[Preparation of P52 recombinant antigenic protein from human cytomegalovirus (HCMV)].
Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.
Susloparov
M A
MA
Department of Molceular Biology, Faculty of Natural Sciences, Novosibirsk State University.
Susloparov
I M
IM
Pliasunov
I V
IV
Bakhtina
M M
MM
Safronov
P F
PF
Grishaev
M P
MP
Blinov
V M
VM
Ivan'kina
T Iu
TIu
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D003587:Cytomegalovirus / Q000276:immunology*; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D011994:Recombinant Proteins; D014764:Viral Proteins