日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及阳性克隆分析  

Screening of T7 Phage-display cDNA Library from Liver of Microtus fortis with Extracts of Schistosomulum and Characterization of the Positive Clones

作　　者：贾人初[1,2] 孙毅[1] 刘金明[1] 傅志强[1] 苑纯秀[1] 石耀军[1] 陆珂[1] 孙焕[1] 李浩[1] 金亚美[1] 林矫矫[1] 

机构地区：[1]中国农业科学院上海兽医研究所国家防治动物血吸虫病专业实验室,农业部动物寄生虫学重点开放实验室,上海200232 [2]上海师范大学生命与环境科学学院,上海200234

出　　处：《生物工程学报》2008年第5期733-739,共7页Chinese Journal of Biotechnology

基　　金：上海市科学技术委员会科研计划项目(No.054909002);科技部自然资源平台项目(No.2005DKA21104)资助~~

摘　　要：东方田鼠对血吸虫具有天然抗性。为筛选和分析东方田鼠抗血吸虫抗性相关基因,以日本血吸虫童虫可溶性裂解物为探针,筛选东方田鼠肝脏噬菌体展示cDNA文库。经三轮筛选,特异性噬菌体得到有效富集(375倍)。随机挑取92个克隆进行序列测定,获得了19条有效EST序列。其中13个条EST序列与已知基因或表达序列标签同源,6个EST序列与已知基因或表达序列标签均无同源性,为新的表达序列标签。将19个EST序列的阳性噬菌体克隆和血吸虫童虫共培养,其中4号(GenBank Accession No.:EW968294)、13号(GenBank Accession No.:EW968303)、14号(GenBank Accession No.:EW968304)、15号(GenBank Accession No.:EW968305)、18号(GenBank Accession No.:EW968308)克隆均诱导了显著的杀虫效果。综合生物信息学分析结果及体外杀伤试验结果,编码CASP8和FADD类似性细胞程序性死亡调节蛋白、α-2-HS-糖蛋白、M4蛋白、具有R3H结构域的一种mRNA结合蛋白以及三种未知蛋白的编码基因(14、15、18号克隆)可能是东方田鼠抗血吸虫病抗性相关基因。为进一步研究东方田鼠抗血吸虫机理奠定了基础。Microtus fortis is naturally resisitent to Schistosoma japonicum. In order to find schistosome-resistence-related genes of Microtus fortis, a T7 phage-display cDNA library from liver of Microtusfortis was screened with the soluble lysate of schistosomula. The specific phages were enriched 375-fold after 3 rounds of biopanning. Ninety-two positive clones picked at random were sequenced and 19 ESTs including 6 unreported genes were obtained. Compared with the negative phage clone control, five positive clones, No.4 （GenBank Accession No.： EW968294）, No.13 （GenBank Accession No.： EW968303）, No.14 （GenBank Accession No.： EW968304）, No.15 （GenBank Accession No.- EW968305） and No.18 （GenBank Accession No.： EW968308） could induce significantly higher schistosomula mortality rate when co-cultivated with schistosomula. According to the function analysis and the shistosomula-killing effect in vitro, the genes encoding CASP8 and FADD-like apoptosis regulator isoform protein, alpha-2-HS-glycoprotein, M4 protein, R3H domain （binds single-stranded nucleic acids） isoform 2 and 3 previously unreported proteins （No. 14, No. 15 and No. 18） obtained here, were schistoso related genes of Microtusfortis

关 键 词：日本血吸虫 东方田鼠 肝脏 噬菌体展示cDNA文库 抗性基因 

分 类 号：R38[医药卫生—医学寄生虫学] Q78[医药卫生—基础医学]