Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.
To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.
Guo
X-Y
XY
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China.
Guo
T-Q
TQ
Wang
S-P
SP
Wang
J-Y
JY
Lu
C-D
CD
D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000821:virology; D005088:Exocrine Glands / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D047030:Sericins / Q000235:genetics / Q000378:metabolism; D047011:Silk
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D047030:Sericins; D047011:Silk; D049452:Green Fluorescent Proteins