RECOMBINANT EXPRESSION OF THE VP2-BETA DOMAIN IN ESCHERICHIA COLI, PURIFICATION AND ITS USE FOR DETECTION OF ANTI-INFECTIOUS BURSAL DISEASE VIRUS ANTIBODIES BY DIPSTICK AND LATERAL FLOW STRIP

Abstract

Disclosed herein is the expression of beta-domain (Asp201-Gly339) of the VP2 protein (NCBI Acc. No. KT281984) of infectious bursal disease virus (IBDV), in codon optimized Rosetta 2 (DE3) strain of E. coli followed by its purification by affinity and gel filtration chromatography and use for development of dipstick and lateral flow strip for diagnosis of antibodies produced in chicken upon IBDV infection/vaccination.

Claims

1. A method of diagnosing presence of antibodies to infectious bursal disease virus (IBDV) by reacting a serum sample with beta-domain (Asp201-Gly339) of a VP2 protein of the IBDV conjugated with gold-nanoparticles as a positive indicator of infection.

2. The method of claim 1, wherein the beta-domain of the VP2 protein is expressed in a codon optimized Rosetta 2 (DE3) strain of E. coli and purified by consecutive affinity, ion-exchange and size exclusion chromatography.

3. The method of claim 1, wherein a test device for diagnosis comprises a dipstick further comprising a buffered sample application pad, a fiberglass pad infused with the beta-domain conjugated with gold-nanoparticles in communication with the application pad, and a nitrocellulose membrane pad marked with a test line of beta-domain non-conjugated protein and a control line of anti-IBDV antibodies, in communication with the fiberglass pad.

4. The method of claim 3, wherein a diagnosis is made by adding a serum sample to the sample pad and incubating the dipstick for 5-10 minutes, placed in a vertical or horizontal position, to enable the serum sample to migrate across the nitrocellulose membrane and concluding a positive test if both test and control lines are visible, and negative if only the control line is visible.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0014] FIG. 1: Structural representation (A) and cloning strategy (B) of expression clone of beta domain of the VP2 protein of IBDV

[0015] FIG. 2: Size-exclusion chromatography of the nickel-affinity purified recombinant beta-domain of the VP2 protein. Major peak at around 15 mL position represent major oligomeric form of beta-domain. The inset shows monomer, dimer and the trimeric forms of the beta-domain, analyzed by SDS-PAGE.

[0016] FIG. 3: Determination of protein concentration to produce stable beta-domain and gold-nanoparticle conjugates. Beta-domain concentration 0.6 g/200 L is taken an optimum concentration to produce stable conjugates.

[0017] FIG. 4: Dipstick format: Detection of anti-IBDV antibodies produced in chicken upon infection with IBDV. SPF represents specific pathogen free chicken

[0018] FIG. 5: Lateral flow strip: Detection of anti-IBDV antibodies produced in chicken upon infection with IBDV

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to the development of a dipstick and a lateral flow strip for the detection of the anti-IBDV antibodies using the recombinant beta-domain of the VP2 protein of IBDV. The VP2 protein is highly immunogenic and capable of capturing anti-IBDV antibodies. Being a major structural and antigenic component of IBDV, the beta-domain of the VP2 protein is represented here as a convincing candidate for the recombinant production and development of diagnostic tools. Another important aspect associated with the present invention is to provide antigen (beta-domain) that was produced from the VP2 sequence of a local (Pakistan) pathogenic very virulent IBDV isolate (Acc. No. KT281984). This use results in high antigen-antibody affinity and specificity in correspondence with diagnosis related to the local vvIBDV strains as compared to the diagnostic tools developed using the whole or the parts of IBDV belong to other geographical regions.

[0020] To pursue development of diagnostic assays, several virulent IBDV isolates from a recent outbreak in Punjab were collected and nucleotide sequence of the VP2 gene coding for a viral surface protein was amplified and submitted in GenBank under accession no.KT28 1 984.

[0021] In the proposed project, beta-domain of the VP2 protein was produced in Escherichia coli by recombinant means and purified using combinations of affinity, ion-exchange and/or size exclusion chromatography techniques. Purified protein was used for conjugation with gold-nanoparticles for developing on-site lateral flow immune-strip test or dipstick to detect anti-IBDV antibodies in the infected chicken. It is expected that locally developed diagnostic test targeting the genetic variability of local strains of IBDV will be more effective.

[0022] The preferred embodiment of the present invention is to develop tools in the form of dipstick and lateral flow strip using beta-domain of IBDV for diagnosis of anti-IBDV antibodies produced in chicken upon corresponding viral infection or vaccination. To construct dipstick and lateral flow diagnostic tools, in another embodiment, nucleotide sequence corresponding to beta-domain was cloned in an expression vector under the control of T7 promoter (FIG. 1) and expression conditions for synthesis of beta-domain were optimized in a codon optimized Escherichia coli, Rosetta 2 (DE3) using standard procedures.

[0023] In another embodiment, oligo histidine tagged beta-domain was purified using nickel-affinity and size exclusion chromatographic methods and integrity was verified by sodium dodecyl gel electrophoresis (FIG. 2). Purified protein at an optimized concentration of 0.6 g/200 L was used to produce stable beta-domain and gold nanoparticles conjugates (FIG. 3) using NaCl based standard method (https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_40_nm_Bare_Gold_Passive_Co njugation_Protocol.pdf).

[0024] In another preferred embodiments, to construct dipstick and lateral flow strip, technical aspect for precise interaction of antigen (beta-domain conjugated to gold nanoparticles) and anti-IBDV antibodies produced in chicken were optimized. Further, various components conjugate pad treated with beta-domain-gold nanoparticles conjugates, nitrocellulose membrane printed with test (beta-domain) and control (anti-IBDV antibodies), buffer treated adsorbent and sample pad were assembled to fabricate dipstick and lateral flow strip devices as described somewhere. These tools were tested as point-of-care device for detection of anti-IBDV antibodies FIGS. 4 and 5). Briefly, for the detection of IBDV antibodies, serum samples containing anti-IBDV antibodies (collected from field) or specific pathogen free antisera were added drop wise on to the sample pad (or dipstick immersed in diluted serum sample) and then strip/dipstick was incubated laterally to allow the sample to pass through the NC membrane for 5-10 minutes. According to the standard protocol, positive results correspond to appearance of two lines in the test and control region whereas negative result is denoted by the appearance of only one red line in the control region.