|
Abstract
|
The aim of this study was to construct, expression of a novel recombinant chimeric protein
consisting of Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of
integral membrane lipoprotein P80 of Mycoplasma agalactiae as a potential diagnostic tool. The
full-length sequence of pdhb and a portion of antigenic regions of P80 were selected and
analyzed by CLC main workbench 5.5 software. Several linkers and three dimensional structure
of PDHB-P80 were compared to the native PDHB and analyzed to select a proper one for
expression. The fusion gene sequence was optimized and synthesized in pMAT cloning vector.
The synthetic pMAT-pdhb-p80 was digested using Bam HI and Sal I restriction enzymes and
ligated into pMAL-p5X expression vector. The pMAL-pdhb-p80 construct was transfected into
E.coli BL21 strain cells and expressed protein were purified using amylose resin. and the
purified protein was analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blotting. In silico analysis demonstrated that fusion proteins using
IgG4 middle hinge (CPSCP) with TM-score of 0.99 showed the higher similarity between three
dimensional structure of PDHB before and after fusion with high antigenic region of P80.
Successful cloning verified by PCR colony, double digestion and sequence analysis. Besides,
SDS-PAGE analysis and Western blotting indicated and confirmed the expression of intact
recombinant chimeric protein MBP-PDHB-P80 along with some truncated forms of the
recombinant protein. it could be concluded that the fusion construct has a potential for
serodiagnostic assay in future studies.
|