猪caspase-1 p20多克隆抗体制备及其在猪繁殖与呼吸障碍综合征病毒感染中的初步应用  

Preparation of anti-swine caspase-1 p20 polyclonal antibodies and primary application in porcine reproductive and respiratory syndrome virus infected PAM

作　　者：刘园园[1,2] 李江南[2] 刘琴芳[2] 张枫[2] 张利杰[2] 黄丽[2] 穆杨[1] 翁长江[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第2期98-101,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31172333;31201883);中国农业科学院院所长基金(0302013004)

摘　　要：为研究猪繁殖与呼吸障碍综合征病毒(PRRSV)在感染细胞中对半胱氨酸蛋白水解酶Ⅰ(caspase-1)的激活作用,本研究以PRRSV HuN4株感染猪肺泡巨噬细胞(PAM)的cDNA为模板,构建caspase-1 p20蛋白的重组表达质粒,以表达的重组蛋白p20为抗原制备了兔抗猪p20的多克隆抗体。利用制备的多抗对PRRSV HuN4株(0.1 MOI)感染的PAM进行western blot检测,结果表明在PRRSV感染PAM 24 h后胞内caspase-1被大量诱导表达,同时caspase-1被水解激活并释放出p20。该研究结果为进一步阐明PRRSV感染细胞后引起IL-1β升高的分子机制研究奠定了基础。Caspase-1 is cleaved into p10and p20 to be activated in cells by virus infection, which in tum activate IL-1β and IL-18 and result in the inflammatory reactions. To investigate caspase-1 activated procedure in porcine alveolar macrophages （PAM） infected with porcine reproductive and respiratory syndrome virus （PRRSV）, the eDNA of caspase-1 p20 was amplified by PCR and cloned into pET-28a（＋） for expression in E.coli. In addition, the polyclonal antibodies against p20 were prepared in rabbits immunized with the purified p20 recombinant protein for detecting the p20 in PRRSV infected PAM, and the result showed that caspase-1 was highly expressed in PRRSV-infected PAM at 0.1 MOI and also cleaved into subunits to form the activated caspase-1, which was proved by the p20 detection in westem blot. These results laid a foundation for further study on IL-1βproduction in PRRSV infected PAM.

关 键 词：猪繁殖与呼吸障碍综合征病毒 肺泡巨噬细胞 半胱氨酸蛋白水解酶I p20 兔源多抗 

分 类 号：S852.65[农业科学—基础兽医学]