Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation

Tingting Li; Qingbing Zheng; Hai Yu; Dinghui Wu; Wenhui Xue; Yuyun Zhang; Xiaofen Huang; Lizhi Zhou; Zhigang Zhang; Zhenghui Zha; Tingting Chen; Zhiping Wang; Jie Chen; Hui Sun; Tingting Deng; Yingbin Wang; Yixin Chen; Qinjian Zhao; Jun Zhang; Ying Gu; Shaowei Li; Ningshao Xia

The novel coronavirus grouped in betacoronavirus genus has become the third serious 41 virus intruder to human in the coronaviridae, after sever acute respiratory syndrome Organization (WHO) has declared the SARS-CoV-2 epidemic as a pandemic of 53 international concern and updates the COVID-19 situation every day. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994152 doi: bioRxiv preprint vaccine design. S is consisted of S1 and S2 subunits and the cleavage on S1/S2 62 boundary by protease during biosynthesis is prerequisite for coronaviruses cellular The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994152 doi: bioRxiv preprint subjected to clinical trials 12 . Here, we cloned S ectodomain and its fragments RBD and 84 S1 into recombinant baculovirus and expressed the proteins in insect cells. We found 85 that S and S1 formed homotrimer in solutions and the three proteins reacted well with 96 To screen a potent immunogen for COVID-19 vaccine development, we designed three 97 constructs-S ectodomain, S1 and RBD-for the SARS-CoV-2 Spike (S) protein 98 expression by aligning the SARS-CoV-2 S gene (Genbank accession no. NC_045512.2) 99 to a SARS-CoV strain (Genbank accession no. NC_004718) S gene sequence in terms 100 of structure-defined domain profile of the SARS-CoV S protein (Fig. 1A) . The gene of 101 SARS-CoV-2 S ectodomain encoding amino acids (aa) 15-1,213 with removal of its 102 original signal sequence was cloned to the downstream of the gp67 signal sequence in 103 pAcgp67B plasmid vector (Fig.1B) . and with its C-terminal addition of a thrombin 104 cleavage site, a T4 trimerization foldon motif and his tag. The segments S1 (aa 15-105 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.16.994152 doi: bioRxiv preprint 680) and RBD (aa 319-541) were cloned similar to S ectodomain, keeping gp67 signal 106 peptide and his tag to facilitate secretory outside cell and affinity purification, 107 respectively, but without the thrombin site and T4 foldon (Fig. 1A) . The three 108 constructed plasmids were respectively co-transfected into Sf9 insect cells with v-109 cath/chiA gene deficient baculovirus DNA for the generation and amplification of 110 recombinant baculovirus, which were then harnessed to infect Hive Five insect cells to 111 eventually produce recombinant proteins, named S, S1 and RBD respectively.