β-Subunit of human chorionic gonadotropin hormone and firefly luciferase simultaneously synthesized in insect cells using a recombinant baculovirus are differentially expressed and transported

Abstract

A recombinant baculovirus vAcβhCG-luc was constructed that carried the cDNAs encoding firefly luciferase (luc) and β-subunit of human chorionic gonadotropin (βhCG) placed under the transcriptional control of individual copies of the baculovirus polyhedrin gene promoter. The simple, rapid, and sensitive detection of LUC expression was used for selecting recombinant viruses that simultaneously expressed βhCG, which was identical in all respects to that synthesized using a recombinant baculovirus carrying the βhCG gene alone. Immunofluorescence staining of virus-infected cells using anti-LUC antibodies revealed that LUC, a nonglycosylated, intracellular protein was retained within the cells whereas βhCG, an extensively glycosylated, secretory protein, was processed and secreted into the culture medium. LUC and βhCG were both immunoreactive on Western blot. βhCG was bioactive, as evident from its ability to associate with ahCG and bind with the receptor and produce testosterone in an in vitro mouse Leydig cell assay system. Comparison of recombinant LUC and βhCG synthesized by the virus-infected insect cells surprisingly revealed that the level of the former was quantitatively higher by at least 10-fold than the latter. A blot of total RNA isolated from vAcβhCG-luc-infected insect cells, when probed with probes corresponding to the 3' region of the βhCG or lue genes, indicated differential transcription of the two genes. Computer-aided sequence analysis indicated extensive secondary structure and stem-loop complex-forming potential of the βhCG gene, which could be responsible for the transcriptional difference observed.