Abstract: 【Objective】 This study was aimed to express Seneca virus A (SVA) VP1 protein in Escherichia coli (E.coli), and establish the detection method of SVA antibody by liquid chip technology.【Method】 According to the SVA VP1 gene sequence (KY747514.1) from GenBank,the VP1 gene was synthesized based on the codon preference of E.coli,and cloned into pCold TF vector to construct the recombinant plasmid pCold TF-VP1.The pCold TF-VP1 was transformed into E.coli BL21 (DE3) competent cells and induced by IPTG.The inducing conditions were studied by optimizing the induction time and IPTG concentration.Recombinant VP1 protein was purified by nickel column.The purified SVA VP1 protein was coupled with fluorescent microspheres,and the microspheres were reacted with SVA negative and positive serum.Luminex 200 system was used to detect the background and the median fluorescence intensity (MFI) of the negative and positive serum.【Result】 The recombinant plasmid pCold TF-VP1 was successfully transformed into E.coli BL21 competent cells and expressed as soluble protein with the molecular weight of 82 ku.The VP1 protein was expressed at the highest yield when inducing at 16 ℃ for 3 h and the final concentration of IPTG was 1.2 mmol/L.The average MFI of negative control was 17 (＜100).The average MFI of the test microspheres was 2 339.5 (＞2 000),indicating that SVA VP1 protein was successfully coupled with the microspheres.The coupled fluorescent microspheres could be used to detect SVA antibody in porcine serum.【Conclusion】 VP1 protein of SVA was successfully expressed in E.coli.The expression conditions were optimized,the target protein was purified and could be used to develop a liquid chip analysis for detection of SVA antibody.

Key words: Seneca virus type A (SVA); VP1 protein; Escherichia coli; protein purification; soluble expression; liquid chip