日本血吸虫7d童虫cDNA文库构建及免疫筛选  

CONSTRUCTION AND IMMUNOSCREENING OF CDNA LIBRARY OF HEPATIC SCHISTOSOMULA

作　　者：韩宏晓[1,2] 洪炀 傅志强[1] 彭金彪[1] 林矫矫[1] HAN Hong-xiao;HONG Yang;FU Zhi-qiang;PENG Jin-biao;LIN Jiao-jiao(Key Laboratory of Animal Parasitology,Ministry of Agriculture and Rural Affairs,Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Minhang Animal Disease Prevention and Control Center,Shanghai 201109,China)

机构地区：[1]中国农业科学院上海兽医研究所农业农村部动物寄生虫学重点开放实验室,上海200241 [2]上海市闵行区动物疫病预防控制中心,上海201109

出　　处：《中国动物传染病学报》2020年第2期92-97,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(81401692);国家重点研发计划(2017YFD0501306)。

摘　　要：为了从7 d童虫cDNA文库筛选出童虫发育阶段特异性表达抗原基因。本研究首先构建了日本血吸虫7 d童虫cDNA文库,其次再采用日本吸血虫感染血清进行筛选。结果显示:建立的文库插入片段为0.5~3.0 kb,文库重组率为98%,初始文库滴度为2.4×10~6 pfu/mL,扩增后滴度为1.6×10~9 pfu/mL,利用感染日本血吸虫10 d和42 d的小鼠血清免疫筛选该文库,初筛和复筛后获得17条有效EST序列,其编码7个基因,分别为复制蛋白(2 ESTs)、肝再生增强因子(3 ESTs)、血小板活化因子(6 ESTs)、钙调理蛋白基因(3 EST)、丝束蛋白(1 ESTs)、核糖体RNA(1 EST)和还原型辅酶脱氢酶基因(1 EST)。该研究结果对今后探讨血吸虫的生长发育机制及筛选血吸虫早期诊断抗原具有重要意义。In this study,we constructed and immunoscreened the cDNA library of 7-day hepatic schistosomula to look for early stage specifi c genes.The cDNA template was synthesized by reverse transcription.The insert fragment length of the cDNA library ranged from 0.5 to 3.0 kb,and the recombination effi ciency accounted for about 98%.The titers were 2.4×106 pfu/mL for the primary library and 1.6×109 pfu/mL for the amplifi ed library.The cDNA library of 7-day hepatic schistosomula was screened with sera of infected mice for 10 and 42 days.Total 17 positive clones were obtained and identifi ed by PCR and DNA sequencing.The sequence analysis showed that 17 ESTs encoded 7 unique genes with similarity to the reported sequence of Schistosoma,including the replication protein(2 ESTs),augmenter of liver regeneration gene(3 ESTs),platelet-activating factor acetylhydrolase(6 ESTs),calmodulin-3(CaM-3)(3 ESTs),fi mbrin gene(1 EST),large subunit ribosomal RNA(rrnL)(1 EST),and NADH dehydrogenase(1 EST).The construction and screening of schistosomulum cDNA library and expression products of those early stage specifi c genes may be useful for screening of new drugs and vaccine candidates for controlling schistosomiasis.

关 键 词：日本血吸虫 童虫cDNA文库 免疫学筛选 

分 类 号：S852.735[农业科学—基础兽医学]