Expression And Neutralization Activity Of SARS-CoV-2 Neutralizing Nanobody In Silkworm

Coronavirus disease 2019（COVID-19）is caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）.The receptor binding domain RBD on the virus S protein binds to the cell receptor ACE2 to allow the virus to enter the cell.As a member of the small molecule antibody,nanobody has greater advantages compared to other antibodies.Its smaller molecular weight makes it less immunogenicity,stronger penetration,better stability,and higher affinity.Nanobody against S protein can specifically target the receptor binding domain RBD of novel coronavirus S protein,and prevent it binding to the receptor ACE2 of cell membrane.Ferritin can self-assemble to form a natural protein nanocage,which has a good application prospect in protein display,antigen presentation,drug targeted delivery and other aspects.By fusing nanobody with ferritin,using its self-assembly characteristics,nanobody can be displayed on the surface of ferritin in the form of 24 polymer,which can improve the targeting affinity of nanobody to S protein and thus improve its neutralization activity.In this study,the neutralizing nanobody H11-D4 of SARS-Cov-2 was selected and connected with the C-terminal of human ferritin heavy chain through linker.The recombinant proteins H11-D4 and Fe-H11-D4 were expressed by E.coli and Bombyx mori baculovirus expression system respectively,and the neutralizing activity of the target protein was detected by pseudovirus neutralization test.The specific research contents are as follows:After optimized synthesis of SARS-Cov-2 neutralizing nanobody H11-D4 gene and human ferritin heavy chain gene,it was cloned into prokaryotic expression vector p ET28a and transformed into Rosetta（DE3）strain for expression to obtain the target proteins H11-D4and Fe-H11-D4.The yields of H11-D4 and Fe-H11-D4 obtained after purification were25.16mg/L and 8.70mg/L respectively.After dialysis renaturation,the self-assembly of ferritin fusion H11-D4 was observed by transmission electron microscope as a spherical structure of about 20 nm,and H11-D4 was displayed on the surface of ferritin.The results showed that H11-D4 and Fe-H11-D4 could prevent pseudovirus from entering cells and had the ability of pseudovirus neutralization.The IC₅₀ were 171.1 nmol/L and 20.87 nmol/L respectively.The results showed that the neutralizing activity of the nanobody fused with ferritin was significantly higher than that expressed alone,which proved that the poly nanobody on the surface of ferritin improved the targeting affinity to S protein.H11-D4 and Fe-H11-D4 genes were cloned into baculovirus transfer vector p BR and co-transfected with inactivated rescue baculovirus DNA into silkworm bm-N cells to obtain recombinant baculoviruses rebambac-H11-D4 and rebambac-Fe-H11-D4.The hemolymph of silkworm was collected after the recombinant baculovirus was infected with silkworm and the expression of Fe-H11-D4 was 101.23μg/m L detected by ELISA.The ferritin nanobody fusion protein Fe-H11-D4 was self-assembled into a 20nm globule-like structure in bombyx mori by electron microscopy after overspeed centrifugal and purification,and H11-D4 was displayed on the surface of ferritin.The neutralization titers of H11-D4 and Fe-H11-D4 in silkworm blood were 1:1226 and 1:2080 respectively.According to the concentration of Fe-H11-D4 protein in silkworm blood,the IC₅₀ of Fe-H11-D4 expressed in silkworm blood is calculated to be 1.46 nmol/L,and the IC₅₀ is significantly higher than that expressed in prokaryotic Fe-H11-D4,which proves that the neutralizing activity of the fusion protein expressed in silkworm blood is higher than that of the ferritin fusion protein purified and refolded in prokaryotic,reflecting the advantage of silkworm baculovirus in post-translational processing and modification of foreign proteins.In this study,the nanobody and ferritin were fused to expressed.on the basis of self-assembly characteristics of ferritin and using E.coli and baculovirus expression system,the nanobody was displayed on the surface of ferritin in the form of 24 polymer.The experimental results show that the nanobody displayed by ferritin can significantly improve the targeting affinity to S protein,so as to improve its activity.Baculovirus expression system has the function of post-translational processing and modification.Therefore,the function of ferritin nanoscale display polyprotein can be utilized to the maximum extent,which lays a foundation for establishing a low-cost,high active drug for diagnosis and treatment of COVID-19.