Construction And Immunogenicity Analysis Of S-RBD Engineering Cells

Severe acute respiratory syndrome coronavirus 2,severe acute respiratory syndrome and coronavirus caused by SARS-Co V-2,people who have recovered from the COVID-19 pandemic in large numbers are also at risk of re-infection,and while the situation is still severe,The most effective way to prevent and control COVID-19 is to establish a herd immunity barrier through the development and use of COVID-19 vaccines to protect vulnerable populations and cut off the spread of the virus.SARS-Co V-2 recognizes SARS-Co V-2 by RBD on spike and binds to host cell receptor angiotensin-converting enzyme 2 to mediate viral conversion to target cells.Moreover,RBD sequence is immunogenic and can induce specific antibodies to neutralize SARS-Co V-2.Thus blocking the virus from entering the host cell.Therefore,RBD is considered to be a major target for COVID-19 vaccine development.In vaccine development,there is still no unified standard for the selection of expression system.In this study,based on the properties that RBD sequence can form multiple pairs of disulfide bonds,and has two potential N-glycosylation modification sites and multiple o-glycosylation sites,a eukaryotic expression system that can maintain the natural conformation and activity of the protein is selected to express recombinant RBD.In this study,firstly,BY46.9L(signal peptide +RBD+Ig G light chain)sequence was digested and cloned into GS p327.7 double expression vector,and the GS p327.7-BY46.9L plasmid was constructed.BY46.9H(signal peptide +RBD+Ig G heavy chain)sequence was cloned into GS p327.7-BY46.9L vector,and the recombinant GS p327.7-BY46.9L-BY46.9H dual-expression plasmid was successfully constructed,and transfected into CHO cells to secrete and express CHO-RBD fusion protein.Recombinant expression plasmid p PIC9K-RBD was constructed and electrotransfered into Pichia pastoris expression vector to secrete recombinant yeast RBD protein.Secondly,the recombinant proteins expressed from two sources were purified by affinity chromatography.The purity of the two target proteins was identified by SDS-PAGE electrophoresis,and both of them were successfully expressed in supernatant.Thirdly,CHO-RBD antigen,yeast-RBD antigen and commercial Jinno-Rbd antigen were used as coated antigens by indirect ELISA method,and the titers of Ig G specific antibodies in serum of positive mice immunized with 2019-n COV whole virus inactivated stock were 1:16 000,1:80 and 1:16000,respectively.Finally,CHO-RBD antigen and Gino commodity RBD antigen were combined with aluminum oxide adjuvant to prepare recombinant subunit vaccine.BLAB/c mice were immunized by peritoneal approach.After immunizing mice with CHO vaccine,the mice produced Ig M antibody titer of 1:320 and Ig G antibody titer of 1 against SARS-Co V-2.20480 The titer of Ig M antibody and Ig G antibody was 1:1280 and 1:520 in mice immunized with the vaccine.In conclusion,this study successfully prepared two recombinant RBD proteins with immune reactivity against SARS-Co V-2,in which CHO-RBD combined with adjuvant can induce mice to produce Ig G and Ig M specific antibodies against SARS-Co V-2,providing the direction for the preparation of SARS-COV-2 vaccine based on different expression systems.