Immunoreactivity Studies Of KGH-R1-ScFv Single-chain Antibody And P21Ras Protein

ras gene,as one of the classical gene,was concerned since it has been found.The research fever isn't still cut down abou ras right now.The main reason is that the Ras protein plays an important role in many signaling pathways,such as regulate cell proliferation,differentiation,and survival.In addition,about 33%of human cancer cells have ras gene mutations.Interventions of ras genes or p21 Ras proteins have a very broad application prospects in the field of cancer trastment.Currently,ras gene as a target for therapeutic strategies mainly include antisense oligonucleotides and RNA interference technology,and to p21Ras protein as a target for the treatment of immune-based therapy major.Immunotherapy targeting due to a highly efficient and secure when essentially kill cancer cells and without damaging normal tissue,it is considered the most promising future in the treatment of cancer treatment techniques.By using anti-Ras antibody can block Ras signaling and may be treat the cancer.Our laboratory have built a broad-spectrum anti-Ras monoclonal antibody by using the wild Hp21Ras protein as an antigen,and on the basis of monoclonal antibody we have constructed a broad-spectrum anti p21Ras KGH-R1-ScFv single chain antibody.Early laboratory work has demonstrated KGH-R1-ScFv single chain antibodies can combind with many kind of p21Ras protein with highly express,but protein of p21Ras types are unclear.In order to maximum play the role of the single-chain antibody in the treatment of Ras associated tumors we must clarify the immune response spectrum.The purpose of this experiment is to study the immunoreactive between KGH-R1-ScFv single chain antibodies and various p21Ras protein isoforms from the levels of protein and cell.1,immunoreactivity with the three wild-types of p21Ras protein;2,immunoreactivity with seven kinds of mutant p21Ras protein;3,immunoreactivity with four kinds of the tumor cells which their p21Ras styles are known.The first step,prokaryotic expression wild-H,N and K-p21Ras protein and mutant Kp21RasG12S,Kp21 RasG12V,Kp21 RasG 13D,Np21 RasQ61K,Np21RasG12C,Np21RasQ61R,Hp21RasG12V protein;the second step,prokaryotic expression KGH-R1-ScFv single chain antibody;the third step,enzyme linked immunosorbent assay(ELISA)to detect the immunoreactivity of KGH-R1-ScFv single chain antibody to the wild and mutant p21Ras proteins;the fourth step,immunofluorescence assay of KGH-R1-ScFv to the tumor cells(MD-MB-231 human breast cancer cells,A549 human lung cancer cells,SW480 human colon cancer cells,HuH7 human hepatoma cells);the fourth step,Western Blotting assay further verification the immunoreactivity between KGH-R1-ScFv single chain antibody and tumor cells p21Ras protein.The results of ELISA method showed that KGH-R1-ScFv single chain antibody combined with the wild-type H,N,K-p21Ras protein and mutant p21Ras protein(Kp21RasG12S,Kp21RasG12V,Kp21RasG13D,Np21RasQ61K,Np21RasG12C,Np21RasQ61R protein),but Hp21RasG12V protein was negative results.Drawing a ELISA four-parameter curve fitting according to protein concentration and absorbance by using microplate reader,the results to showed KGH-R1-ScFv single chain antibodies immune binding mutant Kp21Ras protein was higher than mutant Np21Ras protein,all the wild-type Hp21Ras,Kp21Ras Np21Ras protein have a high immune binding force.Tumor cells immunofluorescence showed KGH-R1-ScFv single chain antibody bind with DMD-MB-231 human breast cancer cells(Kp21RasG13),A549 human lung cancer cells(Kp21RasG12S),SW480 human colon cancer cells(Kp21RasG12V)and HuH7 human hepatoma cell(mutant NRas).Tumor cell lysis and protein in tumor cells were collected,followed by Western Blotting experimental results has once again proven KGH-R1-ScFv single chain antibody combined with tumor cells p21Ras proteins.The experiment improved the KGH-R1-ScFv single chain antibody and p21Ras protein immune response spectrum and provides a theoretical basis the for the follow-up of the single-chain antibody combined with other drugs treatment of cancer.in addition,this experimental results provides strong evidence to the KGH-R1-ScFv single chain antibody therapy type of tumor specific mutations.