High-level expression of human beta-interferon gene in the silkworm with new constructed BmNPV vector.
Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori cells as well as silkworm larvae were used successfully for the production of biologically active recombinant proteins. There are only a few types of BmNPV general vectors. Here a new type of vector plasmid pBm92 was constructed in this experiment. The translational initiation codon ATG of the polyhedrin gene in Pbm92 was changed into ATT, and then five cloning sites of a foreign gene were ligated after the +12 bp site of the polyhedrin gene. Human beta-interferon (HuIFN-beta) gene was cloned into Pbm92 to construct pBmIFN +12; meanwhile we constructed the transfer vector Pbmifn-3 in which HuIFN-beta was cloned after the -3 bp site of the polyhedrin gene. BM-N cells were cotransfected with the two types of transfer vector plasmid DNAs and BmNPV genomic DNA. Recombinant viruses that were screened did not produce polyhedrin inclusion bodies in the virus plaque assay, and were identified by the hybridization of recombinant virus DNA with HuIFN-b gene probe. IFN activity of the culture media of Bm-N cells infected with recombinant virus BmIFN +12 was 2.0 x 10(6) IU/mL, and IFN activity of hemolymph of silkworm larvae infected with BmIFN +12 was 5.0 x 10(7) IU/mL. Expression level of BmIFN +12 was two to four times more than that of BmIFN -3. The rHuIFN-beta produced by BM-N cells and silkworm larvae has an antigenicity identical to that of the native HuIFN-beta.
Deng
J
J
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China.
Wang
S
S
Yang
Q
Q
Cheng
X
X
Li
L
L
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D017344:Genes, Insect / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D007814:Larva / Q000235:genetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D004279:DNA, Viral; D011994:Recombinant Proteins; D016899:Interferon-beta