Jul 2, 2005

Prokaryotic expression of DnaJ-homologous chaperon PBP and preparation of rabbit antibody against PBP

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
Cheng-gang LiuPei-de Xiang

Abstract

To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP. 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. 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. 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.

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Mentioned in this Paper

Shuttle Vectors
DNAJB6 wt Allele
Alkalescens-Dispar Group
DNAJA2 gene
expression vector
Inclusion Bodies
Western Blotting
SDS-PAGE
Immune Sera
Reverse Transcriptase Polymerase Chain Reaction

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