Analysis Of Immunoglobulin Repertoire At Different Developmental Stages In Pigs And Preliminary Screening Of Classical Swine Fever Virus Antibody Gene

Classical Swine Fever(CSF)induced by classical swine fever virus(CSFV)is an important disease causing swine death,leading serious economic losses to the pig breeding industry.At present,vaccine immunization is the most effective preventive measure.However,with the virus mutation,the change of epidemic strains and others,the traditional attenuated vaccine and inactivated vaccine have been gradually unable to meet the demand.The attenuated vaccine has good immunization effect,but it is dangerous to disperse the virus.The humoral immune response caused by the inactivated vaccine is not complete and the immune efficiency is not high.Therefore,the cellular immunity plays a more important role in defence the invading pathogens of the organism.In the process of cellular immunity,there are B lymphocyte activation,a variety of gene recombinant and Ig M,Ig G type antibody(mainly Ig G)generation binding antigen to mediate the immune response.Therefore,screening of CSFV genes and antibodies is of great importance for the development of immunotherapy.The study consists of two parts.In the first part,pig Ig antibody profiles were analyzed: IGH,IGK and IGL genes at different developmental stages of pigs were obtained by PCR,and the frequency and diversity of Ig gene were analyzed using IMGT / V-QUEST online comparison,R language and Graph Pad Prism tools at developmental stage in pigs.The second part is the preliminary screening of CSFV antibody genes: CSFV neutralizing epitopes(aa693-716,aa824-844 and aa844-865)were used by screening the classical swine fever virus antibody-secreting cells(ASCs)and analyzed by flow cytometry sorting.Antibody genes of single ASCs were amplified by Smart-seq2 and nested PCR,cloned into eukaryotic expression vector and transfected into human 239 T cell line.The expressed recombinant protein was purified.The results showed that: 1)IGHV1-4,IGHV1-10 and IGHV1S2 genes accounted for more than 80% of the IGHV gene repertoire after birth,with individual development,the frequency of IGHV1-6 gene was significantly reduced,while the frequency of IGKV1-11 gene gradually increased,and the 70-day pigs prefer to use the IGLV8 family genes;2)The IGH-IGK gene is successfully obtained from a single CSFV antibody-secreting cell and get the CSFV antibody recombinant protein.Antibodies and genes play an important role in the research of anti-virus reserach and are also important tools in the research of new vaccines and antiviral drugs.Therefore,the study laid the foundation for the prevention and treatment classical swine fever.