重组人隐花色素蛋白Ι的表达纯化及其在制备放疗保护剂中的应用

Expression, purification of recombinant human cryptochrome Ι and its application in preparation of protective agent for radiotherapy

摘要:

放疗是治疗肿瘤的最常用手段之一，为寻找能够保护肿瘤组织周边正常细胞的放疗保护剂，通过原核表达质粒的构建，利用大肠杆菌BL21 (DE3) 诱导并成功表达出人隐花色素蛋白Ⅰ (hCRY1)。采用包涵体溶解、梯度透析复性、镍柱亲和纯化以及超滤浓缩的蛋白纯化工艺，1 L菌液可收获10?15 mg纯度超过95%的hCRY1。在HaCaT细胞中通过细胞DNA损伤修复中所产生的H2A.X foci荧光强度的定量检测，验证了纯化所得的hCRY1具有射线损伤防护功能；通过对经过hCRY1以及对照蛋白处理后的细胞凋亡情况的检测排除了hCRY1的毒副作用对H2A.X foci荧光强度的影响。这些为进一步研究hCRY1提供了便利条件，也为将hCRY1开发成为新的放疗保护剂提供了理论基础。

Abstract:

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10?15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaTcell was also detected, and the repression of H2A.X foci formation was not due to hCRY1’s cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.