Construction Of Single Chain Antibody Against CTGF And Its Role On Pancreatic Cancer Cells

Introduction：Connective tissue growth factor (CTGF), is a member of the CCN family, also knownas CCN2, which is isolated from human umbilical vein endothelial cells by Bradham in1991. It iscystein-rich, matricellular secreted, heparin-binding proteins, composed of349amino acid residues with arelative molecular mass of38kd and it is secreted by the Golgi apparatus. CTGF molecule contains fourhighly conserved structural regions which are termed insulin-like growth factor binding area (IGFBP),C-type vascular repeat region of the von Willebrand factor (VWC), thrombospondin1repeat region (TSP1)and cysteine-containing carboxyl end (CT). CTGF is an important cellular factor which regulates cellsurvival, proliferation, differentiation, migration, angiogenesis, wound healing,chondrogenesis and boneformation. But when it is over expressed, CTGF promoting scar formation, fibrosis, tumorigenesis. It isconsidered as the candidate target to cure pancreatic carcinoma in American Society of Clinical Oncology2011. So the antibody to CTGF will play an increasingly larger role in the future of the life sciences field.Phage display technique is first explained in1985, through this technique, the gene encoding targetprotein were inserted into ithe genome which encoded protein capsid of the phage, and then target proteinwould be displayed on the protein capsid of the phage. The target protein would be enriched with thepropagation of phage, and collect by bio-span. Phage display technique is widely used in the developmentof Vaccine, medical diagnosis and treatment, screening of inhibitors ofenzyme, peptide drug development,protein interaction studies, and shows a good prospect.In this experiment, cDNA library of single chain Fv antibodies against CTGF was constructed byphage display technology, single chain antibodies against CTGF was obtained by affinity selection. Affinityselection overcome disadvantage of genetic engineering antibodies that may have low affinity to targetprotein. cDNA library from immunized mice has lower false positive rate than random library. Cellexperiment confirmed the yield inhibiting the proliferation of PANC-1cells, promoting the apoptosis ofPANC-1cells, which supply a new method to synthesize drugs to pancreatic cancer.Objective: This study aimed to construct single chain Fv (scFv) library of human connective tissuegrowth factor (CTGF) by assembling the gene from immunized mice to phagemid PAK100, and to obtainthe anti-human–CTGF scFv by phage display technique. And examine the biological activity of the yield by added into PANC-1cells.Methods: mice were immunized with recombinant human CTGF (rhCTGF).Total RNA was extractedfrom splenocytes of the immunized mice.Complementary DNA was got by reverse transcription PCR withthe extracted RNA. Then the fragment of (variable heavy) VH and (variable light) VL were obtained byPCR. And they were assembled into scFv, which was inserted into phagemids vector PAK100throughSfiⅠsites. The phagemids containing scFV were transferred into E.coli. XLE-Blue bacterial cells toconstruct the scFv library of anti-human CTGF antibody. Four round of bio-panning were performed to getspecific anti-human CTGF scFv from the library. The affinity was examined using ELISA assay. The clonewith highest affinity was amplified and the yield was used to treat PANC-1cells. Through MTT and FCMassay, the influence of scFv antibody on the proliferation and apoptosis was examined.Results: mice were successfully immunized, RNA of immunized mice was reverse transcript intocDNA,which was used to get the gene of scFv antibody. Phage display library of scFv antibody wassuccessfully established, through4round bio-panning, high affinity anti-human CTGF scFV antibody wasobtained. The anti-human CTGF scFv antibody inhibits the proliferation of PANC-1cells, promotes theapoptosis of PANC-1cells.Conclusion: CDNA library derived from immunized mice includes more target gene than randomlibrary, which enhances the possibility to get special antibody. Bio-panning enhance the affinity of yield totarget protein. That the yield has available activity to PANC-1cells, supplies a new method to synthesize drugs to pancreatic cancer.