流行于我国的优势基因型弓形虫株毒力及其棒状体蛋白ROP5和ROP18蛋白序列差异分析

The virulence and putative amino acids difference of the main ROPs virulence factor proteins in the dominant genotype of Toxoplasma gondii in China

目的分析流行于我国的优势基因型弓形虫株(WH3株和WH6株)毒力差异以及棒状体蛋白ROP5和ROP18蛋白序列差异,为研究其致病机制奠定基础。方法使用弓形虫WH3、WH6、RH和PRU虫株分别感染SPF级BALB/c小鼠和昆明鼠,观察小鼠存活时间和存活率,分析毒力强弱。同时PCR扩增4株弓形虫毒力相关效应分子棒状体蛋白ROP5和ROP18基因片段,核苷酸测序后将其转换为氨基酸序列,在蛋白水平上比较其序列差异。结果此4株弓形虫株中,WH3和RH株对BALB/c小鼠和昆明鼠均有较强的毒性,两种小鼠分别在感染后5~7 d和9~12 d均100%死亡;而WH6和PRU株对BALB/c小鼠和昆明鼠的毒性均较弱,两种小鼠在感染后45 d均100%存活,且在脑内检出包囊。棒状体蛋白家族毒力相关分子ROP5和ROP18的氨基酸序列分析发现,Chinese 1型虫株WH3和Wh6与PRU型虫株(Ⅱ型虫株)差异较小,而与毒力较强的RH型虫株(Ⅰ型虫株)差异较大。结论我国流行优势基因型弓形虫株的毒力相关分子ROP5和ROP18,与传统Ⅰ型、Ⅱ型弓形虫株存在差异,为我国弓形虫病的防治提供理论基础。

Objective To analyze the putative amino acids sequence difference of the virulence factor proteins of the dominant genotype of Toxoplasma gondii in China.Methods Forty SPF BALB/c mice and forty Kunming mice were infected with WH3,WH6,RH and PRU strains of Toxoplasma gondii respectively,which were preserved in our laboratory.The virulence of Toxoplasma gondii strains were analyzed using the measurements of survival time and survival rate.ROP5 and ROP18,the virulence-related gene fragments,were amplified by PCR method.After DNA sequencing of the obtained DNA fragments,their nucleotide sequences were translated into putative amino acid sequences to compare the protein sequence differences of four Toxoplasma gondii strains.Results Among these four strains of Toxoplasma gondii,WH3 and RH strains exhibited strong virulence,leading to 100%animal death of BALB/c mice and Kunming mice within 5~7 days and 9~12 days after infection.However,the virulence of WH6 and PRU strains were weak,all animals of both BALB/c mice and Kunming mice survived 100%within 45 days after infection with the detected cysts in mice brains.The amino acid sequence analysis of ROP5 and ROP18 revealed that there were little difference between WH3(Chinese 1)and WH6(Chinese 1)with PRU strain(typeⅡ),whereas much more difference were found between WH3(Chinese 1)and WH6(Chinese 1)with RH strain(typeⅠ).Conclusion There are some difference in virulence-associated-molecule genes of ROP 5 and ROP18 among Chinese 1 strain and the traditional typeⅠ,typeⅡstrains in Toxoplasma gondii.These results may contribute to the basic theory research on toxoplasmosis in china.