Study On The Expression Of GPC3-N Protein And The Preparation Of Its Antibody

Liver cancer is the most common primary cancer in the liver,with the fifth highest incidence rate and the third highest mortality rate in China,seriously endangering the lives and health of Chinese people.Hepatocellular carcinoma（HCC）accounts for 90%of liver cancer.The screening,early diagnosis and treatment of liver cancer need to be improved.Glypican-3（GPC3）is a membrane protein fixed on the cell surface,which is closely related to the occurrence,development and prognosis of liver cancer.It is not only present in the serum of HCC patients,but also expressed specifically on HCC tissues.GPC3 is a biomarker specific for the diagnosis of HCC and an important potential biomarker for the treatment of HCC.The preparation of high-affinity and high-specificity antibody against GPC3 is very important for studying the mechanism of GPC3 in the process of hepatocarcinogenesis,establishing a more efficient detection method,improving the early diagnosis rate and accuracy of HCC,and improving the deficiencies of the existing treatment methods.This thesis is mainly divided into four parts as follow.PartⅠ Prokaryotic expression and identification of GPC3-N proteinThe GPC3-N gene was obtained by polymerase chain reaction（PCR）amplification using the human GPC3 gene as a template,which was digested with Eco R I and Xho I,and integrated into the p ET-22b prokaryotic expression vector.The recombinant plasmid was transformed into E.coli BL21（DE3）p Lys S competent cells by heat shock.IPTG induced its expression and its expression conditions（temperature,time,inducer concentration）were optimized and identified by SDS-PAGE electrophoresis and Western blot.The pET-22b-GPC3-N expression vector was successfully constructed.The GPC3-N protein was expressed in E.coli BL21（DE3）p Lys S.The optimal induction temperature was37℃,the optimal final concentration of IPTG was 0.1 m M,and the optimal induction time was 10 h.The purified protein was immunogenic with a concentration of 1 mg/m L and a purity of 90%.PartⅡ Preparation of rabbit polyclonal antibody against GPC3-N proteinThe GPC3-N protein prepared in the previous stage was used to immunize female New Zealand white rabbits,and the rabbit polyclonal antibody against GPC3-N was obtained by the octanoic acid-ammonium sulfate method.The affinity of the anti-GPC3-N rabbit polyclonal antibody and eukaryotic GPC3 protein or GPC3 expressed by Hep G2 cells was detected by ELISA,Western blot and immunofluorescence techniques.The immunized rabbit serum had a relatively high titer,and its titer can reach more than1:256,000.The rabbit polyclonal antibody purified by the octanoic acid-ammonium sulfate method had high purity,which had good immunoreactivity and immunoactivity.Not only could it recognize commercial full-length GPC3,but it could also specifically recognize the GPC3 protein specifically expressed on Hep G2 cells.PartⅢ Construction and screening of phage display nanobody libraryAfter six rounds of immunization of llama with GPC3-N protein prepared in the previous stage,lymphocytes were extracted from the peripheral blood of llama.m RNA was extracted from lymphocytes and reverse transcribed into c DNA.Using c DNA as a template,designing appropriate primers,and performing two rounds of PCR to obtain the nanobody gene sequence.The target gene was cloned into the vector pComb 3XSS,electroporated into E.coli ER2738competent cells,and infected with helper phage M13K07.With GPC3-N protein as the target protein,four rounds of enrichment panning were performed by phage display technology to screen single clones.The titer of the llama serum after six immunizations reached 1:32,000.The nanobody library was obtained against GPC3-N with a good sequence diversity of 6×10~7 cfu.The phage library titer after amplification by helper phage M13K07 was about 1×10¹³ pfu/m L,meeting the screening requirements.After four rounds of harsh bio-panning,two nanobody phagemids against GPC3-N protein were obtained.PartⅣ Expression and application of nanobodyThe strain containing the nanobody gene was cultured.The plasmid was extracted and introduced into E.coli TOP10F’competent cells by heat shock.IPTG was added for induction of expression and the anti-GPC3-N protein nanobody was obtained by affinity chromatography purification.The affinity of the anti-GPC3-N nanobody and eukaryotic GPC3protein or GPC3 expressed by Hep G2 cells was detected by ELISA,Western blot and immunofluorescence techniques.The expressed protein was identified by SDS-PAGE electrophoresis that have a molecular weight of 19 k D.The bioactive nanobody with a purity of 95%had good immunoreactivity and immunoreactivity.Not only could it recognize commercial full-length GPC3,but it could also specifically recognize the GPC3 protein specifically expressed on Hep G2 cells.In summary,in this study,GPC3-N protein with high purity was obtained,polyclonal antibody against GPC3 protein was obtained,a phage immune nanobody library with a capacity of 6×10~7 cfu was constructed,and nanobody specific against GPC3 protein was obtained.These provide a necessary experimental basis for the later modification of nanobody to establish an early diagnosis method for liver cancer and to achieve molecular targeted therapy.