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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。
The aim was to find genes encoding the secreted protein of salivary glands in Anopheles. Methods Total RNA was extracted from salivary glands of Anopheles stephensi using RNeasy kit. Double - stranded cDNA was reverse transcribed by OrientExpressTM Oligo (dT) cDNALibraryCtruction system. After digestion, EcoRⅠ / HindⅢlinker was added and digested with EcoR Ⅰ / Hind Ⅲ linker. After size fractionation, Connected in vitro packaging lambda phage particles, constructed cDNA expression library; in E. coli ER1647 infected after the detection of cDNA expression library size; anti-salivary gland serum using a portion of the library immunoscreening, and cDNA fragments containing positive λ SCREEN The cloned strain was transformed into pSCREEN plasmid, and after digestion with EcoRI / HindIII, the cDNA fragment was analyzed. Results The cDNA expression library capacity was 4 × 10 6 / pfu. Three anti-salivary gland serum proteins were screened to obtain three positive clones. The length of the digested cDNA fragment was about 500 pb. Conclusion The positive clones that reacted with anti-salivary gland glycoprotein were screened and the gene fragment encoding salivary gland protein antigen was obtained and transferred into pSCREEN plasmid for the further study of the function of coding secreted protein gene in mosquito salivary glands The foundation.