肿瘤归巢肽-近红外荧光蛋白融合蛋白的制备及其荧光特性  

Preparation of tumor homing peptides-near-infrared fluorescent protein miRFP670-LyP1 fusion protein and its fluorescence characteristics

作　　者：杨馥旭 胡楠楠 郭冲 穆业腾 薛晗 范宇鑫 郭峰霖 关新刚 YANG Fuxu;HU Nannan;GUO Chong;MU Yeteng;XUE Han;FAN Yuxin;GUO Fenglin;GUAN Xingang(Key Laboratory of Pharmaceutics and Bioengineering,School of Medical Technology,Beihua University,Jilin 132013,China;Department of Basic Medicine,College of Medical Sciences,Taizhou University,Taizhou 318000,China)

机构地区：[1]北华大学医学技术学院医药生物工程重点实验室,吉林吉林132013 [2]台州学院医学院基础医学系,浙江台州318000

出　　处：《吉林大学学报（医学版）》2022年第6期1455-1461,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划自然科学基金项目(20180101213JC);吉林省卫健委卫生与健康技术创新项目(2020J023);北华大学研究生创新计划项目(北华研创合字[2021] 029);台州学院高层次人才科研启动项目(T20220101026)。

摘　　要：目的:构建肿瘤归巢肽(THPs)-近红外荧光蛋白(NIRFP)miRFP670-LyP1融合蛋白的原核表达载体,纯化融合蛋白,研究融合蛋白的近红外荧光特性。方法:采用限制性核酸内切酶EcoRⅠ和NotⅠ对pmiRFP670-N1质粒和pET-28a质粒进行双酶切,构建pET-miRFP670原核表达载体,通过点突变引入LyP-1的DNA序列,构建重组表达载体pET-miRFP670-LyP1;将测序正确的重组表达载体转化至大肠杆菌BL21细胞中,SDS-PAGE电泳法检测不同温度(16℃和37℃)、不同异丙基硫代半乳糖苷浓度(0.1、0.5和1.0 mmol·L^(-1))诱导下融合蛋白原核表达量;采用Ni-NTA树脂亲和纯化融合蛋白,检测miRFP670-LyP1蛋白原核表达量;采用荧光显微镜观察乳腺癌4T1细胞中miRFP670-LyP1融合蛋白的细胞内吞形态表现。结果:检测到长度约为5343和973 bp的DNA条带,与pET-28a载体及miRFP670基因片段大小相符。DNA测序,LyP1序列成功插入至pETmiRFP670表达载体中。在16℃时miRFP670-LyP1融合蛋白的可溶性蛋白表达量较37℃时更高。采用Ni-NTA树脂纯化得到了高纯度的miRFP670-LyP1融合蛋白。荧光成像,miRFP670-LyP1融合蛋白可被乳腺癌4T1细胞高效内吞。结论:成功构建了pET-miRFP670-LyP1原核表达载体,融合蛋白在低温(16℃)较常温(37℃)诱导的可溶性蛋白表达量更高,亲和层析得到了高纯度的融合蛋白,融合蛋白被乳腺癌4T1细胞高效内吞并显示出近红外荧光。Objective:To construct the prokaryotic expression vector of tumor homing peptide(THPs)-near-infrared fluorescent protein(NIRFP)-miRFP670 LyP1 fusion protein,and to purified fusion protein,and to investigate the near infrared fluorescence characteristics of the fusion protein.Methods:The pmiRFP670-N1 plasmid and pET-28a plasmid were doubly digested with restriction endonucleases EcoRⅠand NotⅠto construct the pET-miRFP670 prokaryotic expression vector.The LyP-1 DNA sequence was introduced through point mutation to construct the recombinant expression vector pET-miRFP670-LyP1;the recombinant expression vector with correct sequence was transformed into the E.coli BL21 cells and the prokaryotic expression amounts of fusion proteins induced under different temperatures(16℃and 37℃)and different concentrations of isopropyl-β-D-thiogalactopyranoside(IPTG)(0.1,0.5,and 1.0 mmol·L^(-1))were detected by SDS-PAG electrophoresis;the fusion protein was purified by Ni-NTA resin affinity,and the prokaryotic expression amount of the miRFP670-LyP1 protein was detected;the endocytosis morphology of the miRFP670-LyP1 fusion protein in breast cancer 4T1 cells was observed under fluorescence microscope.Results:The double digestion of recombinant plasmid showed that two DNA bands of about 5343 and 973 bp were obtained,which was consistent with the sizes of the pET-28a vector and miRFP670 gene fragment.The DNA sequencing results showed that the LyP1 sequence was successfully inserted into the pET-miRFP670 expression vector.The soluble protein expression amount of miRFP670-LyP1 fusion protein was higher at 16℃than that at 37℃.The miRFP670-LyP1 fusion protein with high purity was obtained by purification with Ni-NTA resin.The fluorescence imaging results showed that the miRFP670-LyP1 fusion protein could be efficiently endocytosed by the breast cancer 4T1 cells.Conclusion:The prokaryotic expression vector pET-miRFP670-LyP1 is successfully constructed,the soluble protein expression amount of the fusion protein is higher at low

关 键 词：近红外荧光蛋白 肿瘤归巢肽 融合蛋白 原核表达 细胞内吞 

分 类 号：R394.3[医药卫生—医学遗传学]