Study On Inhibiting PRRSV Infection Into Host Cells In Vitro Of Recombinant Porcine Lung Surfactant Protein A

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most serious infectious diseases of swine. It may lead to pregnant sows’fever and abortion, and the respiratory problems of piglets. It has brought huge economic losses to China’s pig industry. Both domestic and abroad scholars have done a lot of research on PRRS, and they have a deeply recognize about the pathogenesis of PRRS.PRRSV was defined as RNA virus. The main way of PRRSV’s infection is respiratory tract, and the host cells of PRRSV are porcine alveolar macrophages. And PRRSV have great variation between different parentals. Because there is no effective vaccine to control the infection of PRRSV, so the animal’s innate immune system became very important. Innate immune system can not only be timely removal of the alien virus, but also can promote or activate the body’s acquired immune system. Studies have shown that recombinant porcine lung surfactant protein A (SP-A) have antiviral activity, because it has some domains which can identify the surface polysaccharide of virus. As there is no effective PRRS vaccines, we can do some research on anti-PRRSV infection into host cells in vitro of SP-A. And this may provide us a new way to study anti-PRRSV infection from innate immune system.Therefore, to investigate the antiviral activity of SP-A to PRRSV in vitro, we prepared recombinant porcine SP-A protein and do the research on the interaction between SP-A and PRRSV in vivo. The SP-A gene was amplified by PCR from the plasmid containing porcine SP-A gene, and subcloned into pcDNA3.1A-CD5 vector containing the human CD5 signal peptide to generate SP-A eukaryotic expression vector pcDNA-CD5-SPA/MH. The recombinant expression vector was transfected into HEK293T cells mediated with calcium phosphate. The expressed recombinant SP-A was identified by Western-blot and purified from culture medium by Ni-NTA-Agarose beads. The binding activity of SP-A with PRRSV was identified by ELISA. The antiviral activity of SP-A to PRRSV was analyzed by viral titer reduction assays on MARC-145 cells and porcine alveolar macrophages (PAM).The results showed that the eukaryotic expression vector of SP-A gene could mediate SP-A expression in HEK293T cells, the expressed SP-A could bind PRRSV in a dose dependent manner. The PRRSV incubated in advance with SP-A showed the lower infective activity compared with no-SP-A-incubated PRRSV on both MARC-145 cells and porcine alveolar macrophages. The SP-A-treated PRRSV titers in MARC-145 cells and PAM cells were significantly lower than that of SP-A-untreated PRRSV at 72 h post-infection. So we can get the conclusion that recombinant porcine SP-A significantly inhibit the infection of PRRSV to the host cells in vitro, and this indicates that recombinant SP-A posseses anti-PRRSV activity.