关键词: 猪瘟病毒, E2蛋白, 悬浮细胞系, 吸附, 内化

Abstract:

【Objective】 It has been shown that the entry of classical swine fever virus (CSFV) into host cells is mediated by clathrin-mediated endocytosis. However, the viral protein involved in entry stage remains to be elucidated. This study aimed to clarify the role of the E2 protein in the entry of CSFV. 【Method】 Lentivirus carrying the E2 gene were packaged by transient transfection of HEK-293T cells. A cell line stably expressing the E2 protein was established through transducing the suspension 293 cell with the lentiviruses. The expression of the recombinant E2 (rE2) protein was optimized to achieve higher production, and the rE2 protein was purified by affinity chromatography. The expression level and reactivity of the rE2 protein was identified by SDS-PAGE and Western blotting. The effects of the E2 protein on CSFV infection, attachment and internalization were determined by respective assays. Polyclonal anti-E2 antibodies were prepared by immunizing BALB/c mice with the rE2 protein, and its blocking rate was determined by blocking ELISA. Attachment and internalization assays were performed using the prepared polyclonal antibodies to demonstrate the role of the E2 protein in virus attachment and internalization. 【Result】 The cell line 293 was successfully transduced with the lentivirus vector. The SDS-PAGE produced the anticipated band size of rE2 no matter the sample was treated with the reducing agent or not. Western blotting results showed that the rE2 protein could be recognized by the anti-E2 monoclonal antibody WH303. The results indicated that suspension 293 cell line could stably express the rE2 protein. Under the optimized expression condition, the concentration of the rE2 protein in the supernatants of the established suspension 293 cell line was up to 5.84 μg·mL^-1. Soluble protein blocking assay results showed that the expressed rE2 protein exerted antiviral activity during the process of CSFV infection. CSFV infection were significantly inhibited by the rE2 protein treated in the entry step; Blocking ELISA and serum neutralization test showed that the anti-E2 polyclonal antibodies could neutralize CSFV infection; at the same time, attachment and internalization assays demonstrated that CSFV internalization could be inhibited by the rE2 protein and viral attachment was blocked by the polyclonal antibodies. 【Conclusion】 The E2 protein was involved in attachment and internalization of CSFV.

Key words: classic swine fever virus, E2 protein, suspension cell line, attachment, internalization