The Prokaryotic Expression Extracellular Structure Domain Of Sialoadhesin And Study The Antibody Activity

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of main pathogens of porcine reproductivea.In pig body, the target cell of PRRSV is porcine alveolar macrophage (PAM). PRRSV infection PAM cells through three main receptors: Sialoadhesin(Sn), Heparin su1fate (HS) and CD163 molecules. Sialoadhesin can adsorption and mediat virus particle internalization.In this writing, Sn extracellular domain structural domain recombinant protein was expressed through prokaryotic vector and anti recombinant protein antibody was detected. for the study of Sn receptors with PRRSV extracellular domain function field structure to lay the foundationObtained PAM cells by clysis lung and extraction total RNA of PAM cells. Analysis Sn receptor extracellular domain structural domain. Amplification three structural domain gene fragment by RT-PCR.The size 1755bp,1773bp,1620bp,respectively.Three gene fragment was cloned into PMD18-T vector and sequened.Use DNAStar software analysis homology of splicing sequence with Sn extracellular domain structural domain in Genbank ,The result is 99.1%～99.5%. We design 3 pairs specificity primers which 5'ends with EcoRⅠenzyme locus and 3'ends with NotⅠenzyme locus. Amplification three gene fragment based on three pairs primers. The size of them 1755bp,1773bp,1620bp,respectively.The three nucleotide fragments was cloned into prokaryotic expression vector pET32a to constructing recombinant plasmid pET32a - dSn1, pET32a-dSn2 ,pET32a-dSn3.Three plasmid was expressed successfully in IPTG induction.Obtain three protein rdSn1,rdSn2 and rdSn3 and the molecular weight of them approximated 58KD, 59KD, 54KD, respectively, consistent with the expected protein molecular weight.Through further optimized conditions of expression and expression of recombinant protein was extensively. Renaturation and purification recombinant protein by urea. The renaturation recombinant protein was identified by SDS-PAGE electrophoresis. The purity of three recombinant protein 85%,76%,87%. 3 kinds of renaturation protein innoculation mice to obtain the murine antibodies against Sn1, Sn2,Sn3 recombinant fusion protein. The murine antibodies were tested by indirect ELISA. The results shows that the titer of murine antibodies titer was 1:400 ventually. Application of fusion protein purification by sds-page electrophoresis and Western blotting exprement use specificity polyclonal antibody.The result show that specificity polyclonal antibody could binding their recombinant protein , identification that recombinant protein have good immunogenicity.The PAM cells were cultured In the 24-hole cell culture plate, after 24 h,the mouse anti rdSn1,rdSn2,rdSn3 serum and the mixed serum add to the PAM cells, Respectively. the PAMs were closed about 45min and inoculated PRRSV, cultivating 72h and the PRRSV in PAMs were detected by RT-PCR. While the PAM cells was closed with healthy mouse serum as negative control, the PAM cells was closed with healthy mouse serum as positive control. Results showed that all three serum and mixture can not effectively prevent virus infection, demonstration that the process of PRRSV enter PAM cells may not rely on Sn receptor extracellular structure domain.