摘要
通过对多房棘球绦虫(Echinococcus multiloculais)主要卵抗原(Em MEA)基因的表达、抗体制备及血清ELISA检测,初步评价其作为诊断抗原的价值。根据WormBase数据库Em MEA基因序列设计引物,利用RT-PCR扩增Em MEA基因并构建重组表达质粒,诱导表达、纯化重组蛋白Em MEA;制备Em MEA多克隆抗体并进行免疫组织定位;ELISA方法检测Em MEA重组抗原的敏感性和特异性。结果表明,Em MEA基因长度为957 bp,编码319个氨基酸,与其它绦虫中同源蛋白的氨基酸相似性为90.85%~96.93%。Em MEA重组蛋白约为40 ku,制备的多克隆抗体可识别虫体天然Em MEA蛋白,抗体效价达1∶25 600;Em MEA蛋白仅分布于原头蚴体壁。ELISA检测表明,Em MEA重组抗原和虫体天然抗原的特异性均为100%,Em MEA重组抗原的敏感性(100%)优于虫体天然抗原的敏感性(95.83%)。结合以上研究结果,Em MEA抗原具有作为多房棘球蚴病诊断抗原的潜能。
The value of major egg antigen of Echinococcus multilocularis(Em MEA) as a diagnostic antigen molecule is preliminarily evaluated via their expression,preparation antibody,and serum ELISA detection.A pair of primers was designed according to the sequence from Worm Base.Em MEA gene was amplified by RT-PCR and a recombinant expression plasmid was constructed,The recombinant Em MEA protein was induced expression and purified.An Em MEA polyclonal antibody was prepared and immunofluorescence localization was performed.The sensitivity and specificity of the recombinant Em MEA antigen was detected by ELISA.The results showed that the Em MEA gene is 957 bp in length and encodes a protein of319 amino acids.The amino acid sequence similarity of the homologous proteins in the other species of tapeworms is from 90.85% to 96.93%.The recombinant protein Em MEA is about 40 ku.The native Em MEA antigen can be recognized by the prepared polyclonal antibody.The titer of polyclonal antibody against Em MEA protein was up to 1 ∶ 25 600.Em MEA protein is mainly distributed on the body wall of E.multilocularis metacestodes.ELISA showed that the specificity and sensitivity of both Em MEA recombinant antigen and natural antigen were 100%,and the sensitivity of Em MEA recombinant antigen(100%)was higher than that of native antigen(95.83%).Taken together,Em MEA has the potential as a diagnostic antigen for alveolar echinococcosis.
作者
张军梅
李瑞
陈晓倩
何学东
王正荣
郑亚东
胡俊杰
郭小腊
ZHANG Jun-mei;LI Rui;CHEN Xiao-qian;HE Xue-dong;WANG Zheng-rong;ZHENG Ya-dong;HU Jun-jie;GUO Xiao-la(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding,Institute of Animal Husbandry and Veterinary Science,Xinjiang Academy of Agricultural Reclamation,Shihezi 832000,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第5期588-593,共6页
Chinese Veterinary Science
基金
国家自然科学基金-青年科学基金项目(31702224)。
关键词
多房棘球绦虫
主要卵抗原
免疫组织定位
免疫诊断
Echinococcus multiloculais
major egg antigen
immunological localization
immunodiagnosis
