Expression of BRSV nucleoprotein in a prokaryotic system.

摘要

Bovine respiratory syncytial virus (BRSV) and respiratory syncytial virus (RSV) cause severe respiratory disease in calves and young children, respectively. Commercial vaccines against BRSV have been available for nearly two decades; however, none have proven to provide satisfactory protection. Currently, there are no commercially available vaccines against RSV. It is therefore important to evaluate various vaccine strategies for development of an effective vaccine against BRSV and RSV. BRSV infected calves develop similar clinical disease and lung lesion pathology as children do to RSV infections, making the development of an effective vaccine against BRSV also beneficial in developing an effective vaccine against RSV. Data from previous studies have demonstrated that eliciting a strong initial cell mediated immune response is essential in developing a successful vaccine against BRSV and RSV. The administration of a vaccine based on the nucleocapsid protein (NP) has been previously shown to stimulate a robust cell mediated immune response without elicitation of an IgE response in calves; undesirable IgE responses have occurred in calves vaccinated with formalin killed BRSV vaccines. In this study, the expression of the nucleocapsid gene of BRSV using a prokaryotic expression system was investigated to determine if sufficient quantities of soluble NP could be produced to use in a subunit vaccine. The nucleocapsid gene was first cloned into the GEX-4T-1 plasmid and expression of NP was induced in E. coli DH5 alpha; cells. However, expression of NP was only detected in the insoluble fraction of the DH5 alpha; cells, making the use of another prokaryotic expression system necessary. The nucleocapsid gene was then cloned into the pET100/D-TOPO plasmid and expression of NP was induced in E. coli BL21 cells. NP was preferentially expressed in the insoluble fraction of E. coli BL21 cell lysates and a detectable amount of soluble NP was expressed. However, purification of the soluble fraction of pEt100-NP transformed E. coli BL21 cells did not yield sufficient quantities of NP to be used in a subunit vaccine for BRSV to merit further use of this expression system.