弓形虫虎源分离株ROP10基因的克隆、序列分析及其表达研究

CLONING,SEQUENCE ANALYSIS AND EXPRESSION OF ROP10 GENE OF TIGER'S TOXOPLASMA GONDII WILD STRAIN

采用RT-PCR技术从弓形虫虎源分离株中扩增出ROP10基因,将其克隆入pMD18-T载体中进行测序和生物信息学分析,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。该基因全长1761bp,编码586个氨基酸,其中前28个氨基酸残基构成信号肽序列。与GenBank中报道的RH株相比,16个核苷酸存在变异,导致7个氨基酸发生改变,但两者N-联糖基化位点的数量和位置没有差异,两虫株核苷酸和推导氨基酸序列的同源性分别为99.2%和98.8%。转化重组质粒pETROP10的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出分子量为67.6 kDa的重组蛋白,表达量占菌体蛋白的13.8%。

The ROP10 gene of tiger＇s Toxoplasma gondii was amplified by RT-PCR and sequenced by cloning into pMD18-T. Then the target gene was subcloned into pET28a for expression. The full length of ROP10 gene was 1 761 bp, encoding 586 amino acids. The initiative 28 amino acids consist of signal peptide. Compared with RH strain reported in GenBank, there were 16 sitevariations in the nucleotide sequence, which result in the changes of 7 amino acids, but without the difference in number and site for N-glycosylation. The identities of nucleotide sequence and deduced amino acid sequences between RH and the wild strain were 99.2% and 98.8%, respectively. The E. coli strain BL21 （DE3） transformed by pETROP10 can express the recombinant ROP10 protein with a molecular mass of 67.6 kDa, which amounts to 13.8% in the total protein of the induced bacteria.