Molecular cloning and expression of S1 glycoprotein gene of infectious bronchitis virus (IBV) isolate IRFIBV32 in Lactococcus lactis

Avian infectious bronchitis virus (IBV) is a highly infectious pathogen in chickens causing acute, highly contagious respiratory disease [1]. S1 glycoprotein protein, a majorstructural protein of IBV, is responsible for IBV pathogenesis [2]. Lactococcus lactis bacteria can be used to create mucosal recombinant vaccines [3]. In this study, the S1 glycoprotein gene of infectious bronchitis virus isolate IRFIBV32 was amplified by RT-PCR using two pairs of primers and ligated to pTZ57R/T and pTG-19T vectors. Then, the resulting plasmids (pTZ57-S1 and pTG-19-S1) were transformed to competent E.coli GM2163. pNZ8110 and pNZ8148 L. lactis expression vectors for secretory and cytoplasmic heterologous protein were selected. Then, S1 gene fragment cloned into these vectors and pNZ8110-S1/S and pNZ8148-S1/C plasmids were yield for secretory and cytoplasmic protein production, respectively, in E. coli MC1061. After preliminary transformation into E. coli MC1061, the plasmids were extracted and transformed to L. lactis NZ9000 by electroporation. During each step the location and accuracy of genes sequence were confirmed by PCR, restriction enzyme analysis and sequencing. SDS-PAGE of purified intra and extracellular expression of S1 protein from recombinant bacteria revealed that the S1 was successfully expressed in cytoplasmic and secretory forms.

کلیدواژه ها:

Molecular cloning ، Expression ، S1 protein ، IRFIBV32 virus ، L. lactis NZ9000.