The Preparation Of ScFv And Immunotoxin ScFv-TCS And Anticancer Activities In Gastric Cancer

ObjectiveTo study an optimal expression method for the scFv antibody and to construct a single-chain immunotoxin (scFv-TCS) composed of scFv and trichosanthin (TCS) for the treatment of gastric cancer.Part I:Expression of single chain antibody (scFv) in E. coli and P. pastorisThe gene of the scFv antibody was cloned from phage-displayed scFv library. Firstly, the scFv gene was cloned into prokaryotic expression vector PET30a, and expressed in E. coli using 1 mM IPTG for 3 hours at 37℃. After collected, the cells were sonicated, and the scFv protein was expressed as inclusive body. The scFv protein was denatured with 8 M urea and purified by a Ni-NTA column. The scFv was renatured through dialysis against PBS. In order to get the soluble expression, a yeast expression system was introduced. The scFv gene was cloned into yeast expression vector pPIC9, and expressed in P. pastoris with induction of methanol for 120 hours.Part II:Expression of immunotoxin (scFv-TCS) in E. coli and P. pastorisThe genes of scFv and TCS were linked with GGGGS by gene engineering. Firstly, the scIT gene was cloned into plasmid PET30a, and expressed in E. coli induced with 1 mM IPTG for 3 hours at 30℃. The cells were harvested and sonicated. The scIT protein was expressed as inclusive body. The scIT was denatured with 8M urea, purified by Ni-NTA column and subsequently renatured through dialysis step. To get the soluble expression of the immunotoxin, the scIT gene was cloned into vector pPIC9, and expressed by inducing with methanol for 120 hours.Part III:Anti tumor evaluations in vitro and in vivoThe cytotoxicities of recombinant scFv and scIT in gastric cancer cell MGC-803 were analyzed by MTT assay. Nude mice bearing MGC-803 xenograft were intravenously injected with scFv, scIT and TCS for six times. The size of tumor was observed and measured.Part IV:The detection of anti-HER2 antibody to HER2 over-expressing gastric cancerNew Zealand rabbits were immunized to gain anti-HER2 polyclonal antibody. The affinity of the antibodies with HER2 over-expressing human gastric cancer cell NCI-N87 was measured by using ELISA. The immunohistochemistry assay was used to detect the expression of HER2 in 27 specimens of gastric cancer patients.Results1. The scFv with MW 27kD was expressed as inclusive body in E. coli BL21(DE3) after induced with 1 mM IPTG at 37℃ for 3h. The recombinant scFv was obtained after purification and renaturation. However, in P. pastoris GS115, the scFv expressed as a soluble form with low concentration.2. Immunotoxin (scFv-TCS) with MW 55kD was expressed as inclusive body in E. coli BL21(DE3) after induced with 1 mM IPTG at 30℃ for 3h. The scIT was obtained after purification and renaturation. However, in P. pastoris GS115, the scIT expressed as a secretory protein with low concentration.3. Both the scFv and immunotoxin (scFv-TCS) showed cytotoxicity to MGC-803 cells, and the effect was in a dose dependent manner. And both of they had the inhibitory effect in vivo test.4. Anti-HER2-ECD antibody could specifically bind to the HER2 over-expressing gastric cancer cell NCI-N87. The expression of HER2 in 27 cases are IHC 0(14.8%), IHC 1+(25.9%), IHC 2+(40.8%), IHC 3+(18.5%).ConclusionThe scFv and immunotoxin (scFv-TCS) were successfully expressed in E. coli BL21(DE3), and showed inhibitory effects to gastric cancer. High specificity antibody against HER2 was prepared which could be used for detection of HER2 over-expressing gastric cancer.