Optimization of Rough Lipopolysaccharide Extraction from Brucella abortus by Phenol – Chloroform - Petroleum ether

Document Type : Original Article

Authors

MSc / Razi Vaccine and Serum Research Institute

10.35066/J040.2019.234

Abstract

Brucellosis is an important zoonotic disease which threatens public health and livestock industry. The aim of this study was to optimize extraction of rough lipopolysaccharide from Brucella abortus which can be used as the antigen for the development of serological tests to diagnose vaccinated animals. This applied study was performed in the Brucellosis Research and Production Laboratory. For this purpose, the rough strain was cultured in Brucella agar specific medium and final diagnosis was performed by PCR method. The rough lipopolysaccharide of Brucella abortus was extracted by phenol-chloroform-petroleum ether method and the results of rough LPS extraction were approved by LAL and SDS - PAGE methods. Extraction and precipitation of Lipopolysaccharide with cold methanol and sodium acetate was performed by using Phenol – Chloroform - Petroleum ether method. Then Lipopolysaccharide was identified by Limulus Amebocyte Lysate (LAL) test and formation of clots indicated presence of Lipopolysaccharide.  Also SDS – PAGE (14 % Polyacrylamide gel) followed by silver nitrate staining showed a 12 KDa band which indicates Rough Lipopolysaccharide. According to the results of this study, it seems that Phenol – Chloroform - Petroleum ether method is an excellent method for extraction of rough LPS of antigen for the development of serological tests and ELISA Kit to diagnose vaccinated animals.

Keywords

References

1- Avila-Calderon E D, Merino A L, Sriranganathan N, Boyle S M, Rodriguez C. A History of the Development of Brucella Vaccines. BioMed Research International. 2013; 8.
2- Diaz-Aparicio E, Arellano-Reynoso B, Herrera E, Hernandez M, Suarez-Games F. Characterization of the Transitory Immune Response in Cows Immunized with RB51 and its Implication on Diagnosis within Brucellosis EndemicZones.  International Journal of Dairy Science. 2007; 2(4): 364-371.
3- Galanos C, Luderitz O, Westphal O. A New Method for the Extraction of R Lipopolysaccharides.  European Journal Biochem. 1969; 245-249.
4- Kreutzer D, Buller C S, Robertson D C. Chemical Characterization and Biological Properties of Lipopolysaccharides Isolated from Smooth and Rough Strains of Brucella abortus.  Infection and Immunity. 1979; 23(3): 811-8.
5- Moreno E, Sherry S L, Jones L, Berman D. Immunochemical Characterization of Brucella Lipopolysaccharides and Polysacharides.  Infection and Immunity. 1981; 222-241.
6- Moreno E, Pitt M, Jones L, Schurig G, Berman D.  Purification and Characterization of Smooth and Rough Lipopolysaccharides from Brucella abortus.  Journal of Bacteriology. 1979; 361-369.
7- Nielsen K, Smith P, Conde S, Draghi de Benitez G, Gall D, Halbert G. and et. al. Rogh Lipopolysaccharide of Brucella abortus SRB51 as a common Antigen for Serologicsl Detection of B. ovis, B. canis, and B. abortus SRB51 Expoure Using Indirect Enzyme Immunoassay and Flurescence Polarization. Journal of Immunoassay. 2004; 25(2):171-182.
8- Robles C A, Nielsen K, Willems P. Evaluation of three different antigens in an indirect enzyme- linked immunoassay for the detection of antibodies, against, Brucella, abortus SRB51 in vaccinated heifers. Veterinary Immunology and Immunopathology. 2009; 127(1-2): 153-155.
9- Ramesh V, Yongqun H, Larissa S B, Schuring G G. Complementation of Brucella abortus Strain RB51 with a functional WboA Gene Results in O – Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation. Infect Immun. 2000; 68(7): 3927-3932.
10- Wang Z, Wu Q. Research progress in live attenuated Brucella vaccine development. Curr Pharm Biotechnol. 2013; 14(10): 87-96.
11- Xinghang Y. and et al. Progress in Brucella Vaccine Development. Front . Biol. 2013; 8: 60-77.
12- Zowghi E. Proceeding of Second Iranian National congress of Brucellosis,Shahid Beheshti. University of Medical Sciences, 2007 ]in Persian[.

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History

  • Receive Date: 27 September 2019
  • Revise Date: 16 October 2019
  • Accept Date: 11 November 2019

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