江苏大学学报（医学版）2024，Vol.34Issue(1)：44-51,66,9.DOI:10.13312/j.issn.1671-7783.y230183
ELP-SOD融合蛋白的纯化及其脂质体制备
Purification of ELP-SOD fusion protein and liposome encapsulation
朱月蓉 1陈星宇 2冯娇燕 2周奔 2樊燕蓉2
作者信息
- 1. 东部战区总医院检验科,江苏南京 210002
- 2. 南京理工大学环境与生物工程学院,江苏南京 210014
- 折叠
摘要
Abstract
Objective:The ELP-SOD fusion protein was expressed by recombinant E.coli cells,and purified ELP-SOD enzyme with high activity was obtained by three rounds of inverse transition cycling(ITC).ELP-SOD liposomes with long half-life and enzyme activity were prepared by liposome encapsulation technology,which laid a foundation for the application of recombinant human SOD in medical,health care,cosmetics and other fields.Methods:The purity of ELP-SOD was evaluated by SDS-PAGE,Tanon3500 gel imaging system and Image studio software;the enzyme activity of SOD fusion protein was measured using an improved pyrogallol autooxidation method(325 nm method);by adjusting different pH values and temperature gradients,the pH and thermal stability of the fusion protein were assessed;adding Cu2+/Zn2+increased ELP-SOD enzyme activity;in vivo half-life experiments was simulated using rat plasma;cytotoxicity was evaluated by incubating HUVEC and L929 cells with different concentrations of ELP-SOD in culture media;preparation of ELP-SOD liposomes by thin film hydration method and determination of their encapsulation efficiency;mouse skin in vitro was prepared and in vitro release experiments were conducted using TP-6 transdermal diffusion apparatus.By using Franz diffusion cell the transdermal efficiency of ELP-SOD liposomes was to be investigated in carrying ELP-SOD.Results:The highest expression level of ELP-SOD fusion protein was 10 mg/L,with a purity of 97.8％and an enzyme specific activity of 4 894.5 U/mg;its stability was higher at pH 5.0-8.0 and temperatures below 60 ℃;the access of ELP had a certain extension effect on the half-life of SOD and has good biosafety;the encapsulation efficiency of ELP-SOD liposomes reaches over 80％,which could further prolong the half-life of ELP-SOD,and its transdermal efficiency was significantly higher than that of ELP-SOD.Conclusion:The purification process of ELP-SOD fusion protein was simplified through reversible thermal denaturation technology,and combined with lipid preparation technology,the biological half-life of the enzyme protein was extended and its bioavailability was improved.关键词
超氧化物歧化酶/类弹性蛋白多肽/可逆相变循环/脂质体Key words
superoxide dismutase/elastin-like polypeptide/inverse transition cycling/liposome分类
医药卫生引用本文复制引用
朱月蓉,陈星宇,冯娇燕,周奔,樊燕蓉..ELP-SOD融合蛋白的纯化及其脂质体制备[J].江苏大学学报（医学版）,2024,34(1):44-51,66,9.
