论文部分内容阅读
目的建立弓形虫速殖子与缓殖子体外转化体系。方法纯化弓形虫RH株速殖子,按一定比例和条件接种于COS-7细胞内进行体外培养。分别在接种后第1、2、3、4、5、6天观察细胞和虫体的形态并提取TotalRNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAG1基因和SAG2C基因的表达。结果随着速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓;虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。RT-PCR检测显示:速殖子期特异性蛋白SAG1基因在培养的第1～6天均有表达,且表达量呈递增趋势;缓殖子期特异性蛋白BAG1基因从第2天开始出现表达,随时间延续表达量逐渐增加;缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。以上结果表明有越来越多的速殖子转化为缓殖子。改变培养条件,缓殖子也能向速殖子转化。结论成功构建弓形虫速殖子与缓殖子体外相互转化体系,为其转化机制的研究奠定了基础。
Objective To establish the in vitro transformation system of tachyzoites and bradyzoites in Toxoplasma gondii. Methods Toxoplasma gondii RH strain tachyzoites were purified and inoculated into COS-7 cells under certain conditions and conditions for in vitro culture. The morphology of cells and parasites were observed on the 1st, 2nd, 3rd, 4th, 5th and 6th day after inoculation, Total RNA was extracted and the tachyzoite specific protein SAG1 was detected by RT-PCR. Sexin BAG1 gene and SAG2C gene expression. Results As tachyzoites cultured in COS-7 cells continued for a long time, the number of parasites gradually increased, but the rate of proliferation slowed down gradually. The arrangement of parasites in the cells was also changed continuously. The paired arches, Rose-shaped or clustered, into a semicircular shape of the final round-like cyst-like structure. The results of RT-PCR showed that the tachyzoite specific protein SAG1 was expressed on the 1st to 6th day of culture, and the expression increased gradually. The gene of bradyzoite specific protein BAG1 began to express on the 2nd day , And the expression gradually increased with time. The gene expression of the mitochondrial-specific gene SAG2C began to express on the 5th day and increased on the 6th day. The above results indicate that more and more tachyzoites are converted to bradyzoites. Changing culture conditions, bradyzoites can also transform tachyzoites. Conclusion The in vitro transformation system between Toxoplasma gondii and bradyzoites is successfully constructed, which lays the foundation for its transformation mechanism.