弓形虫不同分离株ROP1和P30基因的体外扩增

通过体外扩增弓形虫ＲＨ、ＺＳ１、ＺＳ２、ＧＴ１４个分离株的编码棒状体蛋白（ＲＯＰ１）和主要表膜蛋白（Ｐ３０）抗原的基因片段，比较虫株间差异，为克隆及其ＤＮＡ免疫的研究做准备。特定引物的设计；弓形虫虫株的复苏、接种、收集、纯化；提取弓形虫ＲＨ、ＺＳ１、ＺＳ２及ＧＴ１分离株的基因组ＤＮＡ，并以此为模板进行ＰＣＲ扩增，扩增产物经１％琼脂糖凝胶电泳分析。结果从４个分离株基因组ＤＮＡ中均扩增出编码ＲＯＰ１、Ｐ３０抗原的基因片段，其大小ＲＯＰ１约７５６ｂｐ，Ｐ３０约１０２５ｂｐ，电泳条带未见虫株间的明显差别。研究表明，所扩增４个弓形虫分离株的ＲＯＰ１和Ｐ３０基因片段均与理论预测值相符，并具有高度保守性。

IN VITRO AMPLIFICATION OF ROP1 AND P30 GENES FROMDIFFERENT ISOLATES OF TOXOPLASMA GONDII

In order to make preparation for cloning and immunological study of DNA immunity with ROP1, P30 genes, we amplified the genes of coding rhoptry protein 1 (ROP1) and major surface protein 1 ( SAG1 or P30) from the genomic DNA groups of 4 different isolates of Toxoplasma gondii, RH, ZS1, ZS2 and GT1; compared the similarities and differences between the genes and the isolates. These included designing the specific primers, recovering, inoculating, harvesting and purifying the T. gondii isolates, extracting genomic DNA from tachyzoites of those isolates as templates, amplifying ROP1 and P30 gene fragments by means of polymerase chain reaction (PCR) and identifying the amplification products with 1% agarose gel electrophoresis. As a result, both ROP1 and P30 genes were amplified out from the genomic DNA of those four T. gondii isolates and the amplified fragments size of ROP1 and P30 were about 756 bp, 1 025 bp respectively. No distinct gene differences were observed among those parasitic isolates. The sizes of the amplified gene fragments were in accordance with the calculated ones. The present study indicated that ROP1 and P30 genes among the detected T. gondii isolates were highly conservative.