Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I

To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vecto...

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Published inXi bao yu fen zi mian yi xue za zhi Vol. 23; no. 11; p. 1028
Main Authors Shi, Hong, Wang, Jie, Dong, Fu-Sheng, Yu, Li-Jie, Wang, Xu, Gu, Hong-Tao
Format Journal Article
LanguageChinese
Published China 01.11.2007
Subjects
Amino Acid Sequence
Antibody Specificity
Base Sequence
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Epitopes - chemistry
Epitopes - immunology
Genetic Vectors - genetics
Genetic Vectors - metabolism
Humans
Immune Sera - analysis
Immune Sera - immunology
Molecular Sequence Data
Pentosyltransferases - biosynthesis
Pentosyltransferases - chemistry
Pentosyltransferases - immunology
Pentosyltransferases - isolation & purification
Polymerase Chain Reaction
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - immunology
Recombinant Fusion Proteins - isolation & purification
UDP Xylose-Protein Xylosyltransferase
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Summary:To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibo
ISSN:1007-8738