Wang Feng, Wang Riyuan, Wang Yuancheng, Zhao Ping, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
College of biotechnology, Southwest University, Chongqing, China.
Sci Rep. 2015 Nov 16;5:16323. doi: 10.1038/srep16323.
With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a "safe harbor" locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.
随着每年临床对功能性治疗蛋白的需求不断增加，对大量生产具有生物活性的重组人酸性成纤维细胞生长因子（r-haFGF）的要求也日益提高。在本研究中，我们精心探索了一种在转基因蚕茧中大量生产具有生物活性的r-haFGF蛋白的策略。将序列优化的haFGF插入增强型丝胶蛋白-1表达系统中，以产生原始转基因蚕品系，然后将其与PIG启动菌株进一步杂交，使表达盒重新定位到基因组中的“安全港”位点，以实现r-haFGF的高效表达。结果，突变品系中r-haFGF蛋白的表达量与原始品系相比提高了5.6倍。高含量的r-haFGF便于其纯化和大规模生产。此外，r-haFGF蛋白具有生物活性，可促进NIH/3T3细胞的生长、增殖和迁移，表明r-haFGF蛋白具有天然的促有丝分裂活性和伤口愈合潜力。这些结果表明，蚕的丝腺可能是在蚕茧中重组生产具有生物活性的haFGF的一种有效生物反应器策略。
