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目的 构建抗华支睾吸虫噬菌体抗体库 ,以期筛选出抗循环抗原的抗体组合。方法 应用噬菌体表面呈现技术 ,从感染华支睾吸虫病人淋巴细胞中提取总 RNA,逆转录成 c DNA后 ,用相应引物 PCR扩增出重链 Fd和轻链基因 ,经 Xho 和 Spel、Sac 和 Xba 双酶切 ,先后克隆入噬粒载体 p Comb3,再电转化大肠杆菌 XL1- blue菌株 ,辅助噬菌体 VCSM13超感染。结果 从感染华支睾吸虫病人淋巴细胞中扩增出约 70 0 bp重链 γ1 、γ3Fd段和轻链 κ、λ基因 ,分别和 p Com b3连接后成功导入 XL 1- blue,得到滴度为 7.5× 10 1 0 cfu/ ml,库容量为 5 .6× 10 6 噬菌体抗体库。结论 抗华支睾吸虫 Fab段噬菌体抗体库的构建 ,为进一步筛选抗华支睾吸虫循环抗原单克隆抗体奠定了基础。
Objective To construct the phage antibody library of Clonorchis sinensis to filter out the anti-circulating antigen antibody combinations. Methods Total RNA was extracted from the lymphocytes of Clonorchis sinensis infected by phage display technique and then reverse transcribed into c DNA. The heavy chain Fd and light chain genes were amplified by PCR with Xho and Spel, Sac and Xba double digestion, successively cloned into the phagemid vector p Comb3, and then electroporation E. coli XL1-blue strain, helper phage VCSM13 superinfected. RESULTS: Approximately 70 0 bp of heavy chain γ1, γ3Fd and light chain κ, λ genes were amplified from lymphocytes infected with Clonorchis sinensis, and were successfully introduced into XL 1-blue after ligated with p Com b3 respectively. The titer was 7.5 × 10 10 cfu / ml, with a library capacity of 5.6 × 10 6 phage antibody library. Conclusion The construction of Fab phage antibody library against Clonorchis sinensis lays the foundation for the further screening of monoclonal antibodies against Clostridium sinense.