The Preparation And Study On Anti-human-gastrin McAb And ScFv

Objective: To establish hybridoma cell lines that can secreteanti-human gastrin McAb (monoclonal antibody) with high affinity and specificity. Based on the identificaton of their affinity, specificity and function, clone the variable region genes of the McAb and construct recombinant anti-gastrin single chain Fv (ScFv) gene. Then transform and express the ScFv gene in E. coli, and identify theresulting protein. Methods: The hybridoma cell lines that can secreteanti-human gastrin McAb with high affinity were established by hybridoma technique. Purified McAbs were prepared by salt precipitation and protein G chromatography. The molecular weight and purity of McAbs were identified by SDS-PAGE, the titer of McAbs was assessed by indirect ELISA, the class and subclass of McAbs were detected by double immuno-diffusion, the affinity of McAbs was examined by non-competitive enzyme immuno-assay, and the specificity of McAbs was identified by dot-immunobinding assay and immunohistochemistry. The inhibitory effect of McAbs E8 on the binding between gastrin and its receptor and on the growth of human gastric tumor cell line SGC-7901 were also examined by fluroscencestaining technique and MTT chromatometry. Based on above studies, the total RNA of hybridoma cell line E8 was isolated with TRIZOL reagent, and the variable region genes were amplified with variable region primers by RT-PCR. The variable region genes of heavy chain and light chain were strung together by SOE-PCR (splicing by overlap extension) and the ScFv gene was constructed. After cloned into pGEM-T vector, the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing. The recombinant expression vector pET-gastrin-ScFv was constructed by endonuclease digestion and ligation, and then transformed into E. coli BL21(DE3) strain. After screened on LB plate with Kanamycin and identified by endonuclease digestion and PCR, the positive strain was cultured under IPTG. The inclusion body of the induced bacteria was collected, denaturalized with urea and renaturalized by dialysis with renaturalizing solution. The molecular weight and immuno-activity of expressed protein wereidentified by SDS-PAGE and competitive inhibition ELISA. Results:Three hybridoma strains that can secrete anti-human gastrin McAb were established. The anti-gastrin McAbs with high purity were prepared. Their protein concentration ranges from 3mg/ml to 8mg/ml, with titer of 1: 12,800-1: 51,200 and affinity of 109~1010L/mol. Three strains of McAb show high specificity to both gastrin peptide and gastrin in cells as verified by dot-immunobinding assay and immunohistochemistry. It was also verified that McAb E8 could block the binding between gastrin and its receptor, and significantly inhibit the growth of tumor cells. The variable region genes were amplifiedfrom hybridoma cell line E8 by RT-PCR, and recombinant ScFv gene with about 75Obp was constructed. The sequencing result shows that the ScFv gene consists of 720bp, including 348bp heavy chain variable region gene. 327bp light chain variable region gene and 45bp linker gene. Then the ScFv gene was cloned into expression vector pET-28a ( + ) by the direction of VH-linker-VL. The recombinant ScFv gene was expressed successfully in E.coli under the induction of IPTG. The anti-gastrin ScFv protein was expressed mainly in the form of inclusion body and with a molecular weight of.about 30KD. After denaturalization and renaturalization treatment, the immuno-activity of anti-gastrin ScFv protein was verified as it can competitively inhibit the binding between McAb E8 and gastrin with inhibitory rate of 28%.Conclusions: Three strains of hybridoma cell lines that can secreteanti-human gastrin McAb with high affinity and specificity were establised. The variable region genes of the McAb were amplified and ScFv gene was constructed. The recombinant anti-gastrin ScFv gene was then transform and successively express in E. coli as a fusion protein with immuno-activity.