Publication: Erişkin insan normal hemoglobinine karşı faj gösterim yöntemi ile rekombinant antikor geliştirilmesi
Abstract
Bu çalışmada, kolon kanserinin bir işareti olabilecek dışkıdaki gizli kanın ve hemoglobinopatilerde populasyon taraması çalışmalarında diğer hemoglobin tiplerinin tespit edilmesinde kullanılmak üzere nihai hedef olan immünokitlerin geliştirilmesi amacıyla faj gösterim teknolojisi kullanarak scFv ( tek zincir değişken parça) formunda rekombinant anti-hemoglobin A elde edildi. Hemoglobin A'ya karşı scFv üretimi için , ticari olarak satılan liyofilize hemoglobin A ile Balb/ CJ soyuna ait fare bağışıklandı ve antikor molekülünün fonksiyonel değişken ağır ve hafif zincir genleri antijenle uyarılmış B lenfosit mRNA'larından elde edildi. İmmunglobulin değişken ağır ve hafif zincirleri PCR yardımıyla scFv formunda bir arayagetirilerek pCANTAB5E vektörüne klonlandı. Beş tur yapılan biopanning işlemi sorasında rastlantısal olarak seçilen 93 adet klon hemoglobine karşı faj ELİSA yöntemi ile tarandı ve beş adet klon pozitif olarak kabul edildi.Faj ELİSA sonuçlarına göre pozitif olan klonlar klonlama bölgeleri ve insert büyüklükleri polimeraz zincir reaksiyonu ve enzim kesimleriyle ile test edildi. BerHb77 olarak isimlendirilen klonun nükleotid dizisinin tespit çalışmaları yapıldı. BerHb77' ye ait olan nükleotid dizisinin nükleotid dizi bankasında( GENBANK) yapılan karşılaştırmaları sonucunda aynı diziye rastlanmadı. Sonuçlar diğer hemoglobin tiplerinin saha tespit çalışmasında kullanılacak olan immüntanı kitlerinin geliştirilmesinde faj gösterim teknolojisinin kullanılabilirliğini göstermektedir. DEVELOPMENT OF RECOMBINANT ANTIBODY AGAINST HUMAN ADULT HEMOGLOBIN BY USING PHAGE DISPLAY TECHNOLOGY
In this study we obtained recombinant anti-human hemoglobin A in the form of scFv ( single chain variable fragment ) by using phage display technology with the ultimate aim of developing immunodiagnostic kits for the detection of occult blood in the feces which could be a sign of the column cancer and other hemoglobin types for population screening studies in hemoglobinopathies. In the production of anti-hemoglobin A scFv, Balb/ CJ mice were immunized with lyophilized commercial human hemoglobin A and the functional variable heavy and light chain genes of antibody were obtained from antigen stimulated B-lymphocyte mRNAs. Variable Ig heavy and light chain genes were assembled together in the form of scFv by polymerase chain reaction and cloned into the vector pCANTAB5E ( purchase from Pharmacia). After the five round of biopanning ,randomly selected 93 clones were tested against human hemoglobin by phage ELISA and five clones were accepted as positive. According to the phage ELISA results ; positive clones were tested with respect to its multiple cloning site and its insert size with polymerase chain reaction and restriction enzyme digestion. The clone coded as BerHb77 was sequenced to identify its nucleotide composition. The nucleotide sequence of BerHb77scfv was compared with nucleotide data bank ( GENBANK) by using BLAST and couldn't be found same nucleotide sequence in data bank. The results show that application of phage display technology is possible for developing immunodiagnostic kits for detection other hemoglobin types for population screening studies in hemoglobinopaties
In this study we obtained recombinant anti-human hemoglobin A in the form of scFv ( single chain variable fragment ) by using phage display technology with the ultimate aim of developing immunodiagnostic kits for the detection of occult blood in the feces which could be a sign of the column cancer and other hemoglobin types for population screening studies in hemoglobinopathies. In the production of anti-hemoglobin A scFv, Balb/ CJ mice were immunized with lyophilized commercial human hemoglobin A and the functional variable heavy and light chain genes of antibody were obtained from antigen stimulated B-lymphocyte mRNAs. Variable Ig heavy and light chain genes were assembled together in the form of scFv by polymerase chain reaction and cloned into the vector pCANTAB5E ( purchase from Pharmacia). After the five round of biopanning ,randomly selected 93 clones were tested against human hemoglobin by phage ELISA and five clones were accepted as positive. According to the phage ELISA results ; positive clones were tested with respect to its multiple cloning site and its insert size with polymerase chain reaction and restriction enzyme digestion. The clone coded as BerHb77 was sequenced to identify its nucleotide composition. The nucleotide sequence of BerHb77scfv was compared with nucleotide data bank ( GENBANK) by using BLAST and couldn't be found same nucleotide sequence in data bank. The results show that application of phage display technology is possible for developing immunodiagnostic kits for detection other hemoglobin types for population screening studies in hemoglobinopaties