Preparation of Polyclonal Antibody Against c-Myc Protein in Sus scrofa

  Objective  To facilitate studies on the functions of the porcine circovirus type 2 Rep protein and its interaction with the host c-Myc protein, a polyclonal antibody against the virus was prepared.

  Method  According to the complete sequence of porcine c-Myc (GenBank No. NM_001005154), primers were designed to amplify the CDS sequence of the gene by reverse transcription PCR. The CDS was double-digested by Nde I/Xho I enzymes to construct the prokaryotic expression vector pET-30a(+) and transformed into bacterium Arctic-Express^TM (Escherichia coli) for induction and expression. The obtained products were denatured, re-natured, and affinity purified by His-band Ni+ chromatography as the antigen to prepare polyclonal antibody by immunizing New Zealand white rabbits. The resulting antibody was purified by the antigen affinity purification chromatography, and the titer and specificity determined by indirect ELISA and western blot.

  Result  The cloned CDS-sequence of porcine c-Myc was 1359 bp. The recombinant protein of c-Myc-His was mainly in the form of inclusion bodies with a molecular weight of approximately 63 kDa. The titer of the polyclonal antibody prepared from the protein was greater than 1∶1093500. It could specifically recognize the target proteins including recombinant c-Myc-His and porcine c-Myc proteins.

  Conclusion  The target polyclonal antibody against the porcine c-Myc protein was successfully obtained for further studies on the functions of the protein as well as its interactions with the PCV2 Rep protein.