摘要
目的 为寻找编码按蚊唾液腺分泌性蛋白质的基因。方法 应用RNeasy试剂盒提取斑须按蚊唾液腺总RNA ;OrientExpressTM Oligo(dT)cDNALibraryConstruction系统反转录合成双链cDNA ,修饰后加上EcoRⅠ /HindⅢlinker,经酶切、大小分馏后 ,与λSCREEN - 1臂相连 ,体外包装为λ噬菌体颗粒 ,构建成cDNA表达文库 ;在感染大肠杆菌ER1647后 ,检测cDNA表达文库的大小 ;应用兔抗唾液腺蛋白血清对部分文库进行免疫筛选 ,并将含cDNA片段的阳性λSCREEN克隆株转化成 pSCREEN质粒 ,经EcoRⅠ /HindⅢ消化后 ,分析cDNA片段。 结果 cDNA表达文库容量为 4× 10 6 / pfu。应用抗唾液腺蛋白血清筛选 ,获得 3个阳性克隆株 ,酶切后cDNA片段的长度约为 5 0 0 pb。 结论 已筛选到与抗唾液腺蛋白的免疫血清反应的阳性克隆株 ,而且获得编码唾液腺蛋白抗原的基因片段 ,并将其转入 pSCREEN质粒中 ,为进一步研究蚊唾液腺内编码分泌性蛋白质基因的作用奠定了基础。
Aim To find the genes which code the secreted salivary proteins.Methods Total RNA from salivary glands of Anopheles stephensi was extracted with RNeasy Midi kit.Using OrientExpress TM Oligo(dT) cDNA Library Construction systems,double-strand cDNA was synthesized and directional EcoRⅠ/HindⅢ linker was added to the cDNA fragment.After EcoRⅠ/HindⅢ digestion and size fractionation,the λSCREEN-1 arm was ligated to the cDNA fragment.The ligated cDNA was in vitro packaged into lambda phage.Thus a cDNA expression library was constructed.After the phage infected E.coil ER1647,the size of the library was determined.After that,the library was screened with rabbit anti-salivary gland sera and the positive recombinant phages were converted to pSCREEN plasmid subclones.Digested with EcoRⅠ/HindⅢ,then cDNA insert fragments were analyzed.Results The size of cDNA expression library was 4×10 6/pfu.Three positive clones were obtained using immunoscreening with rabbit anti-salivary gland sera and after digested with EcoRⅠ/HindⅢ,the size of the cDNA fragments was about 500pb.Conclusion The positive clones were found to react with immunosera,and they may be the genes encoding the salivary antigens of An.stephensi.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2001年第5期24-26,共3页
Chinese Journal of Zoonoses
