Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant viral structural protein with high immunogenicity. Previously, the nonessential sequences for virus infectivity were identified at both N and C terminal ends of N protein. Here, by means of reverse genetics, a marker virus (v7APMa) was generated with a mutant N protein that differs from the wild-type strains (vAPRRS, type 2 PRRSV). v7APMa shows stable inheritance in cell culture and the virologic characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies and robust antibodies against the marker peptide, from 14 days post infection. In addition, a peptide-based ELISA was developed to detect the specific antibodies for the introduced 7APMa peptide. This approach, using a rationally designed marker virus, provides a new potential mutant basis for further development of PRRSV novel vaccines.