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目的制备全人源化食管癌单链抗体scFv基因文库。方法取食管癌病人癌肿周围淋巴结作为B细胞的来源,提取总RNA,用RT-PCR的方法获得抗体可变区基因cDNA文库。首先分别网格筛选确定扩增VH和VL基因片段的引物对,以cDNA为模板扩增VH和VL基因片段,再以它们为模板分别扩增VH-linker与VL-linker,用SOE-PCR技术将它们拼接成scFv,再引入酶切位点SfiI和NotI,胶回收PCR产物获得scFv。将scFv基因克隆入噬菌粒载体pCANTAB-5E后电转入EcoliTG1。PCR法鉴定抗体基因插入率,1.5%琼脂糖凝胶电泳鉴定阳性克隆酶切产物。结果食管癌周围淋巴结的提取总RNA琼脂糖电泳结果中可见清晰的28S、18S条带;VH基因的大小约为450bp,VL基因为350bp,组装后的scFv基因约为850bp。PCR连接产物的转化效率为2×107cfu/μg,scFv的阳性插入率为91.7%(22/24)。结论食管癌相关的人源单链抗体基因文库的构建为进一步筛选单链抗体库奠定了基础。
Objective To prepare scFv gene library of humanized esophageal single chain antibody. Methods The total RNA was extracted from lymph nodes around the cancer of the esophageal cancer patients and the total RNA was extracted. The cDNA library of the variable region of the antibody was obtained by RT-PCR. Firstly, the primer pairs of the VH and VL gene fragments were screened by the grid selection respectively, the VH and VL gene fragments were amplified by using the cDNA as a template, VH-linker and VL-linker were amplified by using the template as a template, and the SOE-PCR technique They were spliced into scFv, and then introduced enzyme sites SfiI and NotI, gel recovery PCR products obtained scFv. The scFv gene was cloned into the phagemid vector pCANTAB-5E and then transformed into EcoliTG1. PCR method to identify the rate of antibody gene insertion, 1.5% agarose gel electrophoresis identified positive cloning products. Results The 28S and 18S bands were clearly observed in the total RNA extracted from lymph nodes around the esophageal cancer. The size of VH gene was about 450bp, the VL gene was 350bp, and the assembled scFv gene was about 850bp. The transformation efficiency of PCR ligation products was 2 × 107 cfu / μg and the positive insertion rate of scFv was 91.7% (22/24). Conclusion The construction of esophageal cancer-associated human single-chain antibody gene library lays the foundation for further screening of single-chain antibody library.