Cloning surface protective antigen A gene from Erysipelothrix rhusiopathiae and its expression in Pichia pastoris

Objective Immunization protection area protein, which lies in the N-terminal protective domain of surface protective antigen A(SpaA) of Erysipelothrix rhusiopathiae, was expressed in Pichia pastoris X-33.

Method Using the swine erysipelas, which was isolated from a pig farm in Guangdong as template, a pair of primers was designed according to the cDNA sequence of Spa gene from NCBI.The amino terminal sequence was achieved by PCR amplification, after being inserted into the expression vector pPICZαC to get the recombinant plasmid of pPICZαC-SpaA-N.Recombinant plasmid of pPICZαC-SpaA-N by Sac Ⅰ enzyme was linearized, and electro transformed it into P.pastoris X-33.The positive transformant, screened by YPDS tablet with Zencin^TM and identified by PCR at different concentrations of Zencin^TM, achieved the high number of copies which were induced and cultured by methanol.Supernatant was collected by centrifugation at 48, 72, 96 h respectively after induction, and SDS-PAGE and Western-blot tests were carried out.

Result and conclusion The SpaA gene was successfully cloned and expressed, and the recombinant plasmid of pPICZαC-SpaA-N was constructed.The immunization protection area protein, which lies in SpaA gene amino terminal of swine E.rhusiopathiae in P.pastoris X-33, was successfully expressed.SpaA gene of swine E.rhusiopathiae has been successfully expressed asimmunization protection area protein in yeast host, which will lay a foundation for the development and the mass production of subunit vaccine of swine E.rhusiopathiae.