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

doi:10.3969/j.issn.1004-1524.2019.09.08

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (9): 1453-1460.DOI: 10.3969/j.issn.1004-1524.2019.09.08

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

张浩杰¹, 刘梅¹, 李春燕¹, 何冉¹, 兰景超², 罗娌², 古小彬¹, 谢跃¹, 杨光友^1,*

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

收稿日期:2019-03-14 出版日期:2019-09-25 发布日期:2019-10-11
通讯作者: *杨光友,E-mail: guangyou1963@aliyun.com
作者简介:张浩杰(1993—),男,四川眉山人,硕士研究生,主要从事动物寄生虫病学研究。E-mail: jazzachieve@outlook.com
基金资助:
成都大熊猫繁育研究基金(CPF2014-09)

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

ZHANG Haojie¹, LIU Mei¹, LI Chunyan¹, HE Ran¹, LAN Jingchao², LUO Li², GU Xiaobin¹, XIE Yue¹, YANG Guangyou^1,*

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China

Received:2019-03-14 Online:2019-09-25 Published:2019-10-11

摘要/Abstract

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。

关键词: 犬恶丝虫, NDPK基因, 克隆, 原核表达, 免疫荧光定位

Abstract: 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) and 38.9% (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.

Key words: Dirofilaria immitis, nucleoside diphosphate kinase gene, clone, prokaryotic expression, immunohistochemical analysis

中图分类号:

S855.99

张浩杰, 刘梅, 李春燕, 何冉, 兰景超, 罗娌, 古小彬, 谢跃, 杨光友. 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[J]. 浙江农业学报, 2019, 31(9): 1453-1460.

ZHANG Haojie, LIU Mei, LI Chunyan, HE Ran, LAN Jingchao, LUO Li, GU Xiaobin, XIE Yue, YANG Guangyou. Prokaryotic expression and immunofluorescence localization of nucleoside diphosphate kinase gene from Dirofilaria immitis[J]. , 2019, 31(9): 1453-1460.

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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

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