The Preparation Of The Monoclonal Antibodis Against Polycystin-1 And Their Application In The Study Of Pathogenesis Of ADPKD

The preparation of the monoclonal antibodis against polycystin-1 and their application in the study of pathogenesis of ADPKD Polycystin-l is the protein product of PKD1(polycystic kidney diseasel)gene, which accounts for 85% of autosomal dominant polycystic kidney disease (ADPKD) . According to PKD1 DNA sequence, polycystin-l is predicted to be a large membrane-bound protein and consists of a large N-terminal extracellular domain, several multiple transmembrane domains and a short cytoplasmic C-terminal region. The intact polycystin-l has yet not been gotten heretofore. Because the intracellular region is encoded by single-copy DNA of PKD1 gene, it has not homology to other proteins. In addition, the intracellular region of polycystin- 1 has important function, so we selected the intracellular region for study. In this study, we cloned a segment of PKD1 cDNA which encoded the intracellular region and expressed polycystin-l intracellular region in prokaryotic cells. The monoclonal antibodis against polycystin-l intracellular region were prepared. Polycystin-l distribution in tissues and cells were studied by these monoclonal antibodis. Combined with alterations of extracelluar matrix in ADPKD, the possible pathogenesis of ADPKD was discussed. The study consists of four parts. 7 l.The expression and purification of polycystin-1 intracellularregionA pair of primers were designed with added cloning sites EcoRI and Sal I to the ends. A segment of PKD1 cDNA encodingpolycystin-l intracellular region was amplified by PCR in vitrofrom the plasmid AH8. The PCR products were inserted intocloning vector pGEM-T by T-A cloning techique, then the cDNAfrom the plasmid pGEM-T was inserted into fusion proteinexpressive vector pProEX Hta which then transformed into E.coliDH5 Q. DNA sequencing verified that the vector pProEX Hta-PKDl contained l2473-l3l20nt of PKD1 cDNA that encoded4088-4302aa of polycystin-l intraceIlular region. After IPTGinduction, fusion protein are soluble and a high level expressionof fusion protein was achieved about 23.9% of the total solubleprotein as detected by SDS-PAGE assay. This product waspurified to a single band in SDS-PAGE by Ni-NTA affinitychromatography easily. The Western-blot indicates that the fusionprotein can bind anti-polycystin-l intracellular region monoclonalantibody with strong specifity. So we successfully expressed andpurified polycystin-l intracellular region, which is useful forstudy of polycystin- l and preparation of the monoclonal antibodisagainst polycystin- 1.2.The preparation of the monoclonal antibodis against polycystin-lBalb/ C mice were immunized with purified recombinantprotein that contains polycystin-1 intracellular region, then thespleen cells were fused with SP2/0 myeloma cells by PEG4000.The fusion celIs were screened by HAT selective culture system.The hybrid cells secreting antibodies against polycystin-lintracellular region were detected by ELISA and cloned bylimiting dilution. Four cell lines of hybrids secreting monoclonalantibodis were established. Subtypes of these antibodis were allIgG,b detected by double immunodiffusion. Antibody titers inasitic fluid and serum were l: 106 and l: l0', Respectively. Thecell lines of hybrids secreting steadily monoclonal antibodisagainst poiycystin-1 intracellular region have been establishedsuccessfully, and will be a useful tool in the studies of ADPKD.3.Distribution of polycystin-l in tissues and cellsDistribution of polycystin-l was investigated in tissues andcells by immunohistochemical methods (standard EnVisionmethod) with self prepared the monoclonal antibodis againstpolycystin-1. The subcellular localization is cytopla...