葛根蛋白分离鉴定及其自组装纳米颗粒性质研究

林岱; 高观祯; 周建武; 柯李晶; 饶平凡

doi:10.13386/j.issn1002-0306.2022100193

葛根蛋白分离鉴定及其自组装纳米颗粒性质研究

doi: 10.13386/j.issn1002-0306.2022100193

林岱^{1, 2,},
高观祯²,
周建武^2,*, ,,
柯李晶²,
饶平凡²

1.
福建医科大学公共卫生学院，福建福州 350122
2.
浙江工商大学食品与生物工程学院，浙江杭州 310018

基金项目: 福建医科大学高层次人才科研启动经费（XRCZX2018013）；福建医科大学启航基金项目（2019QH1001）；福建省自然科学基金面上项目（2021J01724）。

详细信息

作者简介:
林岱（1988−），女，博士，讲师，研究方向：膳食成分的结构、功能与健康风险，E-mail：amber@fjmu.edu.cn

通讯作者:
周建武（1977−），男，博士，副教授，研究方向：蛋白质及活性肽的超分子结构，E-mail：jianwuzhou@zjgsu.edu.cn

中图分类号: TS201.2
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出版历程
- 收稿日期: 2022-10-19
- 刊出日期: 2023-05-01

Isolation and Characterization of a Pueraria lobata Protein and Its Self-assembled Nanoparticles Properties

LIN Dai^{1, 2
,},
GAO Guanzhen²,
ZHOU Jianwu^{2,*
, ,},
KE Lijing²,
RAO Pingfan²

1.
College of Public Health, Fujian Medical University, Fuzhou 350122, China
2.
College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China

摘要

摘要: 目的：纯化并表征一种葛根水溶性蛋白，将该蛋白通过热诱导的方式构建为蛋白纳米颗粒载体。方法：采用阴离子交换色谱High Q纯化获得葛根蛋白，通过SDS-PAGE和蛋白质N端测序方法鉴定蛋白的分子量和氨基酸序列。采用激光粒度分析仪对蛋白纳米颗粒的粒径、光散射强度和Zeta电位进行研究，采用高效液相色谱法测定纳米载体对药物的装载效率。结果：从葛根中抽提并分离纯化获得一种主要的水溶性蛋白，命名为PP。N-端氨基酸序列测得为DFVYDMCGNVLNGGTYYIL，通过NCBI数据库比对和蛋白酶活性测定确定PP为一种新的胰蛋白酶抑制剂，且PP在pH2~10的环境中及20~50 ℃的温度范围内均具有较好的稳定性。PP溶液（0.1 mg/mL，pH6.0）在100 ℃下加热60 min可以形成分布均匀的纳米颗粒PP-NPs，测定其平均粒径为172.78 nm，Zeta电位为−25.40 mV。PP-NPs能有效装载葛根素及小檗碱，测得的药物装载率分别为33.83%和24.61%。结论：从葛根中纯化获得的主要水溶性蛋白PP能通过热诱导构建蛋白纳米颗粒，该颗粒具有成为药物载体的潜力。
- 葛根 /
- 纳米颗粒 /
- 自组装 /
- 胰蛋白酶抑制剂
Abstract: Objective: To purify and characterize a water-soluble protein from Pueraria lobata and to fabricate its nanoparticles by heating-induced assembly. Methods: Pueraria lobata protein was purified by anion exchange chromatography High Q, and its molecular weight and amino acid sequence were determined by SDS-PAGE and N-terminal sequencing. The particle size, optical dispersion intensity and Zeta potential of protein nanoparticles were measured by laser-scattering particle analyzer. The drug loading efficiency of the nanocarrier was determined by chromatography. Results: A major water-soluble protein, named PP, was purified from Pueraria lobata and sequenced with a N-terminal amino acid sequence of DFVYDMCGNVLNGGTYYIL. PP was identified as a novel trypsin inhibitor by NCBI database searching and rypsin inhibitory assay. PP was also well-stabilized in the pH2~10 and 20~50 °C ranges. After heating PP solution (0.1 mg/mL, pH6.0) for 60 minutes at 100 ℃, homogenous nanoparticles (PP-NPs) were harvested. These PP-NPs had a particle size of 172.78 nm and a Zeta potential of −25.40 mV. The puerarin and berberine were effectively loaded onto PP-NPs, with loading efficiency of 33.83% and 24.61%, respectively. Conclusion: The major water-soluble Pueraria lobata protein PP can be fabricated into protein nanoparticles by heating-induced assembly, indicating a potential as drug carriers.
- Pueraria lobata /
- nanoparticles /
- self-assembly /
- trypsin inhibitor

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图 1 葛根蛋白电泳图

注：（a）冷抽提电泳图：1. 标样，2. 冷抽提液；（b）乙醇沉降电泳图：1. 标样，2. 20%，3. 40%，4. 60%，5. 80%。

Figure 1. SDS-PAGE of Pueraria lobata protein

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图 2 葛根蛋白纯化结果

注：a. High Q阴离子分离图谱；b. 洗脱峰电泳图。

Figure 2. Results of purification of Pueraria lobata protein

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图 3 葛根蛋白对胰蛋白酶活性的抑制作用

注：*表示与最大抑制活性比较，差异有统计学意义（P<0.05）。

Figure 3. Inhibitory effects of PP on the active of trypsin

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图 4 pH、加热时间、加热温度对葛根蛋白纳米颗粒组装的影响

注：图a中*表示与pH2组比较，差异有统计学意义（P<0.05）；图b中*表示与20 min组比较，差异有统计学意义（P<0.05）；图c中*表示与40 ℃组比较，差异有统计学意义（P<0.05）。

Figure 4. Effects of pH, heating time and heating temperature on the assembly of Pueraria lobata protein nanoparticles

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图 5 PP-NPs透射电子显微镜图

注：a. 5000×；b. 10000×。

Figure 5. TEM image of PP-NPs

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表 1 葛根蛋白分子量及N端氨基酸序列比对

Table 1. Molecular weight and N-terminal amino acid sequence alignment of PP

名称	分子量（kDa）	N端氨基酸序列
葛根蛋白（PP）	20.73	1 DFVYDMCGNVLNGGTYYIL 19
大豆胰蛋白酶抑制剂（STI）	~20	1 DFVLDNEGNPLENGGTYYIL 20

下载: 导出CSV

表 2 葛根蛋白纳米颗粒装载效果

Table 2. Loading effect of PP-NPs

样品	平均粒径（d.nm）	粒子计数率（kcps）	Zeta电位（mV）	包埋率（%）
PP-NPs	172.78±3.89	3018.17±107.33	−25.40±0.71	−
PP-NPs包埋葛根素	491.33±17.8^*	2546.13±56.04^*	−18.67±0.55^*	33.83±2.71
PP-NPs包埋小檗碱	205.24±4.87^*	2423.33±85.47^*	−14.70±0.87^*	24.61±2.19
注：^*表示与PP-NPs组比较，差异有统计学意义（P<0.05）。

下载: 导出CSV

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