Studies on the synthesis of betahCG hormone in vero cells by recombinant vaccinia virus.
Synthesis of the beta-subunit of human chorionic gonado-tropin (betahCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of betahCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of betahCG, 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. mm(2) betahCG/10(6) cells. h and 1.55 AU. mm(2) betahCG/10(6) cells. h, respectively. The time required to secrete 50% of intracellular betahCG was 210 min. Pulse-chase data also showed that 24% of betahCG was degraded intracelluiarly within 10 h, of which 17% was detected in the autoradiogram. Along with 30 kD betahCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia-derived betahCG 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 betahCG-mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half-life of betahCG-mRNA, as determined by follow-up of decay after blocking transcription initiation, was found to be 6.4 h. The synthesized betahCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native betahCG to form heterodimer betahCG, which competed with (125)I-hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells. (c) 1995 John Wiley & Sons, Inc.
Mukhopadhyay
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National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Mukhopadhyay
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Talwar
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