Production of single chain variable fragments of anti-RBD antibody of SARS-CoV-2 in Saccharomyces cerevisiae and development of COVID-19 diagnostic kit

• Grant number: 21/13013-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2022
• End date: September 30, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Sandro Roberto Valentini
• Grantee: Enzo Corvello
• Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

The new coronavirus pandemic (COVID-19) has caused socioeconomic impacts around the world, generating a setback that highlighted hunger and unemployment. This virus circulates in the air and secretions, so several measures were taken to control its spread. However, aiming for a more assertive control of the contaminated people, it was necessary to develop specific diagnostic tests that are sensitive to SARS-CoV-2, since the standard test (RT-PCR) is costly and demands specialized equipment and personnel. Furthermore, an obstacle to Brazilian autonomy in the testing scenario was the lack of technology that would enable the country to develop and produce inputs for the production of diagnostic kits. This left Brazil at the mercy of the availability of these products from countries that are also facing the pandemic and have a domestic and foreign market to supply. Thinking on that, this project aims to carry out the production of single-chain variable fragments (scFv) of the CC12.1 antibody, which has high affinity to the receptor binding domain (RBD) of SARS-CoV-2, in S. cerevisiae, to purify it and apply it in a diagnostic test prototype. For that, the coding sequence of the variable fragment of the light chain will be linked to the heavy sequence by a linker that encodes a repeat of serines and glycine. Then, this product will be cloned into a galactose-inducible yeast expression vector that allows the insert to be fused to a six histidine tail. After transforming, inducing and confirming the expression of scFv in yeast, the cells will be lysed and the scFv purified by metal affinity chromatography, and then used in a prototype immunoenzymatic test in order to verify its applicability and stability. (AU)