Li Jian, Hou Yiling, Ding Xiang, Hou Wanru, Song Bo, Zeng Yichun
Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), College of Life Sciences, China West Normal University, 1# Shida Road, Nanchong, 637009, Sichuan, China.
Mol Biol Rep. 2014 May;41(5):3529-39. doi: 10.1007/s11033-014-3217-3. Epub 2014 Feb 7.
The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 μg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 μg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 μg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.
核糖体蛋白L22（RPL22）属于核糖体蛋白L22E家族。它位于细胞质中。本文旨在探究大熊猫（Ailuropoda melanoleuca）RPL22的结构和抗癌功能。利用RT-PCR技术成功从大熊猫中克隆出RPL22的cDNA。我们构建了一个包含RPL22 cDNA的重组表达载体，并使用pET28a质粒在大肠杆菌中进行过表达。获得的表达产物通过镍螯合亲和层析进行纯化。结果表明，克隆片段的长度为414 bp，其包含一个387 bp的开放阅读框，编码128个氨基酸。一级结构分析显示，推测的RPL22蛋白分子量为14.74 kDa，理论pI为9.21。RPL22基因能够在大肠杆菌中真正表达，并且与N端带有His标签的蛋白融合的RPL22蛋白，产生了预期的20.1 kDa多肽的积累。数据表明，重组蛋白RPL22对细胞生长抑制率具有时间和剂量依赖性。与对照（未处理）细胞相比，用0.05 - 6 μg/ml的RPL22处理人喉癌Hep - 2细胞24小时，使用MTT法检测显示出显著的细胞生长抑制（p<0.05，n = 8）。数据表明，低浓度的效果优于高浓度，1.5 μg/ml的浓度具有最佳的生长抑制率，为47.70%。用1.5 μg/ml RPL22蛋白处理的小鼠的抑制率可达43.75%。肿瘤器官的组织学检查表明，RPL22组的组织排列比对照组更松散。同时，对心脏、肺和肾等其他器官没有明显损伤。正在进行进一步研究以确定负责其抗癌活性的重组蛋白RPL22的生物活性原理。
