Cloning And Expression Of TRAF1,IKKα And IKKγ Genes From Swine Preparation Of Polyclonal Antibody

Classical swine fever(CSF)is an acute,septic and contagious infectious disease caused by classical swine fever virus(CSFV).CSFV widely spreads all over the world,causes huge economic losses in the swine industry.When CSFV invades host pig,a series of functional interactions and changes will occur in host biological process,which involves in pathogenesis and immune response.In this study,we attempted to explore the host factors such as TRAF1,IKKα and IKKγ involved in the pathogenesis and innate immune response of CSFV.Due to the shortage of commercial antibodies against these proteins,we developed the polyclonal antibodies of these proteins to lay a solid foundation for the continuous study of the pathogenesis of CSFV.Tumor necrosis factor receptor associated factor 1(TRAF1)is a multifunctional intracellular signal adaptor molecule,which affects the survival and death of animal cells.IKKα is a component of IKK protein kinase complex and is necessary for activation of NF-κB pathway.At present,the TRAF1,IKKα and IKKγ genes of human and mouse have been studied deeply,while the studies on TRAF1,IKKα and IKKγin pigs are relatively less.However,there are few reports on the interaction of TRAF1,IKKα and IKKγ proteins in swine during the process of CSFV infection.In addition,these swine-derived antibodies are lack in the biological product market all over the world,so it is difficult to detect TRAF1 from pig biological characteristics,cellular localization of IKKα and IKKγ proteins and their interactions with CSFV will be further explored.Antibody is an important and effective tool in studying the function of viral protein and the interaction mechanism between virus and host.Therefore,the preparation of polyclonal antibodies against TRAF1,IKKα and IKKγ from swine can not only further explain study the biological characteristics of these proteins,but also lay a good foundation for exploring the interaction mechanism between CSFV and TRAF1,IKKα and IKKγ proteins.In this study,we selected TRAF1,IKKα and IKKγgenes from pigs as the research objects,carried out gene cloning,constructed prokaryotic and eukaryotic expression vectors,and prepared polyclonal antibodies with purified prokaryotic expression proteins.The results are as follows.1.Amplification and cloning of TRAF1,IKKα and IKKγ genes from swine.After searching the gene sequence published on Gen Bank,1248 bp complete coding region of TRAF1 gene from swine was successfully cloned,which encoded 415 amino acids(aa)and the predicted molecular weight was about 45 k Da.The hydrophobicity and antigenicity of amino acid sequences were analyzed,and 1-576 bp of them were selected as the target fragment of prokaryotic expression.Using the same method,we successfully cloned the swine IKKα gene with 2235 bp encoding 744 aa,its predicted molecular weight was 84 k Da,and 1-561 bp was selected as the prokaryotic expression target fragment.Finally,the 1356 bp fragment of swine IKKγ was successfully sequenced,which encodes 451 aa,with a predicted molecular weight of 48 k Da;of which 561 bp(226-787 bp)was selected as the prokaryotic expression fragment.Sequence analysis showed that the three gene sequences in this study were consistent with those published on Gen Bank.2.Prokaryotic expression of swine-derived TRAF1,IKKα and IKKγ and preparation of polyclonal antibody.The polyclonal antibodies were prepared by immunizing Kunming mice with purified recombinant proteins.Western-blot was used to detect the reactivity of the three antibodies with the recombinant proteins,respectively.The three recombinant proteins showed good reactivity with the prepared antibodies.3.Constructed eukaryotic expression vectors of TRAF1,IKKα and IKKγfrom swine and verified the titer of polyclonal antibody.The titer of the polyclonal antibodies were detected by indirect ELISA.The titer of TRAF1 polyclonal antibody was 1:64000,and the antibody titers of both IKK α and IKKγ were 1:16000,indicating that the polyclonal antibody titers of the three antibodies were high.The protein specific reaction of HEK-293 T cells transfected with eukaryotic expression vector was verified by Western blot,and the protein expression of PK-15 cells transfected by eukaryotic expression vector was detected by indirect immunofluorescence(IFA).The results showed that the three polyclonal antibodies could detect the expression of the target proteins in HEK-293 T cells,and could observe the specific fluorescence in the transfected PK-15 cells.4.The polyclonal antibody could react with TRAF1,IKKα and IKKγproteins in PK-15 cells that infected CSFV.The results showed that the expression levels of the three proteins in PK-15 cells infected CSFV were significantly increased compared with the blank group.In conclusion,the prokaryotic/eukaryotic expression vectors of swine TRAF1,IKKα and IKKγ genes were successfully constructed,and three polyclonal antibodies were obtained with good specificity,which laid a good foundation for further exploring the pathogenic mechanism and immune response of CSFV.Meanwhile,the expression levels of TRAF1,IKKα and IKKγproteins in PK-15 cells infected CSFV were detected and analyzed.