Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.
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.
JSC "Diaproph-Med", Kyiv, Ukraine. Electronic address: KL2004@ukr.net.
Ludmila
L
Korshun
Mariya
M
Vudmaska
Larissa
L
Moysa
Galina
G
Kovtonjuk
Svetlana
S
Mikhalap
Larissa
L
Ganova
Nikolay
N
Spivak
D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification; D019411:Clinical Laboratory Techniques / Q000379:methods*; D003955:Diagnostic Tests, Routine / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006558:Herpes Genitalis / Q000175:diagnosis* / Q000821:virology; D018258:Herpesvirus 2, Human / Q000276:immunology*; D006801:Humans; D007136:Immunoglobulins / Q000097:blood; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C045464:glycoprotein G, herpes simplex virus type 2
Affinity constant; Enzyme-linked immunosorbent assay (ELISA); Glycoprotein G; Herpes simplex virus type 2; Recombinant proteins