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

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Not Ⅰ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5 343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pET-miRFP670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。

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

Methods The pmiRFP670-N1 plasmid and pET-28a plasmid were doubly digested with restriction endonucleases EcoR Ⅰ and Not Ⅰ to 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 5 343 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 temperature （16 ℃） than that at normal temperature （37 ℃）， and the fusion protein with high purity is obtained by affinity chromatography，and the fusion protein is efficiently endocytosed by the breast cancer 4T1 cells and displays near-infrared fluorescence. 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.