【摘要】 目的克隆弓形虫组蛋白H3.3(Tg H3.3)的编码基因,以研究其在弓形虫病免疫诊断及免疫预防上的作用。方法以弓形虫总RNA为模板逆转录合成cDNA链,设计合成引物,用PCR法扩增弓形虫组蛋白H3.3基因编码序列,将其克隆入pGEM-T载体,并用双酶切和以质粒为模板的PCR进行鉴定。结果 RT-PCR扩增出一条约411bp大小的特异性条带,重组质粒的双酶切和以质粒为模板的PCR均获得了一条与RT-PCR扩增的大小相同条带,序列测定结果表明其具有411bp的开放阅读框。结论成功构建了弓形虫组蛋白H3.3重组pGEM-T克隆载体,并完成了序列测定,为进一步的研究提供了条件。更多还原

【Abstract】 Objective Cloning of histone3.3 protein gene of Toxoplasma gondii in order to study its immunological diagnosis and prevention. Methods T.gondii eDNA first strand synthesis was driven by su- perscript ⅡRT using worm total RNA as template. Specific primers was designed and synthesized. Coding re- gion gene of Tg H3.3 proteins was amplified by RT-PCR. The product from PCR was cloned into pGEM-T vector. The cloning vector (pCEM-Tg H3.3) was identified by restriction analysis and PCR followed by DNA sequencing. Results For RT-PCR, A specific band of around 411bp was amplificated. The same band was obtained by double restriction of recombinant plasmids or PCR using recombinant plasmids as template. Of the sequence generated, the nucleotide sequence has an open reading frame of 411bp. Conclusion pGEM- Tg H3.3 was successfully constructed and it provided the basis for further study on Tg H3.3 proteins expres- sion and its function.更多还原