不同毒力株弓形虫速殖子消减cDNA文库的构建

目的构建不同毒力株弓形虫速殖子cDNA消减文库。方法以弓形虫强毒株RH株速殖子为Tester、弱毒株Prugniaud株速殖子为Driver,进行正向抑制性消减杂交;以Prugniaud株为Tester、RH株为Driver,进行反向抑制性消减杂交。分别将获取的2组抑制性消减杂交产物与pGEM-T载体连接,转化大肠杆菌DH5α,筛选阳性克隆并以PCR扩增鉴定插入片段。结果获得正向和反向cDNA消减文库,每个消减文库分别随机挑取384个阳性克隆,PCR扩增显示插入率为98%,片段长度在200~2000bp之间。结论通过抑制性消减杂交技术成功构建2个消减文库,为进一步筛选和鉴定弓形虫毒力相关基因奠定了基础。

Construction of the subtractive cDNA library for tachyzoites of Toxoplasma gondii in different virulent stains

To construct the subtractive cDNA library for tachyzoites of Toxoplasma gondii in different virulent strains,the suppressive subtractive hubridization(SSH)was performed with tachyzoites of the RH strain as tester and those of the Prugniaud strain with low virulence as the driver.On the contrary,the reverse suppressive subtractive hybridization was done with tachyzoites of the Prugniaud strain as tester and those of the RH strain as driver.The products obtained from these two hybridizations were linked to vector pGEM-T and then transformed to E.coli DH5α.The positive clones screened were amplified and the inserted fragment was identified.It was found that both the forward(RH strain)and reverse(Prugniaud strain)subtractive cDNA libraries were obtained in this way,in which 384 positive clones of random recombinants were included in each group.The size of the inserted fragments were within range of 200-2 000 bps.As demonstrated by PCR amplification,the insertion rate was 98%.It is evident that two subtractive cDNA libraries have been established by suppressive subtractive hybridization technique,thus providing the basis for further screening and identification of the Toxoplasma gondii gene related to virulence.