[The antidiphtheria activity of the pre-EGF fragment].
The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.
Sergienko
V I
VI
Research Institute of Physico-Chemical Medicine, 1st City Infectious Hospital, Moscow, Russia.
Sivov
I G
IG
Korzhenkova
M P
MP
Malyshev
N A
NA
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004165:Diphtheria / Q000517:prevention & control*; D004168:Diphtheria Toxoid / Q000276:immunology; D004353:Drug Evaluation, Preclinical; D004815:Epidermal Growth Factor / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007231:Infant, Newborn; D007668:Kidney; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D019444:Vaccines, DNA / Q000276:immunology
D017931:DNA Primers; D004168:Diphtheria Toxoid; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor
Antidifteriĭnaia aktivnost' fragmenta pre-EGF.