家蚕丝素重链启动子驱动DsRed的瞬时分泌表达.PDF

生 物 工 程 学 报 Chin J Biotech 2009, May 25; 25(5): 761-766 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2009 Institute of Microbiology, CAS CSM, All rights reserved 生物技术与方法 DsRed 潘兴亮, 曹广力, 薛仁宇, 贡成良 , 215123 : 根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性, 克隆了家蚕丝素重链基因(Fib-H)启动子及 其下游的信号肽序列(FibHS), 将 DsRed 基因与信号肽序列融合构建了分泌型瞬时表达载体; 转染细胞实验显示, 该载 体能在家蚕BmN 细胞中瞬时表达DsRed; 家蚕注射载体后, 可在丝腺腔中检测到红色荧光, 表明瞬时表达的DsRed 分 泌到丝腺腔, 推测所克隆的序列具有信号肽的功能。此外, 本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定 了基础。 : 丝素重链启动子, 信号肽, DsRed , 家蚕, BmN 细胞, 丝腺 Transiently secretory expression of DsRed driven by fibroin heavy chain promoter of Bombyx mori Xingliang Pan, Guangli Cao, Renyu Xue, and Chengliang Gong School of Pre-clinical Medicine and Biological Science, Medical College, Soochow University, Suzhou 215123, China Abstract: Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal I/Kpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After i