The aim of this study is to construct the prokaryotic expression vector of capsid scaffolding protein ICP35 of herpes simplex virus type 2 (HSV-2) and to analyze its expression characteristics during HSV-2 proliferation. The UL26.5 gene of HSV-2 encoding capsid scaffolding protein ICP35 was amplified by polymerase chain reaction (PCR) and cloned into the prokaryotic expression vector pET-32a(＋). Then the recombinant plasmid was transformed into Escherichia coli (E.coli) BL21 (DE3) to express capsid scaffolding protein ICP35 which was induced by isopropyl β-D-thiogalactoside (IPTG). The purified ICP35 recombinant protein was used as antigen to immunize rabbits for the preparation of polyclonal antibody specific for ICP35 protein, and the expression characteristics of capsid scaffolding protein ICP35 during HSV-2 proliferation was analyzed by immunofluorescence detection. The results of agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis respectively showed that the PCR target band appeared around 1 065 bp, the relative molecular weight of recombinant protein target band was about 6.3×10⁴, which was in accordance with the expectation. In addition, the serum antibody of immunized rabbit was able to specifically recognize HSV-2 capsid scaffold protein ICP35. Immunofluorescence assay results showed that the HSV-2 capsid scaffold protein ICP35 appeared around the nucleus as early as 8 h post infection. At 12 h post infection, the expression level of ICP35 increased further and ICP35 began to migrate into the nucleus. The fluorescence of ICP35 gradually converged into spots and could be detected in both cytoplasm and nucleus at 16 h post infection. The successful construction of prokaryotic expression vector of HSV-2 capsid scaffold protein ICP35 and analysis of its expression characteristics during HSV-2 infection will be helpful to study the function of UL26.5 gene, the interaction of proteins, the interaction of virus-host and the screening for antiviral drug targets.