期刊文献+

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
下载PDF
导出
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.
作者 韩宏晓 洪炀 傅志强 彭金彪 林矫矫 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)
出处 《中国动物传染病学报》 CAS 北大核心 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)。
关键词 日本血吸虫 童虫cDNA文库 免疫学筛选 Schistosoma japonnicum cDNA library immunoscreen
  • 相关文献

参考文献3

二级参考文献38

  • 1Wyka IM,Dhar K,Binz SK,Wold MS.Replication protein A interactions with DNA:differential binding of the core domains and analysis of the DNA interaction surface[J].Biochemistry,2003,42(44):12909-12918.
  • 2Gomes XV,Wold MS.Structural analysis of human replication protein A.Mapping functional domains of the 70-kDa subunit[J].J Biol Chem,1995,270(9):4534-4543.
  • 3Niu H,Erdjument-Bromage H,Pan ZQ,Lee SH,Tempst P,Hurwitz J.Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro[J].J Biol Chem,1997,272(19):12634-12641.
  • 4Iftode C,Daniely Y,Borowiec JA.Replication protein A (RPA):the eukaryotic SSB[J].Crit Rev Biochem Mol Biol,1999,34(3):141-180.
  • 5Wold MS.Replication protein A:a heterotrimeric,single-stranded DNA-binding protein required for eukaryotic DNA metabolism[J].Annu Rev Biochem,1997,66:61-92.
  • 6Araya R,Hirai I,Meyerkord CL,Wang HG.Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway[J].FEBS Lett,2005,579(1):157-161.
  • 7Yoo E,Kim BU,Lee SY,Cho CH,Chung JH,Lee CH.53BP1 is associated with replication protein A and is required for RPA2 hyperphosphorylation following DNA damage[J].Oncogene,2005,24(35):5423-5430.
  • 8Wu X,Shell SM,Zou Y.Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells[J].Oncogene,2005,24(29):4728-4735.
  • 9Fang F,Newport JW.Distinct roles of cdk2 and cdc2 in RPA phosphorylation during the cell cycle[J].J Cell Sci,1993,106(Pt 3):983-994.
  • 10Oakley GG,Patrick SM,Yao J,Carty MP,Turchi JJ,Dixon K.RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions[J].Biochemistry,2003,42(11):3255-3264.

共引文献6

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部