Abstract: This work aims to express and purify recombinant orf virus encoded protein 035（ORFV035）in Escherichia coli and prepare a polyclonal antibody（pAb）against ORFV035 for immunoassays，as well as lay foundation for the further researches on the replication，assembly，morphogenesis，and mature process . The gene of ORFV035 was amplified by PCR from orf viral genome，and sub-cloned into the expression vector pET-30a（+），and recombinant plasmid pET-30a-035 was constructed by enzymatic digestion and ligation of BamH I and Hind III. Then the plasmid pET-30a-035 after double enzyme digestion and sequencing was transformed into Escherichia coli BL21，the transformants were induced by IPTG，and SDS-PAGE was used to identify the expression of the protein. The His-tagged fusion protein ORFV035 was ultrasonic-treated and then purified through Ni-chelating affinity chromatography. The purified ORFV035 was used to immune into BALB/c mouse for producing anti-ORFV035 pAb. The results showed that the recombinant plasmids pET30a-035 was successfully constructed，and the protein ORFV035 in E. coli BL21 was in inclusion body. The ORFV035-his fusion protein was obtained after washing，dissolving，and purifying the inclusion body，and the pAb anti-ORFV035 was prepared. The Western blot analysis showed that this pAb recognized natural ORFV035.

Key words: orf virus, ORFV035, purification, polyclonal antibody