Yanagihara Kana, Terada Satoshi, Miki Masao, Sasaki Masahiro, Yamada Hideyuki
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan.
Biotechnol Appl Biochem. 2006 Sep;45(Pt 2):59-64. doi: 10.1042/BA20060077.
Adenoviral vectors are extensively used as gene-delivery vehicles in gene therapy. They are usually produced by HEK-293 cell (human embryonic kidney-293 cell) culture, which requires specially formulated serum-free medium, the cost of which is considerable or by supplementation with FBS (fetal bovine serum). The risk of infectious diseases such as BSE (bovine spongiform encephalopathy) and endogenous retrovirus derived from cattle is a serious concern. The present study reports the use of sericin protein derived from silkworm (Bombyx mori) as an effective supplement instead of FBS. Without FBS, HEK-293 cells significantly proliferated in the presence of 0.025-0.4% sericin, especially at 0.1%, but the effect was inferior to that of FBS. When a lower titre [MOI (multiplicity of infection) 0.03] of adenoviral vector pAxCAiLacZ was used as the inoculum, HEK-293 cells in the presence of 0.1% sericin produced a nearly 3-fold higher vector titre than culture in the presence of 5% (v/v) FBS. However, when a higher vector titre (MOI 3.7) was used as the inoculum, HEK-293 cells in the presence of sericin produced a slightly higher vector titre than in the presence of FBS, which might suggest that HEK-293 cells produce a maximum amount when a higher vector titre is used as the inoculum. These increases in vector production with sericin were confirmed by LacZ (beta-galactosidase reporter gene) activity assay. Supplementation with sericin decreased lactate dehydrogenase activity, an indicator of cell death, suggesting that sericin improved cell survival; hence, prolonging the culture period might be one of the reasons for increased vector production. On the basis of these results, sericin peptide seems to be a potent and effective alternative supplement for production of adenoviral vectors without such risks as BSE and retrovirus.
腺病毒载体在基因治疗中被广泛用作基因传递工具。它们通常通过HEK - 293细胞（人胚肾293细胞）培养产生，这需要特殊配制的无血清培养基，其成本相当高，或者通过添加胎牛血清（FBS）。诸如牛海绵状脑病（BSE）等传染病风险以及源自牛的内源性逆转录病毒是一个严重问题。本研究报告了使用源自家蚕（Bombyx mori）的丝胶蛋白作为替代FBS的有效补充剂。在没有FBS的情况下，HEK - 293细胞在存在0.025 - 0.4%丝胶蛋白时显著增殖，尤其是在0.1%时，但效果不如FBS。当使用较低滴度[感染复数（MOI）0.03]的腺病毒载体pAxCAiLacZ作为接种物时，存在0.1%丝胶蛋白的HEK - 293细胞产生的载体滴度比存在5%（v/v）FBS的培养物高近3倍。然而，当使用较高载体滴度（MOI 3.7）作为接种物时，存在丝胶蛋白的HEK - 293细胞产生的载体滴度略高于存在FBS时，这可能表明当使用较高载体滴度作为接种物时，HEK - 293细胞产生量达到最大。通过β - 半乳糖苷酶（LacZ）报告基因活性测定证实了丝胶蛋白使载体产量增加。添加丝胶蛋白降低了乳酸脱氢酶活性，这是细胞死亡的一个指标，表明丝胶蛋白改善了细胞存活；因此，延长培养期可能是载体产量增加的原因之一。基于这些结果，丝胶肽似乎是生产腺病毒载体的一种有效替代补充剂且不存在BSE和逆转录病毒等风险。
