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

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 V_H and V_L fragments were first amplified from the cDNA. Secondly, the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Lastly, SOE-PCR was used to connect the V_H-linker and V_L-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 V_H is about 450bp, and V_L is about 350bp. The size of scFv is about 850bp. After transformation into E. coli TG1, 2 ×10⁷ cfu/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.