IPTG-Inducible ASFV Attenuated Strain for D1133L Gene Control

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Solution Overview

Problem

Current methods are unable to effectively knock out structural and non-structural proteins of viruses like ASFV, making it difficult to understand their role in virus replication and immune modulation, particularly due to the immunosuppressive effects of the D1133L protein which inhibits IFN-β and downstream cytokines.

Innovation Solution

An attenuated strain of ASFV with IPTG-induced deletion of the D1133L gene is created using a recombinant virus constructed by inserting a p72-eGFP-U104L-LacI-p72-LacO screening expression cassette into the ASFV genome, allowing conditional control of D1133L expression, thereby reducing immunosuppressive activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If gene editing technology is used to knock out viral proteins, then the role and assembly mechanism of viral proteins can be clarified, but structural and non-structural proteins of viruses like ASFV cannot be directly knocked out

Engineering Contradiction:

Improveclarification of viral protein role and assembly mechanismVSAvoidability to knock out viral proteins

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent introduces a conditional knockout system using an inducible promoter (tet promoter) as an intermediary mechanism. The D1133L gene is equipped with a tet operator sequence that allows conditional expression control through doxycycline induction. This intermediary system enables researchers to control viral protein expression timing and conditions, overcoming the limitation of traditional gene editing that cannot directly knock out certain viral proteins.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Object-affected harmful factors

If the D1133L gene is knocked out to study viral replication, then the immunosuppressive effect is reduced, but the ability to control gene expression is lost

Engineering Contradiction:

Improveimmunosuppressive effectVSAvoidconditional control of gene expression

Core Design Contradiction:

Object-affected harmful factorsVSAdaptability or versatility

Solution Approach 1:

The patent creates a dynamic gene expression system where the D1133L gene expression can be switched on or off based on doxycycline presence. The tet promoter and operator sequence create a controllable system that allows dynamic adjustment of viral protein levels, enabling researchers to study both the normal function and the immunosuppressive effects by simply adding or removing the inducer.

Inventive Principle:

Principle #15Dynamics

3Ease of manufacture

If traditional gene knockout methods are used, then the process is simple, but the D1133L protein immunosuppressive activity cannot be reduced

Engineering Contradiction:

Improvesimplicity of gene knockout processVSAvoidimmunosuppressive activity

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The patent changes the expression parameter of the D1133L gene from constitutive to inducible expression. By introducing the tet operator sequence and using the doxycycline-inducible tet promoter, the system allows precise control over when and how much D1133L protein is produced. This parameter change enables reduction of immunosuppressive activity while maintaining a relatively simple genetic modification approach.

Inventive Principle:

Principle #35Parameter changes

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 approach enables controlled expression of the D1133L gene, facilitating the study of viral protein functions and immune modulation, and demonstrates reduced immunosuppressive effects on IFN-β and downstream cytokines, aiding in understanding virus-host interactions and viral replication mechanisms.

Implementation Method 1

An enzyme encoded in a Lac operon, under negative regulation of a lac repressor, Lactose operon repressor (LacI), can specifically bind to a lac operator gene (LacO) and further bind to a 21-bp sequence of the gene with a high affinity, repressing enzyme expression.

Methodology Applied

Scientific EffectTranscriptional repression:

Implementation Method 2

The Lactose operon repressor (LacI) can also bind to allolactose or IPTG, thereby reducing the affinity of the repressor to the operon. In this way, IPTG can reduce inhibition of Lac operon transcription, thereby inducing expression of Lac operon.

Methodology Applied

Scientific EffectInduction by allolactose or IPTG:

Data Source

PatentUS11987797B2Attenuated strain of African swine fever virus (ASFV) with IPTG-induced deletion of <i>D1133L </i>gene and use thereof

Publication Date: 2024.05.21 LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)

AI summary

The present disclosure belongs to the technical field of biology, and in particular relates to an attenuated strain of African swine fever virus (ASFV) with IPTG-induced deletion of a D1133L gene and use thereof. In the present disclosure, it is firstly found that the D1133L protein of ASFV can inhibit production of IFN-β and downstream cytokines ISG-15 and ISG-56, and can be used as an immunosuppressant with a relatively strong immunosuppressive effect. In addition, the attenuated strain of ASFV with IPTG-induced deletion of the D1133L gene is constructed. Specifically, a screening expression cassette is inserted into a position before the non-structural protein gene D1133L of the ASFV using an Escherichia coli lac operator-repressor system, to obtain a recombinant virus. In the presence of the IPTG, the recombinant virus has similar characteristics to a wild-type virus; and in the absence of the IPTG, the expression of the D1133L protein is inhibited.