Study Of Preparation Of Antibody Against Human Prostate Stem Cell Antigen And Its Application On Diagnosis And Treatment In Prostate Carcinoma

【abstract】: In an effort to identify potential markers for the diagnosis and treatment of prostate cancer, Reiter has searched for genes up-regulated during prostate cancer progression by using the recently developed LAPC-4 xenograft model of human prostate cancer and identified prostate stem cell antigen (PSCA) up-regulated in the LAPC-4 xenograft model by using representational difference analysis (RDA). PSCA is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy. First part: Cloning and expression of human prostate stem cell antigen To clone the cDNA of human prostate stem cell antigen (PSCA) and express the corresponding protein, total RNA was isolated from cultured human prostate cancer cell lines and mRNA was reversly transcribed into cDNA.Then PCR was used to amplify the PSCA coding region. The PCR product was cloned into pMD18-T plasmid and sequenced ,then subcloned into vector pET32a.The PSCA protein was expressed in 901 级博士论文 抗人 PSCA 单抗的制备及其在 PCa 诊断治疗中的应用研究 英文摘要 E.coli of BL21 as fusion protein with Trx.His tag induced by IPTG.. PSCA coding region(241bp) was cloned into pMD18-T and the sequenced was confirmed;Fusion expressing vector of pET32a/PSCA was successfully constructed and correctly expressed PSCA/Trx.His fusion protein(20.4KD). Human prostate stem cell antigen gene is successfully cloned and pET32a/PSCA fusion expressing vector is constructed.PSCA/His fusion protein is correctly expressed in E.coli. Second part: The expression of human prostate stem cell antigen in E.coli and the production of its antiserum To express human prostate stem cell antigen (PSCA) gene fragment in E.coli and its preparation of antiserum, PCR was used to amplify the PSCA gene fragment,The PCR product was inserted into plasmid pET32a with Trx.His tag to form an new plasmid pET32a/PSCA. The PSCA fusion protein was expressed in E.coli BL21 transformed with pET32a/PSCA and induced with IPTG. The expressed protein was purified by affinity resin Ni-NTA and analyzed by SDS-PAGE.In order to produce anti-serum, Mice were immunized by subcutaneously multi-site injection with PSCA fusion protein. The anti-serum was assayed by ELISA. The titer of the PSCA protein antiserum was more than 1:4000.Human prostate stem cell antigen gene is successfully cloned and expressed.The antiserum of PSCA protein has been produced. Third part: Preparation of monoclonal antibodies against human prostate stem cell antigen To obtain specific monoclonal antibody (McAb) against human PSCA and establish specific, sensitive, correct methods in the diagnosis of cancer, BALB/c mouse was immunized by purified PSCA to prepare McAb. One positive hybridome cell line which contained McAb against PSCA was obtained. McAb was IgG1 according to the 1001 级博士论文 抗人 PSCA 单抗的制备及其在 PCa 诊断治疗中的应用研究 英文摘要 examination of immunoglobulin classification and it was named D3. Western-blot test showed that the McAb reacted specifically with PSCA without other crossing reactions. Fourth part: An immunohistochemical study of prostatic carcinoma with the use of mon...