猪唾液素黏附素胞外区在PRRSV感染PAM细胞中的作用

Research the roles of the extracellular domains of Sn in PRRSV infection PAMs

采用新的试验方法制备出纯度较高的抗猪Sn的鼠纯化IgG,并证实此种抗体可以阻碍PRRSV感染PAM,为研究猪Sn各结构域的作用提供参考,为进一步研究PRRSV侵入宿主的作用机制奠定基础。本试验分8个片段克隆猪Sn胞外区功能结构域（第2-17结构域）,并分别构建重组表达质粒,转化到BL21进行诱导表达。通过优化蛋白表达条件,制备其重组蛋白,将8种重组蛋白混匀后制成混合抗原（SnE）免疫小鼠以制备其多克隆抗体血清,用间接ELISA的方法检测高免血清的效价,分离并收集血清。采用硫酸铵盐析与DE-52离子交换层析相结合的方法提取并纯化血清,得到纯度较高的鼠抗猪SnE抗体。无菌灌冲猪肺脏收集原代PAM至24孔培养板中,培养6h后,用抗Sn150（第1结构域）、SnE和Sn150＋SnE等抗原的鼠纯化IgG以及鼠阴性IgG和PBS分别处理PAM,1h后用200CID50PRRSV感染PAM,分别在感染24、48h后用已建立的PRRSV实时荧光定量PCR方法检测感染细胞的病毒拷贝数。结果显示,PRRSV感染24h和48h时后,鼠抗猪Sn150抗体组的PRRSV mRNA拷贝数与阴性IgG组相比较均差异较小（P〉0.05）;而鼠抗猪SnE抗体和Sn150＋SnE混合抗体组的PRRSV mRNA拷贝数均显著低于对应的阴性IgG组,且鼠抗猪SnE抗体组24h和48h时PRRSV mRNA拷贝数分别是阴性IgG组的0.75倍（P〈0.001）和0.74倍（P〈0.001）,Sn150＋SnE混合抗体组24h和48h时的PRRSV mRNA拷贝数分别是阴性IgG的0.83倍（P〈0.01）和0.76倍（P〈0.001）。结果表明,鼠抗猪SnE抗体和Sn150＋SnE混合抗体封闭后PRRSV感染PAM的能力降低,且效果显著,提示PRRSV的吸附和内吞作用可能与pSn整个胞外区都有密切的关系。

In this study,a new method was used for preparing a high purity mouse anti-pig IgG,which could block PRRSV infection PAM.which provided the basis for study the function of each domain of Sn,and could help to learn more about molecular mechanism of the invasion way of PRRSV.In this test 8fragments of the extracellular functional domains of Sn（2-17domains）were cloned,and 8recombinant plasmids were constructed respectively,and were transformed into BL21 to induce expression.The 8recombinant proteins were abundantly expressed by optimizing expression conditions,mixed antigen（SnE）of 8kinds of recombinant proteins mice were immunized to get its polyclonal antibody serum.The hyperimmune serum titer was detected by indirect ELISA method.The high purity mouse anti-pig SnE antibody was extracted and purified by the method of ammonium sulfate precipitation combining with DE-52 ion exchange chromatography.The primary PAMs collected from piglet＇s lungs by aseptic washing were inoculated in 24-well plates and cultured for 6h.Then PAMs were respectively treated with purified anti-Sn150,antiSnE and anti-Sn150＋SnE IgG,along with mouse negative IgG and PBS.After 1h,using 200TCID50 PRRSV infected cells,the mRNA copies of PRRSV were detected by established real-time PCR method after infecting PAM 24 and 48h.The results showed that the copies PRRSV mRNA were a little differences between mouse anti-pig Sn150 antibody group and negative IgG at 24and48h（P0.05）;those of mouse anti-pig SnE and Sn150 ＋ SnE groups were significantly lower than the corresponding negative IgG groups at 24 and 48h,and those of mouse anti-pig SnE groups at 24 his 0.75 times of the negative-IgG groups（P0.001）,and 0.74 times at 48h（P0.001）,those of mouse anti-pig Sn150＋SnE groups at 24 and 48hwere respectively 0.83times（P0.01）and 0.76times（P0.001）of the negative-IgG groups.The results showed that the ability of PRRSV infect PAM was reduced by mouse anti-pig antibodies SnE and Sn150＋SnE block,suggesting that adsorption and endocytosis of PRRSV might have a close relationship with entire extracellular regions of Sn.