【摘要】 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。更多还原

【Abstract】 Objective To construct human single-chain variable fragment (scFv) antibodies gene library associated with esophageal cancer. Methods Metastatic periesophageal lymph nodes of esophageal cancer were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. Firstly, we screened gratefully two pairs of primers of the heavy and light regions separately, then the VH and VL fragments were first amplified from the cDNA. Secondly, the VH-linker and VL-linker were amplified from the VH and VL fragments. Lastly, SOE-PCR was used to connect the VH-linker and VL-linker to ScFv,the Sfi I and Not I restriction site was inlet in the scFv. The gel purified scFv gene repertoires are digested by Sfi I and Not I separately. The ligation mixes of scFv and pCANTAB-5E are transformed into competence E.coli TG1. The insert ratio of scFv antibodies library was identified by PCR. The products of Sfi I/Not I double digestion reaction positive insert clone were identified by 1.5% agarose gel electrophoresis. Results Total RNA is good. The size of VHis about 450bp, and VL is about 350bp.The size of scFv is about 850bp. After transformation into E.coli TG1, 2×107cfu/μg ampicillin resistant bacteria colonies grow after overnight culture. Randomly digestive reaction showed that the positive insert ratio was 91.7% (22/24). Conclusion These results are bases to further phage antibody library construction.更多还原