摘要
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
Objective:To construct a ScFv phage library that can neutralize agkistrodon acutus guenther venom specially, from which positive clonies would be gotten. Methods:BALB/C mice were immunized by the venom.3 mice who had the highest antibody titer were selected and the total RNA of the spleenic cells were extracted from the spleen tissue of the murine.VH and VL were amplified by RT-PCR, and were joined into ScFv(single chain variable fragment) through a linker.ScFv was recombined to pCANTAB 5E vector and was transformed to TG1.Results:The library capacity reached 4×10 8 cfu/L after 4 rounds of absorb-elute-enrich panning.90 clones from it were selected at random and 16 positive clonies with ELISA were gotten.Conclusion:The special ScFv phage library was constructed successfully.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2004年第5期338-340,共3页
Chinese Journal of Immunology
关键词
五步蛇
蛇毒
单链抗体
噬菌体显示文库
Agkistrodon acutus guenther
Venom
Single chain antibody fragment
Phage displaying library
