细粒棘球绦虫3 d幼虫cDNA表达文库的构建及鉴定  

Construction and identification of cDNA expression library of 3rd day larva of Echinococcus granulosus

作　　者：吴小霞 刘晓雷 刘明远 孙树民 赵永军[3] 丁静 WU Xiaoxia;LIU Xiaolei;LIU Mingyuan;SUN Shumin;ZHAO Yongjun;DING Jing(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/Key Labo ratory for Zoonosis Research of the Ministry of Education,Institute of Zoonosis,College of Veterinary Medicine,Jilin University,Changchun 130062,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650000,China;College of Animal Science and Technology,Inner Mongolia Minzu University,Tongliao,Inner Mongolia 028000,China)

机构地区：[1]吉林大学动物医学学院/人畜共患传染病重症诊治全国重点实验室/人兽共患病研究教育部重点实验室,吉林长春130062 [2]云南农业大学动物医学院,云南昆明650000 [3]内蒙古民族大学动物科技学院,内蒙古通辽028000

出　　处：《中国兽医学报》2025年第2期305-311,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2022YFE0114600)。

摘　　要：构建细粒棘球绦虫3 d幼虫cDNA表达文库,并进行初步鉴定。从新鲜带有包囊的患病羊源肝脏内分离采集原头蚴,人工经口感染实验犬,第3天剖检取幽门后小肠约1.5 m,沿纵轴将其剪开置38~40℃生理盐水中稍加摆动使虫体自行脱落,收集细粒棘球绦虫3 d幼虫。采用TRIzol试剂提取总RNA,PolyATtract^(®) mRNA Isolation Systems分离纯化mRNA,反转录合成cDNA,加5′接头后与pBluescript sk-载体连接经电转导入大肠杆菌,构建细粒棘球绦虫3 d幼虫cDNA表达文库。构建的文库初始库容量为1.15×10^(7)CFU/mL,扩增后文库的滴度为1×10^(10)CFU/mL,重组率为100%,插入片段长度平均为1.0~1.2 kb。构建的cDNA表达文库库容量及插入片段大小合适,有望用于细粒棘球绦虫终末宿主犬疫苗候选基因及诊断抗原分子的筛选。This study aims to construct and identify the cDNA expression library of 3rd day larva of Echinococcus granulosus.Echinococcus granulosus protoscolex was isolated and collected from the liver of a fresh infected sheep with cysts,and the experimental dogs were infected manually by mouth.On the 3rd day,approximately 1.5 meters of the duodenal jejunum was taken during the au-topsy,and the worm body was dislodged by swinging it in 38-40℃physiological saline,and then the 3-day larvae of Echinococcus granulosus were collected.Total RNA was extracted by TRIzol reagent,mRNA was isolated and purified using PolyATtract^(®)mRNA Isolation Systems.The cD-NA was synthesized by reverse transcription,ligated with pBluescript sk-vector after addition of a 5'junction and introduced into E.coli by electrotransformation to construct a cDNA expression li-brary for 3-day-old larvae of Echinococcus granulosus.The average length of insert cDNA frag-ments was 1.0-1.2 kb.The initial capacity of constructed library was 1.15×10^(7) CFU/mL,titer of amplified library was 1×10^(10) CFU/mL,recombination rate was 100%.The constructed cDNA ex-pression library and the inserted fragment size were suitable.This library is expected to be used for screening candidate genes and diagnostic antigen molecules of definitive hosts canine vaccines of Echinococcus granulosus.

关 键 词：棘球蚴病/包虫病 细粒棘球绦虫 3 d幼虫 CDNA表达文库 

分 类 号：S855.9[农业科学—临床兽医学]