ISSN 1995-5537
"Biotechnology" journal V. 4, No. 4, 2011
Р. 64-72, Bibliography 27, Ukrainian.
Universal Decimal classification: 57.083.3:616.578.7:616.579.61
L. M. Korshun, L. M. Moysa, G. V. Kovtonjuk, L. O. Ganova,
O. K. Kiselyova, M. J. Spivak
JSC «Diaproph-Med», Kyiv
Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kyiv
The spread of infections caused by herpes simplex virus types 1 and 2 has increased significantly worldwide now. Diseases caused by herpes virus type 2 occurring in more heavy form, with frequent recurrences and complications compared with those caused by agent type 1. When clinical and laboratory tests were carried out, differential diagnosis between these two types of virus became more important. Purpose of the work was optimizing conditions of the bacterial expression of recombinant protein containing immunodominant regions of glycoprotein G of herpes simplex virus type 2 in the Escherichia coli cells and obtaining of the highly purified preparation suitable for use in the immunosorbent composition to the development of diagnostic test kits.
It was found that recombinant protein GST-HSV2gG accumulated both in a soluble form and as insoluble inclusion bodies in bacterial cells. Influence of media composition, expression temperature and IPTG concentration on the level accumulation of the heterologous protein and its immunochemical properties was shown. The maximum level of the soluble protein biosynthesis was reached when the bacteria had been cultivated with usе the TB medium (1,2 % baсtotryptone, 2,4 % yeast extract, 55 мМ glycerol, 17 мМ KH2PO4, 72 мМ K2HPO4, рН 7,2) at 37 0С with decreasing concentration IPTG up to 0,1 mM.
The target protein was purified by affinity chromatography technologies. Under the optimized conditions the yield of the purified soluble protein was up to 78.74 % relative to total recombinant protein.
The immunochemical properties of GST-HSV2gG protein was estimated by ELISA. The results suggested that it might be applicable for the development of the diagnostic kits for the detection of herpes simplex virus type 2.
Key words: recombinant proteins, expression vectors, herpes simplex virus type 2, ELISA test, Escherichia coli.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2008