The Prokaryotic Expression, Polyclonal Antibody Preparation Of DPV GJ Antigen Protein Domain

The bioinformatics feature of the the DPV gJ gene was studied in this essay. We expressed the des-transmembrane and des-signal peptide domain of gJ gene successfully and produced the polyclonal antibody. The results showed that:1. Molecular characterization of DPV gJ geneThe DPV gJ gene contained 1620 basic groups, which encoded a polypeptide of 539 amino acid residues, and its isoelectric point was 6.037. The DPV gJ protein had a signal peptide and transmembrane domains at its N terminus and C terminus, separately. The sequence contained 32 possible sites for phosphorylation,15 on serine,9 on threonine, and 8 on tyrosine residues, when the threshold was defined as 0.5. Secondary structure prediction showed that it contained 50.65% random coil, which provided a favourable space for antigen epitope of gJ(M). In addition, The gJ gene ORF had 62 rare codons which occupied 33.3%, and the sequence contained nine rare codons string with more than two rare codons.2. The prokaryotic expression, polyclonal antibody preparation of DPV gJ(M)pET-32a(+)-gJ and pET-32a(+)-gJ(M) were constructed successfully and transformed to the E.coli BL21(DE3). The results of induction by IPTG showed that the gJ gene couldn’t express while gJ(M) was highly expressed. The unsoluble polypeptide was about 30kDa with an expected size. Western blot results showed that the protein and the rabbit anti DPV serum can react specifically. The optimized condition of expression was inducing 5h at 30℃ after adding O.lmmol/L IPTG. Purified recombinant proteins used to prepare rabbit anti-gJ(M) antibody. Agar diffusion reaction showed that the antibody titer was up to 1:32.