摘要
目的 体外扩增弓形虫RH株致密颗粒蛋白GRA1基因 ,并构建真核表达质粒。方法 从接种了弓形虫RH株的小鼠腹水中收集、纯化速殖子 ,提取基因组DNA ;据已知的GRA1基因列 ,设计合成一对引物 ,并引入EcoRⅠ和BamHⅠ酶切位点。应用PCR技术 ,从弓形虫RH株基因组DNA中扩增GRA1基因片段。扩增目的基因片段经纯化、双酶切后 ,插入真核表达质粒pEGFP -N3中 ,转化大肠杆菌DH5α感受态细胞 ,于卡那霉素阳性LB平板上筛选阳性克隆。重组子经EcoRⅠ和BamHⅠ双酶切、PCR鉴定。结果 从弓形虫RH株基因组DNA中扩增出 785bp的GRA1基因片段 ,构建重组质粒pEGFPN3-GRA1,酶切和PCR鉴定产物大小均与预期值相符。 结论 成功地从弓形虫RH株基因组DNA中获取了GRA1基因 ,并构建了pEGFPN3-GRA1重组质粒。
Aim Amplification of the GRA1gene from Toxoplasma gondii RH strain,and construction of recombinant eukaryotic expression plasmid of pEGFP N3-GRA1 Methods The tachyzoites had been harvested from ascites of mice infected by inoculating RH strain through the intraperitonea,purifying tachyzoites and preparing genomic DNA According to the cDNA sequence of GRA1 gene,a pair of primers were designed and synthesized Using PCR technique,a specific fragment of GRA1 gene was obtained by amplification from the genomic DNA of tachyzoites After having been digested by EcoRⅠ/BamHⅠ,the fragments of PCR products were cloned into a high level expression vector pEGFP N3,then transferred into Escherichia coli(E coli) DH5α,a recombinant pEGFPN3-GRA1 was constructed,it was identified by PCR and EcoRⅠ/BamHⅠdigesting methods Results The size of the amplification GRA1 gene fragment was in accord with the expected one matelly The recombinant pEGFP N3-GRA1 was successfully constructed Conclusions The gene encoding GRA1 was amplified from the DNA of RH strain of Toxoplasma gondii by PCR,and the recombinant plasmid PEGFPN3-GRA1 is constructed The expression of the GRA1 gene of Toxoplasma gondii RH strain will be further investigated
出处
《中国人兽共患病杂志》
CSCD
北大核心
2002年第3期17-19,9,共4页
Chinese Journal of Zoonoses
基金
美国中华医学基金会 (项目号 93 -5 94)
"2 11"重点学科建设课题项目基金资助
