摘要
人角质细胞生长因子-1(human keratinocyte growth factor-1, hKGF-1)属于成纤维细胞生长因子家族(fibroblast growth factor, FGF)的成员之一,且在器官发育、细胞增殖和创面修复以及各种组织损伤恢复中都发挥着非常重要的作用,同时也是目前对皮肤损伤修复最有效且特异性最好的生长因子。hKGF-1体外表达效率普遍很低,因此目前获得重组hKGF-1十分困难。本研究利用家蚕杆状病毒表达系统,旨在建立以经济昆虫家蚕作为生物反应器,高效率、低成本表达生产重组人角质细胞生长因子hKGF-1蛋白的技术平台,实现hKGF-1在家蚕卵巢细胞(Bombyx mori ovary cell line, BmN)中的高水平表达。本研究根据杆状病毒密码子偏好性优化人重组KGF-1基因序列,通过与杆状病毒高水平表达的多角体蛋白融合表达以及添加额外启动子和增强子等方式,大幅提高了hKGF-1在家蚕BmN细胞中的表达水平。结果表明,通过以上策略可以显著提高hKGF-1在家蚕BmN细胞中的表达水平,SDS-PAGE和Western blotting分析表明,多角体蛋白(polyhedrin, Polh)启动子与P6.9启动子串联并与15个氨基酸的多角体蛋白融合表达时,hKGF-1蛋白表达量最高,占家蚕BmN细胞总蛋白的8.7%,且细胞最佳回收时间为120 h。本研究实现了hKGF-1在家蚕细胞中的高效表达,为重组hKGF-1产业化利用提供了较为理想的技术平台,同时也为其他生长因子或复杂蛋白的生产提供了可借鉴的技术方法,对于利用家蚕作为生物反应器表达重组人源蛋白具有一定借鉴意义。
Human keratinocyte growth factor-1(hKGF-1),a member of the fibroblast growth factor(FGF)family,plays crucial roles in organ development,cell proliferation,wound healing,and tissue repair,representing one of the most effective and specific growth factors for skin repair.However,obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro.This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1(hKGF-1)protein,ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line(BmN).In this study,we optimized the hKGF-1 sequence based on the codon preference of baculovirus.By fusing hKGF-1 with polyhedrin(highly expressed in this system)and adding extra promoters and enhancers,we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells.The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells.SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression(accounting for 8.7%of total cellular protein)was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression.The optimal harvest time was determined to be 120 h post transfection.This study achieved the efficient expression of hKGF-1 in Bombyx mori cells,establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1.The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins,but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
作者
李硕豪
王星洋
吴小锋
许裕敬
杨恬
朱新宇
LI Shuohao;WANG Xingyang;WU Xiaofeng;XU Yujing;YANG Tian;ZHU Xinyu(College of Animal Sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China)
出处
《生物工程学报》
北大核心
2025年第7期2634-2646,共13页
Chinese Journal of Biotechnology
基金
政府间科技合作重点研发项目(2022YFE0124400)
国家自然科学基金(32172793)。
关键词
家蚕杆状病毒表达系统
人角质细胞生长因子
家蚕
优化表达
融合表达
Bombyx mori baculovirus expression system
human keratinocyte growth factor
Bombyx mori
optimized expression
fusion expression
