马立克氏病毒被膜蛋白VP16(UL48)的表达及多克隆抗体的制备  

Expression and polyclonal antibody preparation of the tegument protein UL48 encoded by MDV

作　　者：宋敬敬[1] 邓晨[1] 吴山力[2] 郑海南[1] 于佩峰 王梦云[1] 周小露[1] 张玉静[1] 艾永兴[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]吉林大学基础医学院,吉林长春130021

出　　处：《中国兽医学报》2017年第8期1473-1478,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272528);教育部新世纪优秀人才支持计划资助项目(NCET-12-0232);教育部留学回国人员科研启动基金资助项目

摘　　要：UL48蛋白是MDV血清Ⅰ型(MDV-Ⅰ)复制所必需,其与MDV编码的另外一种具有去泛素化酶活性的被膜蛋白UL36以及其他被膜蛋白相互作用,为探究MDV编码的UL48与UL36互作在马立克氏病(MD)肿瘤发生机制中作用,本试验制备了致病型强毒MDV-Ⅰ编码的UL48的抗体。从MDV-Ⅰ基因组中克隆了UL48基因,并将测序正确的UL48基因分别亚克隆到pTYB1和pGEX-4T3原核表达载体,构建成pTYB1-UL48和pGEX4T3-UL48重组质粒。将两重组质粒分别转化到BL21(DE3)E.coli中,分别进行IPTG诱导表达,其中pTYB1-UL48表达的融合蛋白Intein-UL48应用Chitin-Sepharose进行亲和层析纯化,将纯化后的融合蛋白Intein-UL48作为免疫大白兔制备多克隆抗体,其中Intein标签有利于提高UL48蛋白的可溶性并提高抗体效价,pGEX4T3-UL48表达纯化的融合蛋白GST-UL48应用凝血酶进行切割得到UL48蛋白,以此为包被抗原,用于间接ELISA和Western blot抗体效价检测和特异性鉴定。结果显示该抗体效价为1:512 000,成功制备特异性的UL48抗体。Abstract： UL48 plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins. To investigate the interaction between UL48 and UL36 during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study. UL48 gene was amplified from MDV- I genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21（DE3） E. coli cells. Chitin-sepharose and Glu- tathion-sepharose were, respectively, used to purify fusion protein intein-UL48 and GST-UL48. Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days. The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin. Western blot was carried out for specificity analysis of antibody against UL48 protein. The results suggested that UL48 antibody was succesfully prepared,and its titer was 1：512 000.

关 键 词：马立克病毒 UL48 多克隆抗体 INTEIN 凝血酶 

分 类 号：S852.65[农业科学—基础兽医学]