Construction Of Monoclonal Antibody And Single Chain Antibody Fragment Against Secretory Protein SHON

In 2015,China’s cancer statistics show that about 4,292,000 people in China would be newly diagnosed with invasive cancer,which is equivalent to 12 thousand people diagnosed each day.Cancer is a serious threat to the survival and health of modern people.In the late twentieth century,monoclonal antibodies targeting therapy grow rapidly because of the limited efficacy of conventional radiotherapy and chemotherapy.Since the advent of rituximab(the first monoclonal antibody for the treatment of cancer),a large number of monoclonal antibodies as therapeutics have been used in patients and obtain good results.An important factor influencing the therapeutic effect of monoclonal antibody is the choice of antigen protein.SHON is a novel protein that has been reported in recent years in breast cancer,lung cancer,prostate cancer and other cancers.Previous studies in our laboratory have found that the high expression of SHON in breast epithelial cells can promote cell proliferation,invasion and migration,leading to the occurrence of EMT,and participate in the promotion of cancer through the TGF-β signaling pathway.Therefore,SHON is expected to become a new target for diagnosis and treatment of many cancers,especially breast cancer.In this paper,we used affinity purified SHON protein as antigen and prepared several hybridoma cell clones for secreting SHON specific monoclonal antibody.Using ELISA,MTT,Western blotting and migration experiment,we found that antibody 5# can not only specifically bind with SHON in vitro and in vivo,but inhibit SHON carcinogenesis in some extent.We cloned the whole gene of 5# antibody using 5’RACE kit.The length of VH is 415 bp,and the length of VL is 396 bp.The complete sequence were further verified by PCR while the heavy chain is 1425 bp and light chain is 720 bp.Antibody gene was cloned to the eukaryotic expression vector and transfected into CHO cells to test the expression and secretion of the specific antibody.Then we use the SOE-PCR technology to clone a single chain antibody fragment,and successfully constructed a prokaryotic expression vector of single chain antibody with his tag using hydrophilic linker sequence to connected VH and VL in VL-linker-VH order.We obtained a lot of his-scFv-SHON antibody using his tag affinity purification.The capacity of scFv binding antigen was validated by ELISA experiment.Our work thus laid the foundation for next step developing of detection kit and monoclonal antibodies targeting drug of SHON for breast cancer.