Expression Of Porcine Sialoadhesin Epitope In E. Coli And Preparation Of Polyclonal Antibody Against Sialoadhesin

Porcine reproductive and respiratory syndrome (PRRS) is caused by the infection of PRRS virus (PRRSV). It is a kind of acute infection disease, which leads to sow respiratory system disease. PRRSV has been making a huge lost for aquaculture. PRRSV molecular contains a few of structure proteins, and they are closely related with the attachment and internalization processes of the virus. It is clear that the major infection path is to attack macrophages, especially to alveolar macrophages. Normally, the virus attaches and binds to the membrance recepors and infects the cells by internalization. There are three reported receptors on the macrophage membrane, heparan sulphate, sialoadhesin and CD163. These receptors together mediate PRRSV attachment, internalization and uncoating processes. Sn is the primary receptor which mediates the virus internalization. A study showed that a PRRSV non-permission cell stem could be infected by transfected with Sn gene expression vector. Sn is a transmembrane protein made and the functions of most domains are unclear. Therefore, the study on the structures and functions of Sn is required to find out the PRRSV pathogenesis. Moreover, the antibodies against different epitopes or domains are necessary to investigate Sn functions.In this study,the antigenic index of Sn gene sequence was analized by DNAstar software, and a 114-amino acid-peptide fragment (Sn114) at the N-terminal of the Sn extracellular domain was chosen as the expected antigen. The Sn114 gene sequence was codon-optimized based on the E. coli preferred codon to enhance expression level. The gene fragment was connected to multiple cloning site of the prokaryotic expression vector pET-32a(+). The recombinant vector pET-32a-Sn114 is fused with two tags, the thioredoxin (Trx) at the 5′terminal and 6×His-tag at the 3′terminal. The vector was transformed into E. coli BL21(DE3) competent cells to express Sn114 fusion protein indeced with IPTG. Ni-NTA Agarose was used to purify the expression product. The rabbits were immuned with Sn114 protein to prepare antiserum, and the antibody titre was measured by ELISA. In order to investigate the antibody active, western-blot and PAM binding assay was used to find out whether the antibody could recognize or bind intact Sn protein and PAM. The results showed that Sn114 fusion protein was indeed expressed in E. coli, and purified by Ni-NTA Agarose. The antibody titre at 34 d after immunization reached 1: 10 240. The antibody was able to recognized Sn protein and binding to PAM. Overall, the Sn monoclonal antibody was prepared successfully with good active and specificity. This work provides the necessary conditions for further investigation of Sn functions during PRRSV infection.