Prokaryotic Expression, Purification, and Polyclonal Antibody Production of a Truncated Recombinant Rabies Virus L Protein.

BACKGROUND

Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication.

OBJECTIVES

A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody.

MATERIALS AND METHODS

The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET- 28a and transformed into Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively.

RESULTS

The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates.

CONCLUSIONS

Our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well.