华支睾吸虫Rap2B样基因的生物信息学分析及其克隆、表达

目的从华支睾吸虫cDNA文库中筛选Rap2B样基因并分析其结构和功能,初步进行克隆和表达纯化,为进一步研究其功能奠定基础。方法利用多种生物信息学分析软件,分析华支睾吸虫Rap2B蛋白的拓扑学结构、生物学和免疫学功能特征。设计引物,从华支睾cDNA质粒文库中扩增目的基因cDNA序列,构建原核重组质粒并初步表达纯化。结果华支睾吸虫Rap2B样基因长度为847bp,其全长编码序列为426bp,编码141个氨基酸,理论分子量是15851.1。重组的原核表达质粒pET-28a(+)-Rap2B经PCR、双酶切及DNA测序结果均表明重组质粒构建成功。该基因在大肠杆菌中能高效表达。经His亲和层析柱获得了纯化的重组蛋白,Western blotting证实Rap2B重组蛋白能被感染华支睾吸虫的大鼠血清识别。结论华支睾吸虫Rap2B蛋白经生物信息学预测为ras原癌基因家族成员,可能在华支睾吸虫致癌过程中有一定作用。该基因可在原核表达系统中高效表达,并得到了纯化的重组蛋白,为进一步研究该蛋白的功能以及在致病尤其是可能促发肿瘤的作用方面奠定一定基础。

Bioinformatic analysis on the Rap2B-like gene from Clonorchis sinnensis and its cloning and expression

To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein. The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares. Using the full-length cDNA plasmid clone （Noc009e03） as template , the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a（＋） and then expressed in E. coli BL21 through IPIG induction. The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting. It was demonstrated that the size of this gene was 847 bp in length, encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons. As demonstrated by PCR, double enzyme digestion and DNA sequencing, the recombinant expression plasmid pET-28a（＋）-Rap2B was successively constructed and the Rap2B like gene was highly expressed in E. coli BL21. The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C. sinensis. Through these investigations, the Rap2B protein may be predicted as a member of the Ras oneogene family protein and is potential in the carcinogenic process of C. sinensis.