Author/Authors :
Rasti، Mozhgan نويسنده Recombinant Protein Laboratory, Department of Biochemistry, Medical School, Shiraz University of Medical Sciences, P.O. Box 7134-845794, Shiraz, I.R. , , Khatooni، Zahed نويسنده Recombinant Protein Laboratory, Department of Biochemistry, Medical School, Shiraz University of Medical Sciences, P.O. Box 7134-845794, Shiraz, I.R. , , Mostafavi-Pour، Zoherh نويسنده Recombinant Protein Laboratory, Department of Biochemistry, Medical School, Shiraz University of Medical Sciences, P.O. Box 7134-845794, Shiraz, I.R. ,
Abstract :
پروتيين p53 يك پروتيين سركوبگر تومور است كه در بسياري از تومورهاي انساني غير فعال مي شود. دراين مطالعه آنتي بادي پلي كلونال بر ضد پروتيين p53 انساني توليد گرديد. cDNA كد كننده توالي كامل p53 درون حامل PGEX-4T-1 قرار داده شد و پروتيين نوتركيب GST-p53 در باكتري BL21 در اتصال با gluthation s thransferase توليد و خالص سازي شد. سپس اين پروتيين جهت توليد آنتي بادي به خرگوش تزريق شد و سپس به منظور بالا بردن حساسيت آنتي بادي حاصل در تشخيص ، خالص سازي شد. قدرت تشخيص و حساسيت آنتي بادي توليد شده بر روي GST-p53 و پروتيين p53 طبيعي توسط آزمايشهاي اليزا- ايمينوبلات ، ايمينوفلورسنت و كروماتين پرسيپيتاسيون اندازه گيري شد. ميزان ايمني زايي آنتي بادي توليد شده با تيتر بالاي 10000 :1 به روش اليزا تعيين شد. نتايج اين مطالعه نشان ميدهد كه آنتي بادي توليد شده بر عليه GST-p53 به خوبي مي تواند جهت مطالعه بيان پروتيين p53 و همچنين به منظور مطالعه قدرت اتصال اين پروتيين به پروتيين هاي ديگر تومورهاي انساني مورد استفاده قرارگيرد.
Abstract :
p53 is a key tumor suppressor gene that is targeted for inactivation during human tumorigenesis. In this study, we produced and characterized polyclonal anti-human p53 antibody. The cDNA encoding the complete human p53 protein was cloned into pGEX-4T-1 and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST). The rabbits were immunized with the purified p53 recombinant protein. The obtained antisera were purified to increase the specificity of recognition. The sensitivity and specificity of the produced antibody was analyzed by enzyme-linked immunosorbent, immunoblot, immunofluorescence and chromatin immunoprecipitation assays. Enzyme-linked immunosorbent assay showed that immunization with purified GST-p53 produced the high titer (1:10000) polyclonal antibodies with high specificity. Anti-p53 antibody allowed the sensitive detection of native p53 protein in immunoblotting, immunofluorescence and chromatin immunoprecipitation assays. Our results showed that anti-GST-p53 antibody provides a good means for studying the p53 expression pattern and its binding ability to other proteins in tumors.
