ISSN:
0006-3592
Keywords:
Vero
;
vaccinia virus expression
;
MOI
;
βhCG
;
mRNA
;
immunoligical and biological activity
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Synthesis of the β-subunit of human chorionic gonado-tropin (βhCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of βhCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of βhCG, deduced from the pulse-chase experiments were “zero order.” At 30 h postinfection, the relative values of net synthesis and secretion rates were 4.0 AU. mm2 βhCG/106 cells. h and 1.55 AU. mm2 βhCG/106 cells. h, respectively. The time required to secrete 50% of intracellular βhCG was 210 min. Pulse-chase data also showed that 24% of βhCG was degraded intracelluiarly within 10 h, of which 17% was detected in the autoradiogram. Along with 30 kD βhCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia-derived βhCG was 13 kD more that its nonglycosylated form, indicating extensive glycosylation in Vero cells. The mRNA levels in infected Vero cells at different postinfection times were quantified by excess DNA dot-blot hybridization. It appears that the Vero cell possesses some host cell-associated factor(s), which prevented the transcription of early βhCG-mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half-life of βhCG-mRNA, as determined by follow-up of decay after blocking transcription initiation, was found to be 6.4 h. The synthesized βhCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native βhCG to form heterodimer βhCG, which competed with 125I-hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells. © 1995 John Wiley & Sons, Inc.
Additional Material:
12 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260480210
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