弓形虫HT分离株ROP5蛋白基因的克隆、序列分析及其表达研究

Cloning,Sequence Analysis and Expression of ROP5 of Toxophasma gondii Strain HT

目的克隆和表达弓形虫虎源分离株(HT株)ROP5蛋白基因。方法运用RT-PCR技术从弓形虫HT株中扩增出ROP5基因,将其克隆入T载体中进行测序和分析,并将目的基因亚克隆到大肠杆菌表达载体pET28a中进行诱导表达。结果该基因全长1650bp,编码549个氨基酸。其中前24个氨基酸构成信号肽序列。与GenBank中报道的RH株相比,有12个核苷酸有差异,导致7个氨基酸发生改变,两者核苷酸和推导氨基酸序列的同源性分别为99.2%和98.9%。转化重组质粒pETROP5的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出相对分子质量为64800的重组蛋白,并且能与弓形虫抗体发生血清学反应,表达量占菌体蛋白的15.6%。结论成功克隆和表达了弓形虫HT株ROP5蛋白基因,表达的重组蛋白具有良好的反应原性。

Objective To clone and express rhoptry protein 5 （ROP5） gene of tiger＇s Toxophasma gondii strain HT. Methods ROP5 gene was amplified by PCR and cloned into pMD18-T for sequencing. Then the interesting gene was subcloned into pET28a for expression. Results The full length of ROP5 gene was 1 650 bp, encoding 549 amino acids. The N-terminal 1-24 amino acids consist of the signal peptide. ,, Compared with the RH strain reported in GenBank, there were 12 site-variations in the nucleotide sequence, which result in the changes of 5 amino acids. The identities of nucleotide sequence and deduced amino acid sequences between RH and wild strains were 99.2% and 98.9%, respectively. The E. coli strain BL21 （DE3） transformed by pETROP5 can express a recombinant protein with a molecular weight of 64.8 kDa, which amounts to 15.6% in the total protein of the induced bacteria. Conclusion The ROP5 gene of Toxophasma gondii strain HT was successfully cloned and expressed in this study, and recombinant protein is antigenic.