家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

Transiently secretory expression of DsRed driven by fibroin heavy chain promoter of Bombyx mori

摘要:

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。

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 identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS- DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.