白转丽+都慧聪+耿文鑫

[摘要]目的：设计、制备透皮型弹性蛋白样多肽（elastin-like polypeptide， ELP）融合蛋白，探索其作为皮肤美容制剂的潜能。方法：设计具有透皮特性的蛋白转导功能域（protein transduction domain， PTD）和ELP融合蛋白PTD-ELP，联合密码子优化、重叠延伸PCR和定向递归连接等技术构建其原核表达载体。在此基础上将该载体转化至大肠杆菌中，进行异丙基硫代半乳糖苷（isopropyl-β-D-Thiogalactoside， IPTG）诱导表达并对目标蛋白的亚细胞定位和可溶性进行分析。结果：成功构建了携带PTD-ELP读码框架的原核表达载体，携带该载体的大肠杆菌在IPTG诱导5h时PTD-ELP融合蛋白的表达量达到峰值，约占菌体总蛋白的23.6%；亚细胞定位分析表明PTD-ELP融合蛋白主要以包涵体形式在胞内表达。结论：成功设计并构建了能够表达PTD-ELP融合蛋白的原核表达载体并在大肠杆菌中实现了其高效表达，为深入探讨PTD介导的ELP透皮给药在逆转皮肤衰老和预防、治疗光老化中的应用奠定了基础。

[关键词]弹性蛋白样多肽；蛋白转导功能域；原核表达；重组蛋白

[中图分类号]Q591.2 [文献标志码]A [文章编号]1008-6455（2017）10-0064-04

Abstract： Objective To design and express an elastin-like polypeptide （ELP） fusion protein which can be transcutaneously delivered to skin tissues to explore its potential as a skin cosmetics agent in the future. Methods A protein transduction domain （PTD）-ELP fusion protein with transdermal properties was designed and back-translated with E.coli-preferred codons. And its expression vector was constructed by overlap extension PCR and recursive directional ligation. After transforming the vector into Escherichia coli cells， the PTD-ELP protein was expressed by IPTG induction and the subcellular location and the solubility of the recombinant protein were analyzed. Results A prokaryotic vector containing PTD-ELP-coding cassette was successfully constructed as designed. And the E.coli cells harboring the vector could express the fusion protein after IPTG induction， which reached the highest level at 5h post induction with amount of 23.6% of total bacterial proteins. Further analysis demonstrated that PTD-ELP protein was subcellularly located in cytoplasm as insoluble inclusion bodies. Conclusion The recombinant prokaryotic expression vector was constructed successfully and the PTD-ELP fusion protein was efficiently expressed in Escherichia coli cells. These results laid a foundation for further investigating whether PTD-mediated transdermal delivery of ELP could prevent skin aging and reverse photoaging.

Key words： elastin-like polypeptide； protein transduction domain； prokaryotic expression； fusion protein

彈性蛋白是在维持皮肤组织结构和功能中发挥重要作用的细胞外基质蛋白，其约占皮肤总蛋白成分的2%[1-2]。弹性蛋白前体是由成纤维细胞和角质细胞合成并分泌的、分子量约为60～72kDa的可溶性弹性蛋白原，后者可在赖氨酰氧化酶的作用下发生分子内和分子间交联形成不溶性弹性蛋白，进而维持皮肤弹性[3]。弹性蛋白在人体内的合成从出生开始逐渐升高到青春期达到峰值，此后迅速下降。……