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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。
In order to study the genetic variation of GRA14 gene, 9 strains of Toxoplasma gondii DNA from different host and geographic origin were used as templates. The GRA14 gene was amplified by PCR and cloned into pMD18 -T vector after sequencing. The genetic variation of GRA14 gene was analyzed by bioinformatics software and phylogenetic tree was constructed. The structure of GRA14 protein was analyzed and the antigen epitope was predicted by the combination of antigen index and other parameters. The results showed that the length of GRA14 gene of 9 Toxoplasma gondii strains was 1 227 bp, the A + T content was between 46.94% -47.43%, and the nucleotide variation rate of the corresponding sequence of ME49 strain was 0.08% 0.73%. Phylogenetic tree analysis showed that GRA14 could only cluster genotype Ⅰ strains on one branch, but there was no regular clustering in other genotypes. Prediction of protein secondary structure and antigenic epitopes showed that GRA14 protein is composed of three transmembranes, consisting of six α-helices, three β-sheets, eleven β-turns and several random curls, and four B cell epitopes. The results showed that GRA14 protein could be used as a vaccine against Toxoplasma gondii to develop the toxoplasmosis subunit vaccine and epitope vaccine.