Preparation Of Polyclonal Antibody Based On LbCas12a And Study Of Linear B Cell Epitope

CRISPR-Cas is an immune defense system derived from archaea,which has evolved into a powerful tool for gene editing,DNA assembly,whole gene screening,gene expression regulation,and nucleic acid detection since its inception.Among the many CRISPR system types,the mature cr RNA sequence and secondary structure of CRISPRCas12 a are highly conservative,and based on this conservatism,its simple structure and efficient function,researchers have also put more attention and research into it.As a potential medicinal protein,the immunogenicity of Cas12 a protein will become a major problem facing the field of gene therapy.We hope to provide a theoretical basis for the study of the immunological properties of Cas12 a by exploring the B cell epitope.In this study,Lb Cas12 a,which belongs to the source of ND2006,obtained the Lb Cas12 a protein with His tag by inducing the expression of the prokaryotic nucleus system BL21/p ET28a-His-Lb Cas12 a,which was extracted,isolated and purified as immunogen to immunize animal.We obtained a p Ab with high titer and good specificity.Then,by integrating the hydrophilicity,surface accessibility,flexibility,antigenicity,?-turn amino acid properties associated with B cell epitopes,using bioinformatics multiparameter machine learning tools to predict the 9 candidates linear B Cell epitope of Lb Cas12 a.Finally,these candidate epitopes called LBEs was recombined with GST tag,and the GST-LBE series fusion protein was obtained by inducing expression,extraction and purification.We identify their antigenicity in vitro,here are 3 epitopes which have highly antigenicity,namely LBE1,LBE5 and LBE7.Then,Balb/c mice were immunized with them to verify the immunogenicity in vivo,the analysis shows that LBE1 and LBE5 are the most dominant B cell epitopes with both antigenicity and immunogenicity.3D structural analysis revealed that amino acids of LBE1,LBE5 and LBE7 were in close proximity respectively and may be exposed on the surface of the Lb Cas12 a protein.To sum up,we produced Lb Cas12 a polyclonal antibodies,predicted and screened the linear B cell epitope of Lb Cas12 a,and verified its antigen and immunogenicity.The results provide a basis for further analysis of the structure and function of Cas12 a protein,and also provide a new way of thinking for the modification and application of subsequent proteins.