人PRL-3基因真核表达载体构建及相关蛋白生物信息学分析

目的克隆人PRL-3基因并构建其真核表达载体，并运用生物信息学方法对PRL-3的相互作用蛋白进行预测分析。方法运用RT—PCR技术，从人大肠癌细胞系SW480细胞中扩增出人PRL-3 cDNA，经T—A克隆，枸建人PRL-3基因的真核表达载体pcDNA3-PRL-3，通过双酶切鉴定及测序确认，并利用生物信息学方法预测分析其相互作用蛋白。结果成功扩增出人PRL-3全长cDNA，构建pcDNA3-PRL-3真核表达载体，测序结果表明PRL-3全长cDNA与GeneBank中PRL-3序列完全一致。利用模式生物果蝇相互作用蛋白数据库DIP，利用基于保守的蛋白相互作用分析方法，预测认为肌动蛋白家族成员ARP6和功能尚不清楚的小G蛋白家族ARL-3可能是PRL-3的相互作用蛋白，提示PRL-3可能是联系信号分子和细胞骨架系统的重要桥梁，参与构建了一条独特的结直肠癌转移信号途径。结论成功克隆PRL-3基因全长cDNA并构建其真核表达载体，并利用生物信息学方法，初步预测PRL-3的相互作用蛋白，为进一步深入研究PRL-3在大肠癌发生发展中的作用奠定了基础。

Construction of PRL-3 gene eukaryotic expression vector and bioinformatic analysis of PRL-3-interacting proteins

Objective To clone entirely coding human PRL - 3 gene and construct its recombinant eukaryotic expression vector so as to explore its function and roles in metastasis of the human colorectal carcinoma. Methods With total RNA extracted from the human colorectal carcinoma cell sw480 as template, PRL - 3 gene was amplified by RT - PCR with the designed primers based on the public sequence of GeneBank, and then inserted into pGEM - T Easy vector by TA cloning. Recombinant eukaryotic expression vector pcDNA3 - PRL - 3 was then constructed by subcloning technique and confirmed by restriction enzyme digestion analysis and sequencing. Bioinformatical methods were used to predict and analyze PRL- 3 - interacting proteins, and gene expression patterns of the candidate proteins in human tissues or organs were analyzed by Digital northern blot in NCBI online database. Results The entirely coding human PRL - 3 gene was cloned. Recombinant pcDNA3 - PRL - 3 was successfully constructed. Bioinformafics analyses indicated that PRL - 3 was found as an important bridge between ARL - 3 protein and ARP6 protein. Gene expression patterns of PRL- 3 and its related proteins in human tissues or organs were found similar by use of digital northern blot in online NCBI database. Conclusion Human PRL - 3 gene has been successfully cloned and ARL - 3 protein and ARP6 protein are predicted to be PRL - 3 - interacting proteins, which will be useful for further research on its function as well as its implications in the occurrence and progress of colorectal carcinoma.