摘要
为筛选刚地弓形虫的毒力相关基因,以弓形虫为研究对象,构建了弓形虫强弱毒株抑制消减cDNA文库。分别收集弓形虫Ⅰ型RH株和Ⅱ型QHO株速殖子,提取总RNA,纯化后获得mRNA,并反转录为cDNA。用抑制消减杂交技术(SSH)构建弓形虫强弱毒株正向消减(弱毒株消减强毒株)及反向消减(强毒株消减弱毒株)cDNA文库。从两文库中各筛选10个阳性克隆测序及进行在线BLAST分析。结果显示,构建的弓形虫强弱毒株的消减cDNA文库具有较强的特异性;在获得的18个有效表达序列标签中,有12个与已报道的基因有较高相似性,另外6个可能代表新基因。表明,弓形虫强弱毒株差异表达消减cDNA文库的成功构建,为进一步研究弓形虫毒力相关基因的功能奠定了基础。
The objective of present study was to construct subtractive cDNA libraries for tachyzoites of To.roplasma gondii virulent and avirulent strains, for the identification and cloning of virulence-related genes in T. gondii. The subtractive cDNA libraries for RH strain（type Ⅰ ） and QHO strain（type Ⅱ ） were constructed by the technique of suppression subtractive hybridization（SSH） using the PCR-Select cDNA Subtraction Kit. Ten randomly-selected positive clones from the each library were sequenced. The results showed that the two SSH libraries were highly specific. Twelve of the 18 valid expressed sequence tags （ESTs） had significant similarities with those of other known genes. The other 6 ESTs may represent new genes. The successful construction of the subtractive cDNA libraries for virulent and avirulent T. gondii strains provided a foundation for further studies of genes related to the virulence of T. gondii.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第9期733-737,共5页
Chinese Veterinary Science
基金
国家“十一五”重点基础研究发展计划(973)项目前期研究专项(2007CB116301)
国家公益性行业(农业)科研专项经费项目(200803017)
教育部“长江学者和创新团队发展计划”创新团队项目(IRT0723)
广东省高等学校自然科学研究重点项目(06Z004)
关键词
刚地弓形虫
速殖子
抑制消减杂交
消减cDNA文库
Toxoplasma gondii
tachyzoite
suppression subtractive hybridization
subtractive cDNA library
