Autographa Californica Multiple Nucleocapsid Polyhedrosis Research Articles

The homologous region hr4a of Autographa californica multiple nucleopolyhedrovirus specifically enhances viral early promoters

Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) genome contains several homologous regions (hrs) which can be used as origins of viral DNA replication and transcriptional enhancers. Early studies have found that some hrs can not only stimulate viral early promoters, but also enhance gene expression driven by viral late promoters. In this study, by adding hr4a to the upstream of different types of viral promoters and enhanced green fluorescent protein (egfp) reporter gene, we investigated the enhancement effect of hr4a on eGFP expression. The results showed that hr4a increased the gene transcription controlled by early promoters, but had no enhancer effect on late promoters.

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5α-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (Km = 17.9 μM) and placental microsomes (Km = 16.7 μM). The 3β-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 μM) and placental microsomes (Km = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.