Preparation And Inhibitory Effect Of Anti-Porcine CD151Serum On PRRSV Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative gent of porcine respiratory and reproductive syndrome (PRRS), an economically important disease in swine industry worldwide. Previous studies have shown that cellular protein CD151can bind to the3’-untranslated region (UTR) of PRRSV genomic RNA and the anti-CD151serum can block PRRSV infection into MARC-145cells. However, it remains to be tested whether the anti-CD151serum can block PRRSV infection into porcine alveolar macrophages (PAMs).To address this question, the cDNA for porcine CD151was amplified from PAMs using RT-PCR and the extracellular domain was amplified using PCR. The cDNA fragment was subcloned into the prokaryotic expression vector containing the extracellular domain (IgV) of sheep T lymphocyte-associated antigen4(CTLA-4). Sequencing analysis showed that the cloned cDNA had a homology of99.8%or99.2%at nucleotide or amino acid sequence level to the published sequence. The expression vector pET-IgV-CD151was transformed into E.coli competent cells and expression of the fusion protein was induced with IPTG. SDS-PAGE analysis showed an expected protein band in the cell lysate, which was present mainly in the insoluble form. After repeated washing in water and PBS containing2M urea, highly purified inclusion bodies were obtained and the soluble protein was obtained following denaturation and renaturation. ICR mice were immunized for three times with the fusion protein (50ug/animal) and the antiserum was titrated using indirect ELISA, with a antibody title of1:150000. Using the antiserum, an expected29-kDa protein band was detected in CD151+PAMs, but not in CD151-PK-15cells.Two methods were used to test the blocking effect of the antiserum on PRRSV infection. Firstly, MARC-145cell were treated with serialy diluted antiserum and then infected with2different strains of PRRSV. The results showed that the antiserum treatment could bock viral infection within72h after infection with a blocking title of1:20-1:40. Secondly, serialy diluted antiserum was incubated with PRRSV and then added to MARC-145or PAM cell cultures. At different time pints after infection, PRRSV was titrated for TCID50. The results showed that the infected MARC-145cells had no overt CPE and detectable virus within72h after infection with a blocking title of1:40and1:80. The antiserum treatment could reduce viral title in PAM cells, but the difference was not significant compared to normal serum. These data suggest that porcine CD151plays an important role in PRRSV infection into MARC-145cells, but not in PAM cells.