Abstract:
:A recombinant baculovirus vAc beta hCG-luc was constructed that carried the cDNAs encoding firefly luciferase (luc) and beta-subunit of human chorionic gonadotropin (beta hCG) 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 beta hCG, which was identical in all respects to that synthesized using a recombinant baculovirus carrying the beta hCG 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 beta hCG, an extensively glycosylated, secretory protein, was processed and secreted into the culture medium. LUC and beta hCG were both immunoreactive on Western blot. beta hCG was bioactive, as evident from its ability to associate with alpha hCG and bind with the receptor and produce testosterone in an in vitro mouse Leydig cell assay system. Comparison of recombinant LUC and beta hCG 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 vAc beta hCG-luc-infected insect cells, when probed with probes corresponding to the 3' region of the beta hCG or luc genes, indicated differential transcription of the two genes. Computer-aided sequence analysis indicated extensive secondary structure and stem-loop complex-forming potential of the beta hCG gene, which could be responsible for the transcriptional difference observed.
journal_name
DNA Cell Bioljournal_title
DNA and cell biologyauthors
Hasnain SE,Nakhai B,Ehtesham NZ,Sridhar P,Ranjan A,Talwar GP,Jha PKdoi
10.1089/dna.1994.13.275subject
Has Abstractpub_date
1994-03-01 00:00:00pages
275-82issue
3eissn
1044-5498issn
1557-7430journal_volume
13pub_type
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