Ishikiriyama Motoki, Nishina Takuya, Kato Tatsuya, Ueda Hiroshi, Park Enoch Y
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan.
J Biosci Bioeng. 2009 Jan;107(1):67-72. doi: 10.1016/j.jbiosc.2008.11.001.
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.
构建了七组重组表达载体，用于在蚕幼虫和蛹的血淋巴和脂肪体中表达与人抗牛血清白蛋白（BSA）单链Fv片段（scFv）13CG2融合的绿色荧光蛋白（uv）或家蚕素（bx）信号肽C端，使用半胱氨酸蛋白酶缺陷型（BmNPV-CP(-)）和半胱氨酸蛋白酶及几丁质酶缺陷型（BmNPV-CP(-)Chi(-)）杆状病毒载体。当使用BmNPV-CP(-)或BmNPV-CP(-)Chi(-)杆状病毒载体时，在感染后6天，scFv的表达量为16.9-18.9mg/l（11.6-15.0μg/幼虫），而野生型BmNPV杆状病毒载体仅表达4.4mg/l，这可能是由于蛋白质的蛋白水解降解。蚕蛹中scFv的产量仅为0.67-1.0μg/蛹，仅为蚕幼虫血淋巴中产量的5.4%。bx信号肽能够使scFv分泌到血淋巴中。没有信号序列时，融合蛋白在脂肪体中积累并失去其生物学功能。去除绿色荧光蛋白（uv）显著提高了家蚕血淋巴中scFv的产量至188.4mg/l（132.4mg/幼虫），比融合蛋白产量高十倍。
