Role Of Sn Receptor D9 And D10 Domains In PRRSV Infection Of PAM Cells

Porcine Reproductive and Respiratory Syndrome is a contagious disease characterized by clinical manifestations of swine breathing disorder,pregnant sow miscarriage or stillbirth,and high fatality rate of piglets,which is caused by a single strand RNA virus of the arteriviridae family called porcine reproductive and respiratory syndrome virus?PRRSV?.The main target cells of the virus are porcine alveolar macrophages?PAMs?,and PRRSV can also infect monocytes,lymphocytes and other cells.It was shown that porcine sialoadhesin?Sn?and CD163 mediate PRRSV infection in alveolar macrophages,and porcine sialoadhesin plays an important role in mediating the adhesion and endocytosis of PRRSV.Studies had shown that transfection of porcine Sn?p Sn?,human Sn?h Sn?,and mouse Sn?m Sn?into cells that can stably express porcine CD163 can promote PRRSV infection,suggesting that difference in mediating PRRSV infection of the three species receptors was not large,which probably indicated that Sn of these three species may co-exist in a highly conserved functional domain that plays a role in mediating PRRSV infection.In this study,the Sn-conserved domain 10 with high homology was identified by predicting three Sn domains of pigs,humans and mice.At the same time,our laboratory previously divided p Sn into nine segments for competitive binding assays and antibody blocking assays with unrefolded proteins.It was found that Sn6?including domains 9 and 10?may play an important role in PRRSV infection.Therefore,in this study,the porcine Sn domain proteins D9 and D10 were expressed in E.coli prokaryotic expression system,and the role of the recombinant renaturation protein in PRRSV infection and antibody-mediated PRRSV infection of PAM cells was explored and compared with other nine segments of p Sn,so as to selecting the functional domains of porcine Sn in PRRSV infection and antibody-mediated PRRSV infection.In this study,I investigated the function of Sn protein D9 and D10 domains as the following four aspects:This study first searched for the gene sequence of porcine sialoadhesin receptor Sn?p Sn?according to Gen Bank: AF509585.1,the human sialoadhesin receptor Sn?h Sn?gene sequence according to Gen Bank: AF230073.1,the gene sequence of murine sialoadhesin receptor Sn?m Sn?according to Gen Bank: NM₀11426.3,which were then used to predict the domains using the SMART protein prediction online website.The results showed that p Sn,h Sn and m Sn all contained a signal peptide,a transmembrane region,and 18 domains.18 sequences of p Sn and 18 domains of m Sn and h Sn were analyzed by Meg Lign software for gene sequence and amino acid homology analysis.The results showed that p Sn domain 10 shared high homology with m Sn and h Sn,and the gene homology of p Sn domain 10 and h Sn domain 11 is 90.7%,and the amino acid homology is 93.4%,and the gene homology of p Sn domain 10 and m Sn domain 10 is 83.6%,the amino acid homology was 80.3%.According to the Sn homology analysis above and p Sn gene sequence?Gen Bank: NM₀11426.3?,a pair of primers were designed for the gene sequences of domains D9 and D10,respectively,to extract PAM cell RNA and reversely transcribed it into c DNA,which was used as a template.The p Sn domain D9 and D10 were amplified by RT-PCR and the lengths were 504 bp and 381 bp respectively.After cloning D9 and D10 target fragments into the p ET-32 a vector,the recombinant plasmids p ET-32a-D9 and p ET-32a-D10 were successfully obtained.p ET-32a-D9 and p ET-32a-D10 were transformed into BL21?DE3?bacteria,and the recombinant protein was expressed in the optimal IPTG-induced expression concentration and time,and the optimal urea washing concentration was then used to purify the protein.Stored at-40 °C for use.To explore the role of individual domains 9 and 10 in PRRSV infection,the purified D9 and D10 recombinant proteins were renatured,and the BL21?DE3?expressing strains of the nine p Sn fragments that had been constructed in this laboratory were reactivated,expressed and purified,and were also renatured one by one.In this experiment,the experimental group of recombinant refolding proteins of domains 9 and 10,the recombinant refolding protein control group of nine different sections of p Sn,the positive control group of PRRSV and the negative control group were set up,and the PAM cells were collected in a 24-well cell from lungs of healthy piglets that PRRSV antigen and antibody detection were negative.The prepared 11 p Sn recombinant renatured proteins were mixed with an equal volume of PRRSV virus cultured for 1 h,and then inoculated into PAM cells.48 hours later,the total RNA from each well was extracted and reversely transcribed into c DNA.The viral copy number of PRRSV was determined by real-time fluorescence quantitative PCR.The obtained data was analyzed by Graph Pad Prism 5 software.The results showed that the high concentration of p Sn different fragments,separate domains 9 and 10 compared with the PRRSV positive control group all had a certain inhibitory effect,and this degree of inhibition was positively correlated with the concentration of the refolding protein.Although the competitive binding of domain 10 was slightly better than that of domain 9,the contrast was not significant;the inhibitory effects of individual domains 9 and 10 on PRRSV were not significantly different from other fragments of p Sn.To explore the role of individual domains 9 and 10 in antibody-mediated PRRSV infection,we set up the experimental group of recombinant refolding proteins with domains 9 and 10,nine different segments of p Sn recombinant recombination protein control group,PRRSV-Ig G positive control group,PRRSV virus control group and PRRSV negative control group.We collected PAM cells in a 24-well cell from lungs of healthy piglets that PRRSV antigen and antibody detection were negative.The purified porcine anti-PRRSV Ig G was diluted 4-fold and mixed with PRRSV virus for 1 hour at 37°C.The PRRSV-Ig G infected complexes were then mixed with the 11 recombinant renatured p Sn proteins,after 1 hour cultured,inoculating it into the PAM cells.48 hours later,the total RNA from each well was extracted and reversely transcribed into c DNA.The viral copy number of PRRSV was determined by real-time fluorescence quantitative PCR.The obtained data was analyzed by Graph Pad Prism 5 software.The results showed that the individual domains 9 and 10 had a certain inhibitory effect compared with the PRRSV-Ig G positive control group,and the degree of inhibition was positively correlated with the concentration of the refolding protein.