Construction of Gene Library of Human-Originated Single-Chain Variable Fragment Antibody Associated with Esophageal Cancer

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.