犬恶丝虫核苷二磷酸激酶(NDPK)基因的原核表达及其免疫荧光定位  

Prokaryotic expression and immunofluorescence localization of nucleoside diphosphate kinase gene from Dirofilaria immitis

作　　者：张浩杰 刘梅[1] 李春燕[1] 何冉 兰景超 罗娌 古小彬[1] 谢跃[1] 杨光友[1] ZHANG Haojie;LIU Mei;LI Chunyan;HE Ran;LAN Jingchao;LUO Li;GU Xiaobin;XIE Yue;YANG Guangyou(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Chengdu Research Base of Giant Panda Breeding,Chengdu 610081,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]成都大熊猫繁育研究基地,四川成都610081

出　　处：《浙江农业学报》2019年第9期1453-1460,共8页Acta Agriculturae Zhejiangensis

基　　金：成都大熊猫繁育研究基金(CPF2014-09)

摘　　要：对犬恶丝虫核苷二磷酸激酶(NDPK)基因的基本特征进行探究,并评估其诊断价值,旨在为犬恶丝虫病的诊断提供依据。本研究原核表达了Di-NDPK,通过生物信息学、免疫印迹和免疫荧光组化分析了该蛋白的基本特征,通过检测犬恶丝虫阳性血清评估了rDi-NDPK的诊断价值。免疫印迹显示,Di-NDPK具有良好的反应原性,免疫荧光组化显示,Di-NDPK在犬恶丝虫的侧索及肠上皮细胞和肠腔中分布。间接ELISA显示,该方法的敏感性为66.7%(16/24),特异性为38.9%(14/36),其中与犬细粒棘球蚴病阳性血清、犬钩虫病阳性血清以及犬弓首蛔虫病阳性血清发生交叉反应。Di-NDPK是一个分泌蛋白,但不适合作为犬恶丝虫病的诊断候选抗原。In order to provide base of heartworm diagnose,this study analyzed the molecular characteristics and identified the diagnostic potential of Dirofilaria immitis nucleoside diphosphate kinase ( NDPK ) gene. Di-NDPK was cloned and expressed,and the protein was analyzed by bioinformatic characterization,Western blotting and immunohistochemical analysis. Also the diagnostic potential of rDi-NDPK was evaluated by indirect ELISA. The results of Western blotting indicated that Di-NDPK had good reactivity. Immunohistochemical analysis results showed that Di-NDPK was distributed in hypodermis and intestinal epithelial cells of D.immitis . The indirect ELISA results showed that the sensitivity and specificity were 66.7%(16/24) and38.9%(14/36) respectively. The cross reactions were observed with serum samples positive for Echinococcus granulosus,Ancylostoma caninum and Ascaris alata . In conclusion,Di-NDPK might be a candidate vaccine antigen,but not a suitable antigen for the detection of D. immitis .

关 键 词：犬恶丝虫 NDPK基因 克隆 原核表达 免疫荧光定位 

分 类 号：S855.99[农业科学—临床兽医学]