Prokaryotic Expression Of TK Gene From Virulent Duck Plague Virus And Its Cellular Localization In Infected Duck Tissue

A pair of specific primer was designed and synthesized according to the duck plague virus TK gene(GenBank NO.DQ640611) discovered in author's lab.Then PCR amplification,sequencing and bioinformatics analysis of DPV TK gene were studied.Furthermore,prokaryotic expression and immunogenic detection of TK gene were examined,and established the indirect immunoperoxidase histochemical staining method(IISM) and the indirect immunofluorescence(IFA) method to detect the expression products of DPV TK gene.Finally,a series study of expression and orientation of DPV TK gene were conducted by artificial infection in vivo of duck. The results were as follows:1.Molecular characteristics of DPV TK geneThe DPV TK gene was composed 1077bp encoding for a polypeptide of 358 amimo acid residues.The amino acid sequences predicted from the nucleotide contained two functional domains of ATP-binding domain(-DGX XGXG K-) and nucleotide binding (-DRH-).The map of phylogenetic tree demonstrated that DPV TK gene was related to TK of poultry herpes virus(alphaherpesvirinae) more closely.2.DPV TK gene prokaryotic expression and polyclonal antibody preparationThe amplified fragment was inserted into pMD18-T vector,then the pMD18/TK and pET-32a were digested with BamHⅠand HindⅢ.The TK gene was sub-cloned into prokaryotic vector pET-32a.The recombinant plasmid was used to transform into E.coli BL21(DE3) competent cell.A single positive colony was picked and identified by PCR and restriction analysis.The expression product was purified by passing the Ni~+ affinity chromatograph collumn using the recombinant protein with a tag of 6×His.The rabbit was immunized with the purified protein,and ELISA showed that the valence was up to 512000.3.Establishment of IISM and IFA for detection of DPV TK and its location in infected duck tissue The rabbit anti-DPV IgG were purified through High-Q chromotography.IISM and IFA was established and used to detect DPV antigen in paraffin-embedded tissues of ducks which experimentally infected with DPV.IISM and IFA were applied in detection of the DPV antigen in different organs of artificially infected ducks,and the antigen was detected in the heart,liver,spleen,pancreas,lung,kidney,bursa of fabricius,esophagus,trachea,muscle,thymus,duodenum,jejunum,ileum,cecum and rectum.The establishment of IISM and IFA to the detection DPV TK in paraffin-embedded tissues had good specificity,can be applied in diagnosing and studying the distribution of the DPV in duck tissues.