Construction and screening of human-originated phage single-chain antibody library associated with esophageal cancer / 中国药理学通报

ABSTRACT

Aim To construct human phage single-chain antibody library associated with esophageal cancer and to screen the specific scFv against Eca109 cells from the liberary. Methods Metastatic periesophageal lymph nodes of esophageal cancer patients were used as the B cells source, the total RNA of these B cells was extracted and prepared as the template of RT-PCR. First, we screened graticulely 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 by the polymerase chain reaction (PCR). Second, the V_H-linker and V_L-linker were amplified from the V_H and V_L fragments. Last, the V_H-linker and V_L-linker were assembled into scFv gene fragments by SOE-PCR,and then Sfi I and Not I restriction site were inlet in it. ScFv gene was cloned into the pCANTAB-5E phagemid. Phagemids were introduced into E.coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Recombinant scFv phage library was constracted and PCR was used to identify the insert ratio of scFv antibodies library. Results of SfiI/Not I double digestion reaction positive insert clone were identified by 1.5% agarose gel electrophoresis. The phage library was panned with NHEEC and Eca109 cancer cells in suspension for four rounds. Strongly positive recombinant phage clones were used to infect E.coli HB2151. Expression of soluble scFv was induced by IPTG. Soluble scFv from periplasm were purified by affinity chromatography and identified by SDS-PAGE and Western blot. Cell ELISA , immunohistochemical staining and immunocytochemical staining were used to identify the activity of the soluble scFv. Results The result of agarose gel electrophoresis showed that total RNA of these B cells had two bands of 28 S and 18 S. The size of V_H fragment is about 450 bp,V_L fragment is about 350 bp and scFv is about 850 bp. The competence is 108 cfu??g-1 pUC18 DNA. Randomly digestive reac-tion showed that the positive insert ratio was 91.7% (22/24). After four rounds of panning, the fourth phage yield is 141 times as much as that of the first one. SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30 ku and the brand of 30 ku was stained. Immunohistochemical staining showed strong stainning of the tissue of esophageal cancer, but not the liver and gastric cancer tissue. Immunocytochemical staining showed significant staining of the esophageal cancer line Eca109. The result of cell ELISA assay revealed that soluble scFv had highly specific and could combined with Eca109 cells, but not with BGC-823 and NHEEC. Conclusion A human scFv phage display library associated with esophageal cancer has been constructed successfully and the specific scFv antibody against Eca109 has been identified from the liberary.