Cloning And Expression Of Chicken Shp-2,and Its Preparation Of Monoclonal Antibodies And Knockout In LMH Cell Line

Src homology region 2(SH2)protein tyrosine phosphatase 2(Shp-2)is a non-receptor tyrosine phosphatase,and ubiquitously expressed in various vertebrate cells.As a tyrosine phosphatase encoded by the proto-oncogene PTPN11,the expression of Shp-2 and its altered activity have been demonstrated to be closely related with the hematopoietic malignancy,juvenile myelomonocytic leukaemia and lots of human cancer diseases.However,the roles of Shp-2 in avian diseases have rarely been reported.To investigate the function of the chicken Shp-2 and its roles in the associated diseases,in this study,the chicken Shp-2 gene was cloned and expressed,the monoclonal antibodies specific to chicken Shp-2 were generated and the Shp-2-knockout LMH cell lines were established by the technique of CRISPR-Cas9.1.Cloning and expression of chicken Shp-2 geneIn order to obtain the immunogen and screening antigen for the preparation of monoclonal antibody specific to chicken Shp-2,the Shp-2 gene from chicken was cloned into the linearized vectors pGEX-6p-1 and pcDNA3.1,and the recombinant plasmids pGEX-6P-1-Shp-2 and pcDNA3.1-Shp-2 were successfully constructed.The positive recombinant pGEX-6P-1-Shp-2 was transformed into the Rosetta competent state and induced by IPTG.The SDS-PAGE analysis revealed that the GST-Shp-2 protein could be expressed either in the form of inclusion bodies or in the soluble form.Western blot analysis demonstrated that the recombinant protein GST-Shp-2 had good antigenic activity.GST-Shp-2 protein expressed in the soluble form was further purified by GST agarose gel column,which provided materials for the immunogen for the generation of monoclonal antibody specific to chicken Shp-22.Generation and characterization of monoclonal antibodies against chicken Shp-2In order to obtain monoclonal antibody against chicken Shp-2,BALB/c mice were immunized with the purified GST-Shp-2,DF-1 cells transfected with pcDNA3.1-Shp-2 were as screening antigens and the monoclonal antibodies were screened by IFA.Two hybridoma cell lines which secreting monoclonal antibody specific to chicken Shp-2 were obained and designated as 3A10 and 3G12.The subclass of both 3A10 and 3G12 was IgGl,and the light chain of both 3A10 and 3G12 was ? chain.The IFA titers of both 3A10 and 3G12 could reach to 1:12800.Western blot assay showed that both 3A10 and 3G12 reacted specifically with either endogenous or exogenous Shp-2.The preliminary epitope assay indicated that the epitopes recognized by both 3A10 and 3G12 spanned two important functional domains of NSH2 and CSH2.The generation of two monoclonal antibodies specific to chicken Shp-2 laid the foundation for further studying the biological function of the Shp-2.3.Construction of Shp-2-knockout LMH cell linesTo explore the relationship between Shp-2 and avian diseases,the Shp-2-knockout LMH cell lines were developed using CRISPR/Cas9 technology.In brief,five sgRNAs designed for the Shp-2 gene were first cloned into the lentiCRISPRv2 vector,the recombinant plasmids were then transfected into LMH cells,and the transfected cells were screened with puromycin.Western blot analysis showed that the knockout effect of sgRNA-3 was the best one tested.And then the LMH cells transfected with lentiCRISPRv2-sgRNA-3 were subcloned,and the level of protein expression of Shp-2 was detected by Western blot.Finally,Shp-2 in LMH cells was successfully deleted using CRISPR/Cas9 technology.The Shp-2-knockout LMH cell lines obtained were named as LMH-KO-Shp-2-3?LMH-KO-Shp-2-6?LMH-KO-Shp-2-11,which provided a powerful tool for studying on the roles of Shp-2 in avian diseases.