[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].
OBJECTIVE
To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity.


METHODS
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.


RESULTS
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 antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity.


CONCLUSIONS
The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.
Shi
Hong
H
Department of Oral Pathology, College of Stomatology, Hebei Medical University, Shijiazhuang 050017, China.
Wang
Jie
J
Dong
Fu-Sheng
FS
Yu
Li-Jie
LJ
Wang
Xu
X
Gu
Hong-Tao
HT
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000032:analysis* / Q000276:immunology*; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000097608:UDP Xylose-Protein Xylosyltransferase
D000939:Epitopes; D007106:Immune Sera; D010430:Pentosyltransferases; D011993:Recombinant Fusion Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase