaction
Implementation Method 3
a human abzyme which is a light chain of a human antibody for a rabies virus and includes enzyme activity... having a nucleolytic activity
Data Source
AI summary
The present invention provides a human abzyme containing a human antibody κ light chain.