The Prokaryotic Expression, Polyclonal Antibody Preparation, Time Courses Of Transcription And Cellular Localization Of DPV GI Gene

The prokaryotic expression, polyclonal antibody preparation time courses of transcription and cellular localization of DPV gl geneThis paper has carried out a series of researches on the identified gl gene (assigned accession no. EU035298) of the duck plague virus (DPV) in our laboratory, including molecular characteristics analysis, cloning, prokaryotic expression, preparation of polyclonal antibody, the time of transcription analysis and cellular localization of DPV gI gene in infected host cells. The results were as follows:1. Prediction of molecular characteristics of DPV gl geneDPV gI gene open reading frame (ORF) was 1116 bp in length and its primary translation product was a polypeptide of 371 amino acids long. Comparison with other herpesvirus gI homologous genes showed higher similarity, had a close evolution relationship with members of the genus Mardivirus which consisted of MeHV-1, GaHV-2 and GaHV-3, but itself branched and was independent to others. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal peptide,3 putative N-linked glycosylation sites, a C-terminal transmembrane domain. Moreover, gI protein of DPV contained 19 potention phosphorylation sites,13 epitopes, subcellular location analysis demonstrated that the protein principally located in endoplasmic reticulum (55.6%). The analysis on codon usage bias showed that DPV gI gene was strong bias towards the synonymous codons with A and T at the third codon position, and was low codon usage bias gene, presumed lower expression. There were 26 codons showing distinct usage differences between DPV and Escherichia coli,24 between DPV and yeast,31 between DPV and Homo sapiens.The results suggest that codon preference of DPV gI is close to both of the prokaryote and eukaryote. The E. coli and yeast expression system may be suitable for the expression of DPV gI gene.2. Cloning, prokaryotic expression, preparation of polyclonal antibody of DPV gl geneThe special 1221-bp fragment containing complete open reading frame(ORF) of DPV gI gene was amplified by PCR, then inserted into pMD18-T vector, resulting in pMD18-T-gI. The inserted fragment was extracted from plasmid pMD18-T-gI by digesting with BamWⅠand XhoⅠ, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DPV gI gene was successfully expressed by the addition of isopropyl-P-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. The gene was highly expressed with 0.2 mmol/L IPTG at 37℃for 6 h, and found in large amounts in insoluble fractions (inclusion bodies) of cracked cells. The expression product was purified by passing the Ni+ affinity chromatograph collumn using the recombinant protein with a tag of 6×His. Western-blot analysis showed that the purified His6-tagged gI was recognized by the rabbit anti-DPV serum. The rabbitts were immunized with the purified recombinant protein, and the antibody titer was up to 1:32 in agar diffusion reaction. The specificity of the rabbit immuneserum was detected by its ability to react with the recombinant protein His6-tagged gI. 3. Transcriptional analysis of gI gene in DPV infected DEFFQ-PCR was used to detected the transcription of gI gene in DPV infected cells, the results showed transcriptional products of DPV gI gene were low in early phases of infection, presenting slightly increased before 12. Subsequently, signal intensity immediately increased after 12, peaked at 48 h, and then declined. The increasing transcription of DPV gI in the late infection stage suggests that DPV gI protein may closely related to the assembly and maturation of DPV. Moreover, the transcription kinetics of the gI gene was identified in the presence of acyclover (ACV) and cycloheximide(CHX), which are inhibitors of viral DNA synthesis and of protein synthesis, respectively, was detected with RT-PCR. The result showed that the transcription of gI gene was inhibited by ACV or CHX, indicating the gene transcripted depending on the DNA and cell protein synthesis. Thus, it can be presumed from the model of transcription that the DPV gI gene is a late gene.4. Expression and cellular localization of DPV gI in infected DEFIntracellular distribution of DPV gI protein was detected by immunofluorescence technique utilizing rabbit immune serum against expressed gI protein. The results showed that faint fluorescence was detected in cytoplasm as early as 4 hours post infection(hp.i.). As the infection time passed, a strong fluorescence was found intensively distributed in the cytoplasm and especially in the juxtanuclear region at 12 hp.i. and 24 hp.i., this typical pattern of fluorescence indicated gI protein may distribute in some cellular organs as Golgi apparatus. In the late phase of infection, following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DPV-infected cells, fluorescence was gently dispersed. There, it can be infered from the distribution pattern that DPV gI was expressed in cytoplasm.