1. Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.
- Author
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Korshun L, Vudmaska M, Moysa L, Kovtonjuk G, Mikhalap S, Ganova L, and Spivak N
- Subjects
- Enzyme-Linked Immunosorbent Assay methods, Escherichia coli genetics, Gene Expression, Herpes Genitalis virology, Humans, Immunoglobulins blood, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Antibodies, Viral blood, Antigens, Viral genetics, Antigens, Viral isolation & purification, Clinical Laboratory Techniques methods, Diagnostic Tests, Routine methods, Herpes Genitalis diagnosis, Herpesvirus 2, Human immunology, Viral Envelope Proteins genetics, Viral Envelope Proteins isolation & purification
- Abstract
In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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