[Prokaryotic expression of DnaJ-homologous chaperon PBP and preparation of rabbit antibody against PBP].
OBJECTIVE
To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP.


METHODS
The PBP cDNA was amplified from the human fetal brain tissue by RT-PCR. After confirmed by DNA sequencing, the PBP-cDNA was cloned into expression vector pET28a and then the PBP gene was expressed in E.coli under the IPTG induction. The expressed protein was purified through Ni-NTA affinity chromatography column. The rabbit antibody against PBP was prepared by immunizing two New Zealand white rabbits using the purified PBP as immunogen. The titer and specificity of the antisera were determined by Western blot.


RESULTS
The 720 bp PBP gene was amplified, cloned, and expressed in E.coli. The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate. The purified PBP reached electrophoretic purity. The rabbit antibody against PBP was prepared and its titer was about 1:1,600. Western blot analysis showed that the antibody could bind to the expressed PBP protein specifically.


CONCLUSIONS
The PBP protein was expressed in E.coli and rabbit antibody against PBP was prepared successfully, which lays the foundation for further study on the structure and biological function of PBP.
Liu
Cheng-gang
CG
Center of Clinical Molecular Biology, General Hospital of Air Force, Beijing 100036, China. Liucg4336@sina.com
Zhu
Mei-cai
MC
Li
Wen
W
Shi
Ying
Y
Zhan
Zhi
Z
Wang
Yin-jing
YJ
Xiang
Pei-de
PD
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000918:Antibody Specificity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies; D011817:Rabbits
D000907:Antibodies, Bacterial; D018076:DNA, Complementary; D050956:HSP40 Heat-Shock Proteins