Prokaryotic cloning expression and immunologic analysis of suppressor of cytokine signaling -1 gene in rats

Objective To clone and express the suppressor of cytokine signaling-1 gene (SOCS-1)in rats. Methods PCR product of full-length coding sequence of rat SOCS- 1 was cloned into a prokaryotic expression plasmid [pET-28a(+)] to construct the recombinant expression plasmid [pET-28a(+)-ratSOCS-1].Escherichia coli BL-21/DE3 was tranformed into the recombinant plasmid and expressed with isopropyl-β-D-thiogal (IPTG) induction. Recombinant proteins in the expression products were purified by Ni-IDA affinity chromatography. SDS-PAGE was used to verify the expression products and the recombinant protein. The purified recombinant protein was used to sensitized New Zealand rabbits until the last immunization at an samples which were then determined for antibody titers and separated for sera. Total proteins of peripheral blood WBC were prepared with RBC lysis method. Negative serum (from New Zealand rabbits not immunized with recombinant protein), immunized serum and rabbit anti-histidine labeled antibody were transferred to separate bands and the immunologic activities of recombinant proteins were determined with Western blotting. Results PCR, double enzyme digestion and DNA sequencing demonstrated successful construction of pET-28a(+)-ratSOCS-1 plasmid. The results of SDS-PAGE showed formation of an obvious protein band about 26 000 in relative molecular weight by both Escherichia coli transformed with recombinant plasmid and samples of precipitated bacterial debris from ultrasonic degradation. While characteristic single band from purified recombinant proteins obtained by affinity chromatography was shown to be consistent with this band, no protein expression in supernatant of bacterial debris from ultrasonic degradation was observed at the same location. In addition, Western blotting demonstrated the competency of immunized serum as it clearly recognized the recombinant protein, the 24 000 band of lysed protein from rat peripheral blood leukocytes, and rabbit anti-histidine labeled antibody, generating three distinct bands with relative molecule weights of 26 000, 24 000 and 26 000 respectively. Conclusions Rat SOCS- 1 can be successfully expressed in Escherichia coli (BL-21/DE3). The purified recombinant protein of SOCS-1 may have strong immunogenicity and immunocompetence. Key words: Suppressors of cytokine signaling- 1; Plasmids; Gene expression; Chromatography,affinity; Immunocompetence