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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (7): 44-52    DOI: 10.13523/j.cb.2212024
技术与方法     
重组白蛋白结合肽融合人白介素-11的原核可溶表达、纯化、表征及初步活性评价*
孟奂,粟乙梵,王洒,张淑雯,郑永祥,余蓉,张纯**()
四川大学华西药学院 靶向药物及释药系统教育部重点实验室 成都 610041
Prokaryotic Soluble Expression, Purification, Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11
Huan MENG,Yi-fan SU,Sa WANG,Shu-wen ZHANG,Yong-xiang ZHENG,Rong YU,Chun ZHANG**()
Key Laboratory of Drug Targeting and Drug Delivery System (Ministry of Education), West China School of Pharmacy, Sichuan University, Chengdu 610041, China
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摘要：

目的:改善重组人白介素-11在原核表达体系中的可溶表达水平以及在体内的循环半衰期。方法:通过构建白蛋白结合肽融合人白介素-11重组蛋白表达载体[pET-30a(+)-ABD-rhIL11],实现融合蛋白在原核表达体系中的可溶表达;基于疏水层析、离子交换层析和凝胶层析建立从破菌上清液中纯化ABD-rhIL-11的方法;通过圆二色光谱及荧光发射光谱分析等鉴定ABD-rhIL-11空间结构,并进行体内外活性及药效学评价。结果:成功获得纯度高于95%的重组融合蛋白,体外结合实验结果表明,ABD-rhIL-11能够迅速与人血清白蛋白(human serum albumin,HSA)结合;细胞增殖实验结果显示,ABD-rhIL-11与对照品活性相当;药代动力学结果显示,融合蛋白能够显著延长白介素-11在大鼠体内的循环半衰期;血小板计数实验结果显示,ABD-rhIL-11能够有效刺激大鼠血小板增殖。结论:为基于白蛋白结合肽融合策略的白介素-11药物长效设计提供了新的思路。
关键词： 人白介素-11;  白蛋白结合肽;  可溶表达;  血小板增殖    
Abstract:

Objective: To improve the soluble expression level of human interleukin-11 in prokaryotic expression system and enhance its circulating half-life in vivo. Methods: The recombinant protein expression vector (PBV220-ABD-rhIL11) was constructed, and it was successfully expressed in prokaryotic expression system in soluble form. Based on hydrophobic chromatography, ion exchange chromatography and gel chromatography, a method for purification of ABD-rhIL-11 from the supernatant of bacteria was successfully established. The circular dichroism spectrum and fluorescence emission spectrum analysis confirmed that the spatial structure of the purified ABD-rhIL-11 was folded correctly. In vivo and in vitro activity and pharmacodynamic evaluation was carried out. Results: The purity of recombinant fusion protein was higher than 95%. The results of in vitro binding experiments showed that ABD-rhIL-11 could rapidly bind to human serum albumin. The results of cell proliferation experiment showed that the activity of ABD-rhIL-11 was equivalent to that of the control group. Pharmacokinetic results showed that the fusion protein could significantly prolong the circulating half-life of interleukin-11 in rats. The results of platelet count test showed that ABD-rhIL-11 could effectively stimulate platelet proliferation in rats. Conclusion: The results of this study would provide a new idea for the long-term design of interleukin-11 drugs based on albumin binding peptide fusion strategy.
Key words: Human interleukin-11    Albumin binding peptide    Soluble expression    Platelet proliferation
收稿日期: 2022-12-18 出版日期: 2023-08-03
ZTFLH:  Q816  
基金资助: 国家自然科学基金(82003685);四川省科学技术厅项目(2021YJ0187)
通讯作者: **电子信箱:chunzhang@scu.edu.cn   
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引用本文:

孟奂, 粟乙梵, 王洒, 张淑雯, 郑永祥, 余蓉, 张纯. 重组白蛋白结合肽融合人白介素-11的原核可溶表达、纯化、表征及初步活性评价*[J]. 中国生物工程杂志, 2023, 43(7): 44-52.

Huan MENG, Yi-fan SU, Sa WANG, Shu-wen ZHANG, Yong-xiang ZHENG, Rong YU, Chun ZHANG. Prokaryotic Soluble Expression, Purification, Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11. China Biotechnology, 2023, 43(7): 44-52.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2212024    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I7/44

图1  ABD-IL-11诱导表达及可溶性鉴定结果
图2  重组融合蛋白的分离纯化层析图谱及纯度鉴定图
图3  ABD-IL-11与HSA结合能力鉴定
图4  ABD-IL-11结构表征
图5  抗人IL-11抗体相对结合能力与细胞活性实验
图6  IL-11、ABD-IL-11重组蛋白在大鼠体内药物代谢动力学活性评价
图7  大鼠血样(50倍稀释)显微镜100 μm视野
图8  大鼠血液中血小板数量
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