Construction And Screening Of SARS-CoV-2 Ribosomal Display Nanobody Library

COVID19 caused by SARS-CoV-2 is still rampant all over the world,seriously threatening human life and health and public health safety.Currently,covid-19 vaccines are the main control measures for new crown pneumonia.Among them,antibodies have the characteristics of high specificity,strong targeting,low toxicity and side effects and significant curative effect,which provides a new choice for emergency prevention and treatment of diseases.Antibody miniaturization is one of the development directions of antibody drugs.There is a heavy chain antibody with natural deletion of light chain in camels（camels,alpacas,etc.）and sharks.The NBS prepared based on the variable region of heavy chain antibody is also called VHH antibody.The molecular size of nano antibody is about 15k Da,which is only 1/10 of that of traditional whole molecule antibody.It is an antigen binding fragment with complete structure and minimum function.It has the advantages of good water solubility,easy expression,stable structure,strong affinity,large-scale production and easy humanization transformation.It has shown broad development prospects in the field of medicine.The development of nano antibody is different from the preparation of monoclonal antibody by traditional hybridoma cells.It is generally screened by non immune antibody library or immune antibody library.The common construction technologies of nano antibody library include phage display technology,ribosome display technology,yeast display technology and so on.Among them,ribosome display technology combines m RNA and correctly translated protein on ribosomes in vitro by simulating the internal environment of cells to form"m RNA ribosome protein"ternary complex,so as to realize the coupling of genotype and phenotype.Ribosome display technology has the advantages of convenient database construction,easy screening,large library capacity and good diversity.It has been widely used in the screening and evolution of small molecular antibodies,and has emerged in antibody screening,tumor diagnosis and treatment,proteomics and so on.In this study,Australian alpaca was immunized with SARS-CoV-2 specific immunogen.After6 times of immunization,the peripheral blood lymphocytes of Alpaca were isolated,and the total RNA was extracted and reverse transcribed into c DNA.The VHH gene was amplified by nested PCR,and the necessary elements required for ribosomal display nano library were introduced.The phage gene-Ⅲgene was connected with VHH gene by SOE-PCR,and the ribosomal display nano antibody library was constructed and its library capacity was calculated.The library capacity was as high as 1.12×10¹³.In addition,the RBD recombinant eukaryotic expression plasmid p CAGGS RBD of SARS-CoV-2 spike protein was successfully constructed.The recombinant plasmid was successfully expressed in the supernatant of mammalian Expi293F cells and identified correctly by Western blot.The RBD protein expressed in eukaryotic system was purified by twin strep label purification column,and the s SARS-CoV-2 RBD protein with purity of 97.5%and screening quality was successfully obtained.The constructed ribosomal display nano antibody library was further screened with SARS-CoV-2 RBD antigen by solid-phase screening.After screening,the eluted m RNA was reconstructed into a new nano antibody library for the next round of screening.After four rounds of panning,the nano antibody gene specific for SARS-CoV-2 was successfully enriched,and the obtained antibody gene was sent to the company for sequencing.The obtained gene sequence was analyzed by MEGA7 software,The results showed that the VHH gene sequence obtained by ribosome display technology had good integrity and diversity,which laid a foundation for the subsequent acquisition of nano antibodies.