弓形虫致密颗粒蛋白14基因的克隆及序列分析

Cloning and sequence analysis of GRA14 genes of Toxoplasma gondii isolates from different hosts and geographical locations

为研究弓形虫致密颗粒蛋白14(GRA14)基因的遗传变异规律,以来自不同宿主及地理来源的9株弓形虫DNA为模板,分别用PCR扩增了其GRA14基因,并将PCR产物克隆到pMD18-T载体后测序。利用生物信息学软件分析了GRA14基因的遗传变异并构建了系统发育树,分析了GRA14蛋白的结构并结合抗原指数等参数预测了其抗原表位。结果表明,9个弓形虫虫株的GRA14基因长度均为1 227bp,序列的A+T含量在46.94%～47.43%之间,与弓形虫ME49株相应序列的核苷酸变异率在0.08%～0.73%之间;系统发育树分析显示,GRA14仅能将基因Ⅰ型虫株聚在一个分支上,但其他基因型虫株的聚类没有规律。蛋白二级结构和抗原表位预测结果表明,GRA14蛋白经过3次跨膜,主要由6个α-螺旋、3个β折叠、11个β转角和若干个无规则卷曲构成,并预测有4个B细胞抗原表位。表明GRA14蛋白可以作为抗弓形虫病疫苗候选分子研制弓形虫病亚单位疫苗及表位疫苗。

Toxoplasma gondii GRA 14 gene encoding dense granule protein 14 was amplified from 9 T. gondii strains from different geographical locations and hosts by PCR. The 9 PCR products were cloned into the pMD18-T vector and then transformed into Escherichia coli JM109, respectively. After sequen- cing,the relationships of 9 T. gondii strains were analyzed by bioinformatics software and a phylogenetic tree was constructed by Phylip 3.67. The B cell epitopes were predicted by DNAStar software. The length of the complete GRA14 sequences from the 9 T. gondii strains was 1 227 bp,and the A＋T contents varied between 46.94% and 47.43%. GRA14 sequences were conserved among the T. gondii strains,and the vari- ation compared with the ME49 strain was up to 0.73 %. Phylogenetic analysis revealed that GRA14 gene sequence was not a suitable marker for studying genetic relationships of T. gondii isolates. However, the low variation among GRA14 gene sequences indicates it can be a vaccine candidate against toxoplasmosis. The B cell epitopes were most likely to form at 54--62aa,320--360aa,379--385aa and 400--408aa in GRA14protein. The results of the present study have implications for further development of molecular vaccines against T. gondii infection.