Dados do Trabalho

Título

EXPRESSION AND PURIFICATION OF RECOMBINANT NUCLEOCAPSID AND SPIKE PROTEINS OF SARS-COV-2 FOR SEROLOGICAL DIAGNOSIS

Introdução

During the COVID-19 pandemic the measurement of IgG from the population by Enzyme-Linked Immunosorbent Assay (ELISA) was an important tool to determine real disease epidemiology. The expression of recombinant proteins is important for the monitoring of antibodies in human serum against such pathogens.

Objetivo (s)

To express the nucleoprotein (N) and the spike glycoprotein (S) D614G of SARS-CoV-2 as recombinant proteins for application in serodiagnosis methods.

Material e Métodos

The N and S protein sequences were retrieved from GenBank (NC_045512). The design of S protein considered the D614G mutation. The sequences were optimized for protein expression. The sequence of N protein was cloned in the pET21a vector and expressed in Escherichia coli (DE3) pRARE2, while the S D614G was cloned in pCMV vector and expressed by Human Embryonic Kidney (HEK) 293FT cell line. Both proteins were expressed as N-terminal His-tagged protein and purified by affinity chromatography. Serum was collected from people positive or negative for COVID-19 RT-qPCR after 15 days of the diagnosis. The samples were validated for IgG production using a commercial N protein. Human sera from naturally infected people or controls were used to validate the produced recombinant protein by ELISA. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Integrated Manufacturing and Technology Campus (CIMATEC – Salvador, BA, Brazil) (approval number 4,334,505).

Resultados e Conclusão

N and S D614G recombinant proteins were detected by Western blot using an antibody against the C-terminal 6-histidine tag, which revealed bands at 48 and 150 kDa, respectively. Seventy-eight serum samples were used to validate the N protein and 70 to validate the S D614G. By ELISA, the assay using the N protein presented a sensitivity of 73% and specificity of 100%, while the assay using the S D614G presented sensitivity of 100% and specificity of 93%.
The purified recombinant proteins presented satisfactory performance in serodiagnosis, could detect true positive samples based on the RT-qPCR, and presented low false results. These proteins can be used in the monitoring of IgG levels against Sars-Cov-2.

Palavras-chave

Recombinant proteins. SARS-CoV-2. Serodiagnosis.

Agradecimentos

This study was supported by the National Research and Development Council (Grant Number: 424911/2021-3) and the Ministry of Science, Technology and Innovation of the Federal Govern and National Department of SENAI (SENAI DN – Scholarship No. 329266).