| Autosomal dominant polycystic kidney disease(ADPKD) is one of the most common single genetic diseases in humans affecting all ethnic groups worldwide with an incidence of 1 in 500 to 1 in 1,000. The hallmark of the disease is the development of multiple bilateral cysts in kidney, resulting in progressive renal failure in 50% of patients by their sixth decade of age. ADPKD is a systemic disease with many extra-renal manifestations including liver cysts, cerebral aneurysms and cardiac valvular abnormalities.Polycystin-1 is the protein product of PKD1 (polycystic kidney diseasel) gene, it's structural and functional abnormalities account for 85-90% of ADPKD. Polycystin-1 has been detected in the plasma membrane of tubular epithelial cells in kidneys. Polycystin-1 is predicted to be a 4302-residue protein with a large amino-terminal domain(believed to be extracellular), 11 transmembrane domains, and an ~200-residue carboxylterminus. The large, extracellular amino-terminal domain contains a unique array of distinct protein motifs, which may be very important to polycystin-1 's normal physiological function. The polyclonal and monoclonal antibodies were prepared against the amino termini of polycystin-1, exactly against parts of LRR domain and parts of WSC domain. In this study, we try to testify whether the transmembrane protein polycystin-1 undergoes cleavage at its extracellular domain in vivo. If the cleavage is essential for polycystin-1 's biologic activity. We also discussed the significance of quantitative determinatio of the polycystin-1 in body fluid in the clinical diagnosis of ADPKD. The study consists of three parts. 1. Expression and purification of polycystin-1 extracellular region invitroTotal RNA was extracted in normal kidney tissue. Two gene sequences which code parts of LRR domain and WSC domain in polycystin-1 extracellular region(according to GenBank L33243) were amplified with one-step RT-PCR. The fragments(502bp and 471bp) were inserted into prokaryotic expression vector pQE30 , respectively. DNA sequencing verified that the vector pQE30-PKD1e1 and pQE30-PKD1e2 contained the PKD1 cDNA we wanted. Then the expression vector were transformed into competent E.coli M15 cells which contain repressor(pREP4) plasmid. The fusion protein expression was induced by IPTG and purified by Ni-NTA affinity chromatography. The products were identified by SDS-PAGE analysis and Western blot. Fusion proteins of polycystin-1 extracellular region were obtained, with a molecular weight of 19.8kDa and 18.9kDa, respectively. These set up the basement of producing anti-polycystin-1 monoclonal antibody. 2. The preparation of the monoclonal antibodies and polyclonal antibodies against polycystin-1In order to prepare the monoclonal antibodies and polyclonal antibodies, Balb/C mice and New Zealand rabbits were immunized with purified recombinant protein(with a molecular weight of 18.9kDa) that contains polycystin-1 extracellular region. Rabbits were sacrificed and their serum was collected after three months successful immunization, then the polyclonal antibodies against polycystin-1 were purified with Protein B affinity chromatography. The procedure of preparing monoclonal antibodies are below: After the immunization of mice, the spleen cells were fused with SP2/0 myeloma cells by PEG4000. The fusion cells were screened by HAT selective culture system. The hybridoma clones secreting antibodies against polycystin-1 extracellular amino-terminal domain were detected by enzyme-linked immunosorbent assay(ELISA) and cloned by limiting dilution. One cell line(nominated 7B1) of hybridoma secreting monoclonal antibodies was established. Subtype of the antibodies was IgG1 detected by direct ELISA. Western blot analysis showed that the monoclonal antibodies reacted strongly and specifically to polycystin-1 extracellular amino-terminal domain.The hybridoma cell line 7B1 was cultured in peritoneal cavity of Balb/C mice as ascites, and the monoclonal antibody(lgG1 class) was purified with Protein B affinity chroma...
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