[Preparation and characterization of anti-Amelotin polyclonal antibody].
OBJECTIVE
To construct a mouse Amelotin recombinant plasmid, express mouse Amelotin protein and prepare its polyclonal antibody.


METHODS
The cDNA sequence of mouse Amelotin gene was downloaded from GenBank. The coding region, which did not include signal peptide, was amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a and transformed into E.coli BL21(DE3). After induced by IPTG, the recombinant protein was expressed and purified using affinity purification. The polyclonal antibody was obtained from New Zealand rabbit immunized with the recombinant protein and the titer was identified by ELISA. The polyclonal antibody was identified by Western blot and immunohistochemistry assays.


RESULTS
The recombinant prokaryotic expression vector pET32a-Amelotin was constructed and the recombinant prorein was purified successfully. ELISA analysis showed the titer of the generated antiserum was 1:12,800. Western blot and immunohistochemistry analysis demonstrated this antibody bound specifically with Amelotin.


CONCLUSIONS
The anti-Amelotin antibody from the rabbit with high titer and specificity had been prepared with the purified recombinant Amelotin as immunogen, which is helpful for further research into the detection and function of Amelotin.
Sun
Yan
Y
Institute of Stomotalogy, School of Stomotalogy, Weifang Medical University, Weifang 261042, China. yansw2005@yahoo.com.cn
Zhang
Juan-juan
JJ
Han
Ting-ting
TT
Li
Dong-liang
DL
Cao
Xue-mei
XM
Li
Wu-xiu
WX
Gao
Yu-guang
YG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D003743:Dental Enamel / Q000378:metabolism; D003746:Dental Enamel Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism
C507066:Amtn protein, mouse; D000911:Antibodies, Monoclonal; D003746:Dental Enamel Proteins; D007106:Immune Sera; D011994:Recombinant Proteins