Preparation And Characterization Of Polyclonal Antibodies Against Different Isoforms Of OGT

O-GlcNAcylation is a dynamic process in which proteins become posttranslationally modified through the attachment of Nacetyl-D-glucosamine (GlcNAc) moieties to the hydroxyl group of serine and threonine residues. The attachment of GlcNAc is catalyzed by the enzyme O-GlcNAc transferase (OGT), which utilizes uridine diphospho-N-acetylglucosamine produced in the hexosamine biosynthesis pathway from glucose as substrate. Reversely, O-GlcNAc moieties are enzymatically removed from proteins by the glycoside hydrolase O-GlcNAcase (OGA). More than 3,000 kinds of proteins were found to be modified with by OGT, and O-GlcNAcylation is likely linked to many biological proccesses, such as, dynamic interplay with proteasomal degradation, phosphorylation, transcription regulation, nuclear transport, cytoskeletal organization, and cell signaling. In addition, its faulty regulation is also involved in some human diseases such as diabetes, cancer and neurologic disorders.Only a gene encoding OGT exists in the human genome, its products mainly have three kinds of gene expression OGT isoforms, ncOGT(nucleocytoplasmic OGT), mOGT(mitochondrial OGT), sOGT(short OGT) respectively. The three physiological isoforms of OGT differ from primarily in the number of N-terminal TPRs. The longest isoform, with 12 TPRs, is referred to as nuclear and cytoplasmic OGT (ncOGT,116 kD). A shorter form, with 9 TPRs and bearing a mitochondrial targeting sequence, is localized to the inner mitochondrial membrane (mOGT,103 kD). The shortest of the three (sOGT,78 kD), has only 2.5 TPRs and is perhaps the least studied. However, the catalytic structural domains of three isoforms, the C-terminal of OGT. were identical. Detection of the OGT expression levels is vital during the research, and it is thus important to preparation the antibodies against the OGT. However, due to the lack of favorable tool, studies of O-GlcNAcylation and OGT were impeded. Therefore,in order to further explore the physiological and pathological role of OGT, high specificity antibodies aginst OGT remain to be prepared.For all three isoforms contain the same catalytic domain structure, therefore, in order to prepare the effective identification of three OGT isoform antibody at the same time, the OGT catalytic domain was selected as the structure of antigen fragments. Aim to further study the function of the function of mOGT in mitochondrial, we need to produce the antibody aginst mOGT, thus, the specific sequence located in the N-terminal of mOGT was selcted as antigen fragments according to the structure characteristics of OGT. This will provide favorable tools for OGT research.This thesis aimed at OGT-C antibody is mainly on the basis of molecular cloning and polyclonal antibody preparation principle. The DNA sequences of human OGT were from the database of NCBI, the C-terminal of OGT (556-1046 amino acid) was selected as the antigen. First, the DNA sequence corresponding the C-terminal of OGT (556-1046 amino acid) was inserted into the E.coli expression vector pET30-a. Recombinant proteins named as OGT-C were expressed. They were purified by Ni2+ affinity chromatography. Serums were obtained from Wistar rats immunized with OGT-C. The titer and specificity of anti-OGT-C polyclonal antibodies were detected by ELISA and Western blotting. The titer of polyclonal antibody was approximately 1: 80000, and the polyclonal antibody could sensitively detect the recombinant OGT-C by Western blotting. The antibody could specifically recognize ncOGT and mOGT protein in the lysate of H1299 human lung cancer cell. Our data suggest that this antibody has high specificity and titer aginst OGT, which may be used to measure the expression of ncOGT and mOGT in the function study of mOGT.Then, ncOGT, mOGT and sOGT sequences from the GenBank database were compared by using BioEdit for mOGT polyclonal antibody preparation. And the specific sequences located in the N-terminal of mOGT were selcted as antigen fragments named respectively, m-1 and m-2. Synthetic peptides were m-1: MISPSSPPPPNL (mOGT N-terminal 14-25 amino acid). and m-2: SHLLSLTPPkACYL (mOGf N-terminal 42-55 amino acids). The two synthetic peptides derived from mOGT (O-linked N-acetylglucosamine transferase) were designed and conjugated to Keyhole hemocyanin (KLH) and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with the mOGT protein in a panel of different cells lysates was then evaluated by western blot. The titer of polyclonal antibody was approximately 1:100000. and the polyclonal antibody could sensitively detect the recombinant mOGT by Western blotting. The antibody could specifically recognize mOGT protein in the lysate of H1299 human lung cancer cell. Our data suggest that this antibody aginst mOGT has high titer, which may be used to measure the expression of mOGT in the biology study of mOGT.In summary, this paper is mainly on the basis of molecular cloning and polyclonal antibody preparation principle. This antibody aginst OGT has high specificity and titer, and this antibody aginst mOGT has high titer, which may be used in the biology study of OGT.