Abstract
The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.
Highlights
Humanity is faced with an unprecedented challenge—the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes a severe respiratory illness—coronavirus disease 2019 (COVID-19) pandemic
We describe a method of generating stably transfected Chinese hamster ovary (CHO) cell lines, secreting large quantities of monomeric SARS-CoV-2 receptor-binding domain (RBD), suitable for serological assays
Serological assays for detecting seroconversion upon SARS-CoV-2 infection are mostly based on two viral antigens– N and S proteins, or fragments of the S protein, including the RBD
Summary
Humanity is faced with an unprecedented challenge—the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes a severe respiratory illness—coronavirus disease 2019 (COVID-19) pandemic. Countries were sent to lockdown; people could not make informed decisions about the possibility of social contacts; the need for diagnostic tests is very high. At the beginning of the pandemic, polymerase chain reaction (PCR) testing methods dominated since such test systems can be developed urgently, soon after the emergence of a new virus in the population. Serological testing makes it possible to reliably determine whether a person is infected with the SARS-CoV-2, even in the absence of disease symptoms, or long after the event of infection. Serologic tests are needed to detect convalescent plasma of therapeutic interest and assess emerging vaccines’ effectiveness
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