Isolation of a fusion protein containing the antigenic domain 1 of human cytomegalovirus glycoprotein B and its application in ELISA tests.
The glycoprotein B (gB) of human cytomegalovirus represents a dominant antigen for the humoral immune response. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids for antibody binding. In this study, this domain was expressed in Escherichia coli as a fusion protein with beta-galactosidase but yielded insoluble protein aggregates as inclusion bodies. To recover the fusion protein, inclusion bodies were solubilized by two extractions with urea 8 M: and the fusion protein then isolated using gel filtration chromatography. After confirmation of fusion protein antigenicity by Western blotting, the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of viral antibodies in serum samples of pregnant women. A cut-off point of approximately 0.2 absorbance units could discriminate the results of seropositive from seronegative pregnant women. The data indicates the potential usefulness of the fusion protein for the development of immunoassay for detection of the HCMV antibodies.
Sousa
F
F
Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal. fdomingues@ubi.pt
Melo
A
A
Almeida
S
S
Paixão
P
P
Queiroz
J A
JA
Domingues
F C
FC
D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016056:Immunodominant Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D001616:beta-Galactosidase