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

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，将其克隆人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。

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

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.