pComb3H vector

It has been reported that the viral nucleocapsid protein (NP) of SARS-CoV-2 is usually very conserved, highly immunogenic, and abundantly expressed during infection, suggesting that NP can be used for early diagnosis of SARS-CoV and MERS infections. In this study, researchers used phage display technology for the first time to construct a comprehensive human antibody library from five convalescent COVID-19 patients. A panel of human monoclonal antibodies was obtained by panning with purified NPs. Subsequently, the properties of the antibodies were evaluated, including binding specificity, affinity for NPs, targeting epitopes, and application in diagnostics.

In this study, 5 RNA samples extracted from PBMCs of 5 recovered COVID-19 patients were used as templates for cDNA synthesis. The five cDNA samples were mixed evenly to form a template pool. The full-length light chain gene and the Fd fragment of the heavy chain gene (variable region and CH1 domain of the heavy chain) were amplified by PCR from the cDNA template library using the primer pairs 5VK, 7VL, and 8VH gene families. The light chain gene was cloned into the phagemid vector pComb3H between the Xab I and SacI restriction sites to form a light chain gene library. The heavy chain genes were cloned sequentially into the light chain library between the Xho I and Spe I restriction sites in pComb3H. Through two-step cloning, the final anti-SARS-CoV-2 phage antibody library was constructed.

Two independent anti-SARS-CoV-2 Fab fragment antibody libraries were established using the pComb3H vector system, one for the lambda chain and one for the kappa chain. The two phage libraries contained 1×109 and 1.4×109 independent clones and 100% Fab gene diversity, confirmed by sequencing. After three rounds of panning, 192 randomly picked colonies were further screened by ELISA to evaluate binding activity to SARS-CoV-2 NPs. Finally, 178 positive clones were identified.

Figure 1. Generation of human antibodies against SARS-CoV-2 NP by screening phage libraries. (Zhang L, et al., 2020)