弓形虫强毒株和弱毒株致密颗粒蛋白1基因的比较研究

Comparative study of the GRA1 gene of Toxoplasma gondii in virulent and avirulent strains

目的比较弓形虫强毒株RH株、桂弓株和弱毒株B36株致密颗粒蛋白1(GRA1)基因片段的异同。方法分别从接种RH株、桂弓株、B36株弓形虫的小鼠腹水中收集、纯化虫体,提取基因组DNA,PCR扩增3个虫株的致密颗粒蛋白1(GRA1)目的基因片段;构建pMD-18-T/GRA1重组质粒,PCR和双酶切鉴定其准确性;测序并用NCBI中Blast软件和MEGA3.1软件分析比较3种不同毒力株GRA1基因的异同。结果获得3株弓形虫的GRA1目的基因片段并构建了pMD-18-T/GRA1重组质粒,经鉴定目的基因片段大小与预期理论值相符。测序分析3不同毒力株GRA1基因片段相似为100%。结论弓形虫强毒株RH株、桂弓株和弱毒株B36株的GRA1基因片段极其相似,且高度保守。因此认为弓形虫GRA1基因具有较高应用价值。

Objective To compare the GRA1 gene of Toxoplasma gondii in virulent and avirulent stains.Methods T.gondii was collected and purified from ascites of mice infected with the virulent RH and Gui Gong strains and avirulent B36 strain.Genomic DNA was isolated from T.gondii.A specific fragment of the GRA1 gene was specifically amplified from the genomic DNA of different virulent strains of T.gondii using PCR.The pDM-18-T/GRA1 recombinant plasmids of three strains were constructed.They were then identified using PCR and digestion with two types of restriction enzymes.The recombinant plasmids were screened,and DNA sequence analysis was done using the Blast program from NCBI and MEGA3.1.Results Specific fragments of the GRA1 gene were obtained,and exact recombinant plasmids were constructed from three strains of T.gondii.Identified fragments were consistent with those predicted.DNA sequence analysis demonstrated that three specific fragments of the GRA1 gene from RH,Gui Gong,and B36 strains of T.gondii had similarity of 100%.Conclusion Sequences of the GRA1 gene from RH,Gui Gong,and B36 strains of T.gondii are very similar and highly conserved.Results indicated that the GRA1 gene of T.gondii might be of use in the future.