pcDNA3-HBsAg-p30-ROP2真核表达载体的构建与鉴定  

Construction and identification of pcDNA3-HBsAg-p30-ROP2 expression vector

作　　者：魏庆宽[1,2] 王英婷[3] 闫运琴[3] 肖婷[1] 李瑾[1] 徐超[1] 刘功振[1] 刘娟美 仲维霞[1] 尹昆[1] 付斌[1] 闫歌[1] 黄炳成 

机构地区：[1]山东省医学科学院山东省寄生虫病防治研究所山东省医学科学院寄生虫病分子免疫学重点实验室,济宁272033 [2]济南大学山东省医学科学院医学与生命科学学院 [3]山东省济宁市第一人民医院 [4]山东省戴庄医院

出　　处：《中国血吸虫病防治杂志》2014年第1期46-50,共5页Chinese Journal of Schistosomiasis Control

基　　金：山东省自然科学基金(2009ZRC03083、2009ZRC03050);山东省医药卫生科技发展计划项目(2011HW049);山东省济宁市科技计划项目(2013jnwk51);山东省医学科学院科技计划(2013-01)

摘　　要：目的构建pcDNA3-HBsAg-p30-ROP2多基因重组表达载体,并对其进行初步鉴定。方法根据重组体pcDNA3-p30-ROP2酶切位点和乙型肝炎表面抗原(HBsAg)基因序列等因素设计合成引物,扩增HBsAg目的基因片段,再应用酶切、连接等分子生物学技术将HBsAg目的基因克隆至pcDNA3-p30-ROP2表达载体中。应用聚合酶链反应(PCR)初筛,再采用酶切、测序等技术对构建的重组表达载体pcDNA3-HBsAg-p30-ROP2进行鉴定。结果 PCR扩增出HBsAg基因片段,构建了pcDNA3-HBsAg-p30-ROP2多基因真核表达载体。PCR与酶切结果显示,该基因片段大小均与理论值相符;测序结果显示该重组表达载体包含了p30-ROP2和HBsAg目的基因的完整序列。结论成功构建了多基因重组表达载体pcDNA3-HBsAg-p30-ROP2,为进一步研究多基因核酸疫苗奠定了基础。Objective To construct a multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector and identify it preliminarily. Methods According to recombinant pcDNA3-p30-ROP2 restriction sites, HBV HBsAg gene sequences of primers were designed and synthesized to amplify target fragment, and then cloned into pcDNA3-HbsAg-p30-ROP2 expression vector. Af ter sequencing, it was identified finally by restriction enzyme digestion and other molecular biology techniques. Results HBV HBsAg gene segment was amplified by PCR and the muhi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector was constructed and identified to be correct as theoretical values. The PCR and restriction enzyme digestion results showed that HBsAg and p30-ROP2 gene in recombinant plasmid were confirmed by DNA sequencing. Conclusion The muhi-gene recombinant pcD- NA3-HBsAg-p30-ROP2 expression vector is successfully constructed.

关 键 词：弓形虫 表面抗原1(p30) 棒状体分泌抗原2(ROP2) 乙肝表面抗原 基因重组 

分 类 号：R382.5[医药卫生—医学寄生虫学]