猪CD163分子在PRRSV感染PAM细胞中的作用研究  

Roles of porcine CD163 molecule in PRRSV infection of PAMs

作　　者：冯亚琼[1] 杜东颖 王冬梅[1] 崔春晓[1] 秦运杰[1] 李娜娜[1] 杨青原[1] 夏平安[1] 张改平[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《免疫学杂志》2015年第1期22-26,共5页Immunological Journal

基　　金：国家自然科学基金(31172346)

摘　　要：目的研究CD163分子在PRRSV感染PAM细胞中的作用。方法生物学软件分析猪CD163分子的结构,将其胞外区分为4个片段进行扩增,分别克隆到PET-32a原核表达载体中构建4种重组表达质粒并获得4种融合蛋白,用这4种融合蛋白分别免疫小鼠制备多克隆抗体,经间接ELISA方法检测4种多抗克隆抗体效价分别为1∶10 240、1∶12 800、1∶25 600和1∶12 800。饱和硫酸铵盐析法和DE-52离子交换层析技术二步法提取并纯化得到4种纯度较高的特异性抗体。用制备的4种抗体以及4种抗体的混合物分别封闭PAM细胞1 h,经PRRSV感染24 h和48 h,用已建立的绝对荧光定量PCR方法检测感染细胞中PRRSV的m RNA水平,用Graph Pad Prism 5软件进行处理所得到的数据。结果鼠抗猪CD163-P3抗体能够阻碍PRRSV感染PAM细胞,提示CD163-P3(SRCR5-6)可能参与PRRSV感染PAM细胞过程,其他片段不影响PRRSV感染宿主过程。结论用本试验方法所制备和纯化的抗体具有生物学活性,且CD163在PRRSV感染宿主细胞过程中发挥作用,发挥功能作用的部分是CD163-P3(SRCR5-6)。To study the roles of CD163 molecule in PRRSV infection of PAM, the structure of swine CD163 molecule was analyzed using biology software, and CD163 molecular extracellular region was divided into four fragments, which were amplified by RT-PCR. The fragments were then subcloned into the prokaryotic expression vector PET-32 a to construct the recombinant plasmids and expressed in the expression strain BL21. After induction with IPTG, four fusion proteins were obtained. Polyclonal antibodies were prepared by immunizing the mice with the four fusion proteins and then analyzed by indirect ELISA. Indirect ELISA indicated that the titers of four kinds of polyclonal antibodies were 1∶10 240, 1∶12 800, 1∶25 600 and 1∶12 800, respectively. Two-step method of ammonium sulfate precipitation method and the DE-52 ion exchange chromatography technology were used to extract and purify the four specific antibodies. The four prepared antibodies were used to block PAM for 1 h, which was then treated with PRRSV for 24 h and 48 h respectively. Then PRRSV m RNA levels in PRRSV-infected cells were detected with established PRRSV SYBR Green Ⅰ real-time PCR method, and the data we got was analyzed with Graph Pad Prism 5 software. The results showed that the CD163-P3 antibodies prepared could block PRRSV infection to PAM, suggesting CD163-P3（SRCR5 and SRCR6 domains） may be involved in the process of PRRSV infected PAM cells while the other fragments of CD163 did not affect PRRSV to invade host cells, which was consistent with the main function of SRCR5 domain of CD163. In conclusion, the antibodies prepared and purified by the method has immunobiologic activities, and CD163 plays a role in the process of PRRSV infection of host cells, of which main function domain is CD163-P3（SRCR5-6）.

关 键 词：CD163 多克隆抗体 制备和纯化 PRRSV 抗体封闭 

分 类 号：S858.28[农业科学—临床兽医学]