论文部分内容阅读
目的 :构建一个鼠源性的抗内毒素单链噬菌体抗体库 ,从中筛选出对内毒素具有较高亲和力的单链抗体。方法 :从小鼠脾细胞中提取总RNA ,通过RT PCR技术扩增出小鼠抗体重链、轻链可变区基因 (VH,VL) ,用Linker将VH,VL 交联形成单链抗体可变区片段 (ScFv)。经NotⅠ ,SfiⅠ双酶切后与经同样双酶切的pCANTAB5E载体相连 ,转化入大肠杆菌TG1以构建鼠抗内毒素单链噬菌体抗体库。在援救噬菌体抗体库后 ,用内毒素淘筛特异性的ScFv ,富集的噬菌体阳性克隆重新感染TG1。在 96孔板分别援救单个含特异性ScFv的TG1菌落 ,最后随机挑选出 190个菌落经ELISA检测抗内毒素ScFv。结果 :小鼠血清中抗内毒素的效价为 1∶12 80 0。提取的总RNA浓度为 12 .3 813 μg/ml,纯度较好。扩增出的VH 长约 3 4 0bp ,VL 约 3 2 0bp ,ScFv约 80 0bp。转化入TG1后有约 1.9× 10 7个菌落。淘筛一轮过后即有 3× 10 4阳性菌落长出 ,190个菌落经ELISA检测有 2个阳性克隆。结论 :成功地构建了一个库容量为 1.9× 10 7的鼠抗内毒素单链噬菌体抗体库 ,并从中筛选出了 2株抗内毒素ScFv。
OBJECTIVE: To construct a murine anti-endotoxin single-chain phage antibody library and screen out the single-chain antibody with high affinity to endotoxin. METHODS: Total RNA was extracted from mouse spleen cells. The heavy and light chain variable region genes (VH and VL) of mouse antibody were amplified by RT PCR. The Linker was used to crosslink VH and VL to form single chain antibody Zone fragment (ScFv). After double digestion with NotⅠ and SfiⅠ, the recombinant plasmid was ligated into the pCANTAB5E vector with the same double digestion and transformed into E. coli TG1 to construct a murine anti-endotoxin single-chain phage antibody library. After rescuing the phage antibody library, the specific ScFv was panned with endotoxin and the enriched phage-positive clones reinitiated TG1. Respectively in a 96-well plate rescued a single specific ScFv containing TG1 colonies, the final random selection of 190 colonies by ELISA anti-endotoxin ScFv. Results: The titer of endotoxin in mouse serum was 1:12 80 0. The extracted total RNA concentration was 12.3813 μg / ml, the purity was better. The amplified VH is about 340 bp, the VL is about 320 bp, and the ScFv is about 80 bp. Approximately 1.9 × 10 7 colonies were obtained after transformation into TG1. After a round of panning, 3 × 10 4 positive colonies grew and 190 colonies were detected by ELISA with 2 positive colonies. Conclusion: A murine anti-endotoxin single-chain phage antibody library with a capacity of 1.9 × 10 7 was successfully constructed and two anti-endotoxin ScFv were screened out.