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目的 :构建抗五步蛇毒的特异性ScFv噬菌体显示文库 ,并从中筛选出阳性克隆。方法 :用五步蛇毒素免疫BALB C小鼠 ,挑选其中效价最高的 3只小鼠提取脾脏组织 ,抽提细胞总RNA ,经RT PCR分别扩增出VH、VL基因片段 ,经Linker连接成ScFv基因 (singlechainvariablefragment) ,再把ScFv基因重组到pCANTAB 5E载体 ,转化至大肠杆菌TG1中表达 ,经辅助噬菌体 (Helperphage)M13K0 7拯救后建成噬菌体显示文库。结果 :经 4轮吸附 洗脱 富集筛选后 ,库容量达到 4× 10 8cfu L ,随机挑取 90个克隆进行ELISA检测 ,结果 16个呈阳性 ,并进行了重复验证。结论 :成功构建出具有一定库容的特异性ScFv噬菌体展示库
OBJECTIVE: To construct a specific library of ScFv phage displayed by anti-five-step snake venom and screen positive clones. Methods: BALB C mice were immunized with pentobarbital toxin and 3 mice with the highest titer were selected to extract the total RNA of the cells. The VH and VL gene fragments were amplified by RT PCR and linked by Linker ScFv gene. The ScFv gene was then recombined into pCANTAB 5E vector and transformed into E. coli TG1 for expression. After rescue by Helperphage M13K0 7, a phage display library was constructed. Results: After four cycles of adsorption and enrichment screening, the library capacity reached 4 × 10 8 cfu L, and 90 clones were randomly selected for ELISA test. The results of 16 tests were positive and repeated validation was carried out. CONCLUSION: A specific ScFv phage display library with a certain capacity has been successfully constructed