Seminar

 Optimisation Of Recombinant GP41 Protein For Use In HIV Diagnostic Assay

Time: 13:30 - 13:45

Date: 25 April 2018

Theatre: Dodder A

Synopsis

Optimisation Of Recombinant GP41 Protein For Use In HIV Diagnostic Assay

Molecular Diagnostics

School of Biological Sciences, Dublin Institute of Technology, Kevin Street, Dublin 2

Gilligan M, Beale S, Chia H, Beck I, Naughton JA, Goosen C

Human Immunodeficiency Virus (HIV) has been long established as the causative agent for Acquired Immune Deficiency Syndrome (AIDS). Recommendations are that initial testing for HIV should be carried out using an antigen/antibody combination immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) (1). The project objective was to assess the functionality of two variants of the reference recombinant HIV protein Gp41 used in a commercial ELISA kit; a wild-type variant with a poly-His tag fused to its C-terminus and a mutant variant without cysteines produced using splicing by overlap extension PCR.

High-throughput cloning and transformation methods were used to establish the optimal expression vectors for both constructs via expression screening (2). The best expression vectors, determined by SDS-PAGE and Western Blot analysis, were selected for large-scale expression testing. The protein was purified by Immobilised Metal Affinity Chromatography (IMAC) (3). The wild-type and mutant protein products were biotinylated and their reactivity assessed in ELISA.

Although both constructs were observed to be functional in ELISA, neither the wild-type nor the mutant construct performed as well as the reference construct. The mutant construct performed comparatively well with an average reactivity of 54% of the reference standard. The wild-type protein product had an average reactivity determined to be less than 30% of the reference standard. However after use of automated purification techniques and optimisation of the biotinylation process, the mutant product was observed to be more reactive in ELISA than the current reference standard.

References

(1) Branson B, et al. Laboratory Testing for the Diagnosis of HIV Infection Updated Recommendations. Centers for Disease Control and Prevention. 2014: p. 8-14.
(2) Bryksin A, Matsumura I. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques. 2010;48(6):463-465.
(3) De Costa F, et al. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC). Methods in Molecular Biology. 2016;1405:91–97.

Speakers

  • Maud Gilligan DIT

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