Publication: Rekombinant DNA teknolojisi ile rekombinant bir antijene karşı antikor üretimi
Abstract
Son yyllarda klasik hibridoma teknolojisi ile hücrelerin ölümsüzle?tirilmesinin yerini, rekombinant DNA teknolojisi ile faj gösterim tekniklerinin birle?tirilerek uygulanmasy ile genlerin ölümsüzle?tirilmesi almaya ba?lamy?tyr. Monoklonal antikorlar in vitro tany testlerinde, 1980'lerden beri ba?ary ile kullanylmaktadyr. Antikorlaryn tedavi ve in vivo tany testlerinde kullanymy 1990'dan sonra giderek artmy?tyr ve tedavi molekülleri olarak da de?erlendirilmeye ba?lanmy?dyr. Monoklonal antikor üretiminde kullanylan B hücre ölümsüzle?tirme tekni?indeki pek çok zorluk faj gösterim ile çözülmü?tür. 1990'laryn ba?yna kadar monoklonal antikorlar Köhler ve Milstein tarafyndan tanymlanan hibridoma teknolojisi ile elde edilmekte idi. Ymmün sistemin antijene özgül antikorlary üreten B hücrelerinin sayysyny artyrmaya yönelik uyarymy, hücre füzyonu ile bu antikor üreten hücrelerin immortalizasyonu ve geni? bir tarama ile istenen özelliklerde antikor üreten sabit hücre hatlarynyn tanymlanmasy ile özetlenebilen teknik 20 yyldan fazla zamandyr kullanylmaktadyr. 1990'laryn ba?yndan bu yana kaydedilen son geli?melerle; bakteride fonksiyonel antikor parçalarynyn üretilmeye ba?lanmasy, polimeraz zincir reaksiyonu (PZR) (58) kullanylarak antikor genlerinin izolasyonu ve ço?altylmasy, faj gösterimle güçlü tarama sistemlerinin birle?tirilmesi (53), hibridoma tekni?indeki kar?yla?ylan pekçok zorlu?u a?makta hatta immünizasyon ve hayvan kullanymy tamamen i?lem dy?y byrakylmaktadyr. Bu tez çaly?masy rekombinant DNA teknolojisi ve faj gösterim tekni?i uygulanarak yapysal olmayan bir protein antijen olarak kullanylarak rekombinant proteine ait scFv eldesi üzerine yapylan bir çaly?madyr. Model protein olarak kullanylan Tütün Mozaik Virüs'ünün fonksiyonel proteini olan RNA'ya ba?ly RNA polimerazyn (replikaz) antijenik bölgesi, bakteride füzyon protein olarak eksprese edilmi? ve füzyon protein antijen olarak hazyrlanmy?tyr. TMV'nin bitki hücresini enfekte etti?inde ilk sentezlenen proteini olan replikaz membran yapyda bulunan safla?tyrylmasy zor bir proteindir. Füzyon protein olarak hazyrlanan replikaz farelere immünize edilmi? ve fare dalaklaryndan antikorlaryn de?i?ken bölge genleri ço?altylmy?tyr. scFv ?eklinde bir araya getirilen antikor V bölge genleri pCANTAB 5E fajmit vektöre klonlanmy?tyr. scFv-faj kütüphanesi füzyon protein ile elenerek anti-replikaz-b-gal.-scFv kütüphanesi elde edilmi?tir. Bu tez çaly?masy TMV replikazyna kar?y ilk rekombinant antikor çaly?masydyr. PRODUCTION OF ANTIBODY AGAINST A RECOMBINANT ANTIGEN BY USING RECOMBINANT DNA TECHNIQUES
Hybridoma technology is uesd for the immortalization of antibody producing cells, but in recent years by using recombinant DNA and phage display antibody genes are immotalized. Monoclonal antibodies have been used succesfully in vitro diagnostic tests since 1980. In vivo therapeutic and diagnostic use of antibodies have been increasing since 1990. The technical problems seen in B cell immortalization in monoclonal antibody production has not been observed in phage display. Monoclonal antibodies have been obtained by the methods described by Köhler and Milstein till 1990's. Stimulation of immune system to increase antigen specific B cell number immortalization of antibody producing cells by cell fusion, and with wide screening system to detect stable cell lines producing antibodies with desired properties. Hybridoma technology have been used for 20 years succesfully. The improvements from the begining of 1990; production of antibody fragments in bacteria, isolation and amplification of antibody varibale region by using polimerase chain reaction (PCR) (58), combination of phage display (53) and powerful screaning systems, the difficulties in hybridoma technology have been bypassed even without immunization and use of animal. In this thesis, recombinant DNA technology and phage display techniques have been used for obtaining scFv against a recombinant nonstructural protein. Tobacco Mosaic virus nonstructural protein, RNA dependent RNA polymerase (replicase) antigenic site was used as a model protein and expressed in bacteria and used as an antigen. Replicase is a membraneus protein and it is difficult to purify. Replicase is the first protein expressed by TMV after infection of plant cell. Mouse were immunized with prepared replicase fusion protein and V genes were amplified. V genes were assembled as scFv and cloned on pCANTAB 5E phagemid vector. ScFv-phage library selected with fusion protein and an anti-replicase-b-gal.-scFv library has been obtained. This thesis is the first scFv study against TMV replicase.
Hybridoma technology is uesd for the immortalization of antibody producing cells, but in recent years by using recombinant DNA and phage display antibody genes are immotalized. Monoclonal antibodies have been used succesfully in vitro diagnostic tests since 1980. In vivo therapeutic and diagnostic use of antibodies have been increasing since 1990. The technical problems seen in B cell immortalization in monoclonal antibody production has not been observed in phage display. Monoclonal antibodies have been obtained by the methods described by Köhler and Milstein till 1990's. Stimulation of immune system to increase antigen specific B cell number immortalization of antibody producing cells by cell fusion, and with wide screening system to detect stable cell lines producing antibodies with desired properties. Hybridoma technology have been used for 20 years succesfully. The improvements from the begining of 1990; production of antibody fragments in bacteria, isolation and amplification of antibody varibale region by using polimerase chain reaction (PCR) (58), combination of phage display (53) and powerful screaning systems, the difficulties in hybridoma technology have been bypassed even without immunization and use of animal. In this thesis, recombinant DNA technology and phage display techniques have been used for obtaining scFv against a recombinant nonstructural protein. Tobacco Mosaic virus nonstructural protein, RNA dependent RNA polymerase (replicase) antigenic site was used as a model protein and expressed in bacteria and used as an antigen. Replicase is a membraneus protein and it is difficult to purify. Replicase is the first protein expressed by TMV after infection of plant cell. Mouse were immunized with prepared replicase fusion protein and V genes were amplified. V genes were assembled as scFv and cloned on pCANTAB 5E phagemid vector. ScFv-phage library selected with fusion protein and an anti-replicase-b-gal.-scFv library has been obtained. This thesis is the first scFv study against TMV replicase.