Cloning, Prokaryotic Expression And Antiserum Preparation Of Porcine CD163and Sn

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important viral infectious diseases in worldwide swine industry. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped single-stranded positive RNA virus which infects mainly the porcine macrophage lineage. Presently, vaccination is the main strategy for control of the disease, but the prophylactic efficiency remains to be proved due to emergence of highly virulent isolates. Therefore, there is an urgent nedd for developing new strategy for efficient control of the disease.PRRS virus infectes the target cells by receptor-mediated endocytosis. Presently, at least three receptor molecules have been identified on porcine alveolar macrophages (PAMs). Among them, Sn is involved in virus attachment and internalization, while CD163lays an important role in viral uncoating and genomic RNA releasing. The objective of this study is to prepare the recombinant antigens and antisera against porcine CD163and Sn for research on tissue-targeted RNA interference and gene deletion of the viral receptor genes. The1511-bp and1657-bp cDNA fragments for porcine CD163and Sn, respectively, were amplified from PAMs using RT-PCR and their679-bp and384-bp fragments were re-amplified using PCR for sequencing and prokaryotic expression. The cDNA fragments were subcloned into the prokaryotic expression vector containing the extracellular domain of cytotoxic T lymphocyte-associated antigen-4and the fusion protein expressions were induced with IPTG. ICR mice were immunized with50μg affinity-purified antigen for three times and the specific antibody was titrated using indirect ELISA. The specificity of the immune sera was confirmed using Western blotting and immofluorescence. Agarose gel electrophoresis showed that both RT-PCR and PCR products had expected sizes. Sequence analysis showed that the cloned cDNAs had homologies of>99%without amino acid substitution to the published sequences. After IPTG induction, expected43-kDa and32-kDa protein bands were detected in the insoluble form of sonicated recombinant E.coli lysates. After immunization for three times, ELISA titles of the anti-CD163and anti-Sn sera reached to1:300000and1:40000, respectively. Using the anti-sera, expected120-kDa and185-kDa protein bands were detected in porcine alveolar macrophages, but not in PK15cells. Immunofluorescence showed that the anti-sera reacted positively with the3T3cells transfected with CD163and Sn cDNAs. These data demonstrate that the prepared recombinant antigens and antisera were useful reagents for detection of porcine CD163and Sn gene and protein expression.