猪CD151重组抗原表达及其免疫血清的PRRSV感染抑制作用

用RT-PCR从猪巨噬细胞中扩增CD151胞外区编码序列,将其插入含细胞毒性T细胞相关抗原4胞外区序列的表达载体,重组载体转化大肠杆菌,SDS-PAGE检测重组蛋白表达;用包涵体纯化和尿素变性与复性法获得可溶性蛋白,纯化蛋白免疫小鼠,ELISA测定免疫血清抗体效价,Western-blot验证免疫血清特异性;以处理细胞或处理病毒方式,用免疫血清进行猪繁殖与呼吸综合征病毒(PRRSV)感染抑制试验。结果表明:重组菌表达预期的32ku重组蛋白,获得的可溶性重组蛋白纯度较高;免疫血清抗体效价达1∶170 000,在猪巨噬细胞中能检测到预期的29ku蛋白,能抑制PRRSV感染MARC-145细胞,但有毒株差异性,处理病毒的感染抑制作用较强。结果提示猪CD151免疫血清能以结合细胞和结合病毒方式抑制PRRSV感染。

Expression of recombinant porcine CD151 and inhibitory effect of the antiserum on PRRSV infection

The coding sequence of the extracellular domain of pig CD151 was amplified using RT-PCR and subcloned into an expression vector containing the IgV sequence of T lymphocyte-associated antigen 4.Expression of the fusion protein in the recombinant E.coli was detected using SDS-PAGE and the soluble protein was obtained by denaturation and renaturation of the inclusion bodies.After twice immunization of mice with the recombinant protein,the immune serum was titrated using ELISA and its specificity to pig CD151 was confirmed by Western-blotting.The inhibitory effect of the immune serum on porcine reproductive and respiratory syndrome virus(PRRSV) infection of Marc-145 cells was investigated by treating the virus or cells.The results showed that the recombinant E.coli expressed an expected 32 ku fusion protein and that the soluble protein was obtained with high purity.The immune serum had an antibody titer of 1∶17 000 and could detect an expected 29 ku protein in pig alveolar macrophages could be detected.A dose-dependent inhibitory effect on PRRSV infection was detected after treatment of cells or virus with the immune serum.These data indicated that the antiserum against pig CD151 can inhibit PRRSV infection of MARC-145 cells by binding to the cells or virus.