葛根蛋白分离鉴定及其自组装纳米颗粒性质研究

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

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。

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℃ 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.