Identification Two Gene Mutants And Preparation Polyclonal Antibody Of Swine Mhc Class ⅡTransactivator Ciita

Classical swine fever virus (CSFV) is a pathogen for highly infectious disease as swine fever, belongs to Flaviviridae pestivirus genus. In recent study, CSFV is detected in semen, gonads, tonsil and lymph node, confirming that CSFV has been established as persistent infection.Major Histocompatibility Class Ⅱ Transactivator (CIITA) acts as an important co-activator. The carboxyl terminus of the protein binds to multiple transcription factors on the promoter of MHC class Ⅱ gene, and then forms a large transcription complex to regulate the expression of MHC class Ⅱ molecules.Our previous results showed that the major histocompatibility complex Ⅱ (MHC Ⅱ) expression level was significantly lower in peripheral blood lymphocytes of classical swine fever virus infected pigs and PK-15cells. Up to now, CⅡTA is the most pivotal regulator of MHC class Ⅱ gene expression. The aim of this study is to clone CⅡTA gene, and then prepare polyclonal antibody of CⅡTA. Anti-CⅡTA polyclonal antibody was used to detect the expression of CⅡTA protein in PK-15with PCDNA3.0-GFP-CⅡTA4transfection. In this study, total RNA was extracted from lymphocytes of Danish Landrace, and then the CIITA gene was amplified by RT-PCR. Subsequently, sequence alignment revealed that three forms of CIITA were obtained named CIITA4、CIITA8and CIITA-N. The homologous analysis revealed that the homologies of nucleotide and deducted amino acids sequences between the reference sequences and CIITA4were99.4%and98.6%, respectively. Sequence alignment also found that40bp deletions exhibited at the position929-969of CIITA8and CIITA-N gene, and another144bp deletions existed at the position225-368of CIITA-N gene. The deduced amino acid analysis showed that since the absence of some gene fragments, the stop codons of the CIITA8and CIITA-N were advanced, therefore the proteins encoded by CIITA8and CIITA-N were shortened. The three genes CIITA4、CIITA8and CIITA-N were encoded proteins of565aa、312aa and264aa. In our initial speculation, these deletions may be related to the pre-mRNA splicing of CIITA gene.A723bp fragment encompassing the3’terminus of CIITA4gene was subcloned to the prokaryotic expression vector PET-32a+. The PET-CIITA4-C was transformed into E.coli BL-21for inducting expression by IPTG. CIITA4-C recombinant protein was purified by Ni-NTA resin and then refolded. The refolded CIITA4-C protein was used as an antigen to immunize mice. The reactivity and titer of polyclonal antibody against CIITA was determined by Western-blot assay. At the same time, we have constructed and successfully expressed the eukaryotic expression plamid PCDNA3.0-GFP-CIITA4in PK-15 cells.Polyclonal antibody against CIITA was used to identify the CIITA4protein expression in PK-15,and to explore the influence of classical swine fever virus infection in PK-15cells after the expression of CIITA4protein. The results showed that expression of the CSFV E2protein did not change significantly after the expression of CIITA4. The relationship between CIITA4protein and classical swine fever virus requires further study.