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A silkworm based silk gland bioreactor for high-efficiency production of recombinant human lactoferrin with antibacterial and anti-inflammatory activities.
Journal of Biological Engineering ( IF 5.7 ) Pub Date : 2019-07-05 , DOI: 10.1186/s13036-019-0186-z
Sheng Xu 1, 2, 3 , Feng Wang 1, 2, 3 , Yuancheng Wang 1, 2, 3 , Riyuan Wang 1, 2, 3 , Kai Hou 1, 2, 3 , Chi Tian 1, 2, 3 , Yanting Ji 1, 2, 3 , Qianqian Yang 1, 2, 3 , Ping Zhao 1, 2, 3 , Qingyou Xia 1, 2, 3
Affiliation  

Background Silk glands are used by silkworms to spin silk fibers for making their cocoons. These have recently been regarded as bioreactor hosts for the cost-effective production of other valuable exogenous proteins and have drawn wide attention. Results In this study, we established a transgenic silkworm strain which synthesizes the recombinant human lactoferrin (rhLF) in the silk gland and spins them into the cocoon by our previously constructed silk gland based bioreactor system. The yield of the rhLF with the highest expression level was estimated to be 12.07 mg/g cocoon shell weight produced by the transgenic silkworm strain 34. Utilizing a simple purification protocol, 9.24 mg of the rhLF with recovery of 76.55% and purity of 95.45% on average could be purified from 1 g of the cocoons. The purified rhLF was detected with a secondary structure similar with the commercially purchased human lactoferrin. Eight types of N-glycans which dominated by the GlcNAc (4) Man (3) (61.15%) and the GlcNAc (3) Man (3) (17.98%) were identified at the three typical N-glycosylation sites of the rhLF. Biological activities assays showed the significant evidence that the purified rhLF could relief the lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells and exhibit potent antibacterial bioactivities against the Escherichia coli (E. coli) and Bacillus subtilis. Conclusions These results show that the middle silk gland of silkworm can be an efficient bioreactor for the mass production of rhLF and the potential application in anti-inflammation and antibacterial.

中文翻译:

一种基于蚕的丝腺生物反应器,用于高效生产具有抗菌和抗炎活性的重组人乳铁蛋白。

背景 蚕使用丝腺来纺丝纤维以制造它们的茧。这些最近被认为是生物反应器宿主,可用于经济高效地生产其他有价值的外源蛋白质,并引起了广泛关注。结果在这项研究中,我们建立了一个转基因蚕品系,该品系在丝腺中合成重组人乳铁蛋白(rhLF),并通过我们先前构建的基于丝腺的生物反应器系统将它们纺成茧。转基因家蚕品系 34 产生的具有最高表达水平的 rhLF 的产量估计为 12.07 mg/g 茧壳重量。使用简单的纯化方案,得到 9.24 mg 的 rhLF,回收率为 76.55%,纯度为 95.45%平均可以从 1 克蚕茧中提纯。检测到纯化的 rhLF 具有与商业购买的人乳铁蛋白相似的二级结构。在 rhLF 的三个典型 N-糖基化位点鉴定出以 GlcNAc (4) Man (3) (61.15%) 和 GlcNAc (3) Man (3) (17.98%) 为主的八种 N-聚糖。生物活性测定表明,纯化的 rhLF 可以缓解 RAW264.7 细胞中脂多糖 (LPS) 诱导的细胞炎症,并对大肠杆菌和枯草芽孢杆菌具有有效的抗菌生物活性。结论这些结果表明,家蚕中丝腺可以作为一种高效的生物反应器,用于大量生产rhLF,并在抗炎和抗菌方面具有潜在的应用价值。在 rhLF 的三个典型 N-糖基化位点鉴定出以 GlcNAc (4) Man (3) (61.15%) 和 GlcNAc (3) Man (3) (17.98%) 为主的八种 N-聚糖。生物活性测定表明,纯化的 rhLF 可以缓解 RAW264.7 细胞中脂多糖 (LPS) 诱导的细胞炎症,并对大肠杆菌和枯草芽孢杆菌具有有效的抗菌生物活性。结论这些结果表明,家蚕中丝腺可以作为一种高效的生物反应器,用于大量生产rhLF,并在抗炎和抗菌方面具有潜在的应用价值。在 rhLF 的三个典型 N-糖基化位点鉴定出以 GlcNAc (4) Man (3) (61.15%) 和 GlcNAc (3) Man (3) (17.98%) 为主的八种 N-聚糖。生物活性测定表明,纯化的 rhLF 可以缓解 RAW264.7 细胞中脂多糖 (LPS) 诱导的细胞炎症,并对大肠杆菌和枯草芽孢杆菌具有有效的抗菌生物活性。结论这些结果表明,家蚕中丝腺可以作为一种高效的生物反应器,用于大量生产rhLF,并在抗炎和抗菌方面具有潜在的应用价值。生物活性测定表明,纯化的 rhLF 可以缓解 RAW264.7 细胞中脂多糖 (LPS) 诱导的细胞炎症,并对大肠杆菌和枯草芽孢杆菌表现出有效的抗菌生物活性。结论这些结果表明,家蚕中丝腺可以作为一种高效的生物反应器,用于大量生产rhLF,并在抗炎和抗菌方面具有潜在的应用价值。生物活性测定表明,纯化的 rhLF 可以缓解 RAW264.7 细胞中脂多糖 (LPS) 诱导的细胞炎症,并对大肠杆菌和枯草芽孢杆菌表现出有效的抗菌生物活性。结论这些结果表明,家蚕中丝腺可以作为一种高效的生物反应器,用于大量生产rhLF,并在抗炎和抗菌方面具有潜在的应用价值。
更新日期:2020-04-22
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