Human single-chain antibody expression in the hemolymph and fat body of silkworm larvae and pupae using BmNPV bacmids.
Seven sets of recombinant expression vectors were constructed for the expression of the human anti-bovine serum albumin (BSA) single-chain Fv fragment (scFv) 13CG2 fused to the C terminus of GFP(uv) or bombyxin (bx) signal peptide in the hemolymph and fat body of silkworm larvae and pupae, using cysteine protease- (BmNPV-CP(-)), and cysteine protease- and chitinase-deficient (BmNPV-CP(-)Chi(-)) bacmids. When BmNPV-CP(-) or BmNPV-CP(-)Chi(-) bacmids were used, 16.9-18.9 mg/l (11.6-15.0 microg/larva) of scFv was expressed at 6 d.p.i., whereas wild-type BmNPV bacmid expressed only 4.4 mg/l, probably because of proteolytic degradation of the protein. The scFv yield in silkworm pupae was only 0.67-1.0 microg/pupa, which was 5.4% of that in the hemolymph of silkworm larvae. The bx signal peptide enabled the secretion of scFv into the hemolymph. Without the signal sequence, the fusion protein accumulated in the fat body and lost its biological function. The removal of GFP(uv) significantly increased the scFv yield in the hemolymph of silkworms to 188.4 mg/l (132.4 mg/larva), which was ten times higher than that of the fusion protein.
Ishikiriyama
Motoki
M
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan.
Nishina
Takuya
T
Kato
Tatsuya
T
Ueda
Hiroshi
H
Park
Enoch Y
EY
D000818:Animals; D012831:Bombyx / Q000378:metabolism; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006458:Hemolymph / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry
D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D003546:Cysteine Endopeptidases