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Type: Journal article
Title: A novel chimeric recombinant protein PDHB-P80 of Mycoplasma agalactiae as a potential diagnostic tool
Author: Akbarzadeh-Niaki, M.
Derakhshandeh, A.
Kazemipour, N.
Eraghi, V.
Hemmatzadeh, F.
Citation: Molecular Biology Research Communications, 2020; 9(3):123-128
Publisher: Shiraz University Press
Issue Date: 2020
ISSN: 2322-181X
Statement of
Malihe Akbarzadeh-Niaki, Abdollah Derakhshandeh, Nasrin Kazemipour, Vida Eraghi, Farhid Hemmatzadeh
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.
Keywords: Contagious agalactia; Serodiagnostic assay; Antigen; Recombinant fusion protein
Rights: Open Access Policy. This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge.
RMID: 1000024370
DOI: 10.22099/mbrc.2020.37684.1513
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Appears in Collections:Animal and Veterinary Sciences publications

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