摘要
目的构建弓形虫棒状体蛋白18(ROP18)和微线体蛋白2(MIC2)的基因融合的重组真核表达质粒,并在转录和翻译水平进行鉴定。方法利用分子克隆技术构建重组真核表达质粒pBudCE4.1-ROP18-MIC2,经PCR、酶切及测序鉴定正确后,体外转染人包皮成纤维细胞(HFF),采用RT-PCR在48 h时检测转录水平,SDS-PAGE在72 h时检测蛋白表达。结果重组真核表达质粒pBudCE4.1-ROP18-MIC2构建正确,经RT-PCR、SDS-PAGE鉴定,弓形虫ROP18、MIC2可在HFF细胞中瞬时表达。结论成功获得重组真核表达质粒pBudCE4.1-ROP18-MIC2,为进一步研究弓形虫疫苗的免疫保护性奠定基础。
Objective To construct the recombinant eukaryotic plasmid encoding rhoptry protein 18（ROP18） and microneme 8（MIC8） of Toxoplasma gondii and identify it in the transcription and translation level.Methods The recombinant eukaryotic expression plasmid pBudCE4.1-ROP18-MIC2 was constructed by the molecular cloning technology.After being identified by PCR,restriction enzyme cleavage and sequencing,pBudCE4.1-ROP18-MIC2 was transfected into the HFF cells.We then detected the transcription level at 48 h using RT-PCR and examined the protein expression at 72 h by SDS-PAGE.Results Analysis of PCR,restriction enzyme cleavage and sequencing confirmed that the recombinant eukaryotic expression plasmid of pBudCE4.1-ROP18-MIC2 was precisely constructed.The identification of RT-PCR and the appraisal of SDS-PAGE showed that ROP18 and MIC2 of Toxoplasma gondii got the correct transient expression in the HFF cells.Conclusion The successful constrution of the recombinant eukaryotic expression plasmid pBudCE4.1-ROP18-MIC2 has laid foundation for the further development of protective vaccine against Toxoplasma gondii infections.
出处
《山东大学学报（医学版）》
CAS
北大核心
2012年第8期62-67,共6页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81071373)
山东省自然科学基金(ZR2009CM079)
家畜疫病病原生物学国家重点实验室开放基金(SKLVEB2011KFKT005)
山东省优秀中青年科学家科研奖励基金(BS2009SW008)
