乙肝表面抗原和弓形虫棒状体分泌抗原多功能真核表达载体的构建与鉴定  

Construction and identification of multifunctional eukaryotic expression vector of HBsAg and ROP_2

作　　者：魏庆宽[1] 肖婷[1] 李瑾[1] 马丽萍[2] 王林[2] 王英婷[2] 闫运琴[2] 高建丽[2] 朱嵩[1] 仲维霞[1] 尹昆[1] 付斌[1] 张佃波[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,世界卫生组织淋巴丝虫病,绦囊虫病合作中心,济宁272033 [2]济宁市第一人民医院

出　　处：《中国临床新医学》2013年第3期191-195,共5页CHINESE JOURNAL OF NEW CLINICAL MEDICINE

基　　金：山东省自然科学基金资助项目(编号:2009ZRC03083;2009ZRC03050);山东省科技攻关项目(编号:2010GWZ20232);济宁市科技发展计划项目(编号:济科2009-56-21)

摘　　要：目的探讨通过体外扩增乙肝表面抗原(HBsAg)基因,构建真核表达载体pcDNA3-HBsAg-ROP2的可行性。方法根据乙肝S基因序列及pcDNA3-p30-ROP2酶切位点等情况设计、合成引物,应用聚合酶链反应(PCR)技术,扩增HBsAg基因片段;应用回收、纯化、酶切、连接等技术,将HBsAg基因替换pcD-NA3-p30-ROP2中的p30基因,并进行酶切、PCR扩增及测序鉴定。结果 PCR扩增出约0.7 kb的目的基因HBsAg,成功构建pcDNA3-HBsAg-ROP2重组载体。PCR、酶切结果与理论相符,重组体包含了HBsAg和ROP2基因的完整序列。结论利用体外扩增乙肝表面抗原,可成功构建乙肝和弓形虫多功能重组载体pcDNA3-HBsAg-ROP2。Objective To construct eukaryotic expression vector pcDNA3-HBsAg-ROP2 by amplifying HBsAg by PCR.Methods According to the specific sequence of HBsAg and restriction sites of pcDNA3-p30-ROP2,the primers were designed and synthesized.HBsAg was amplified by PCR,then purified HBsAg gene was inserted into vector instead of p30 by enzyme digestion and ligation,and the construct was identified by enzyme digestion,PCR and sequencing.Results 0.7 kb of HBsAg was aquired,and the pcDNA3-HBsAg-ROP2 eukaryotic expression recombinant plasmid was constructed.The results of PCR and digestion showed that HBsAg and ROP2 in the recombinant plasmid contained the complete genes sequences.Conclusion The recombinant plasmid pcDNA3-HBsAg-ROP2 was successfully constructed.

关 键 词：乙肝表面抗原(HBsAg) 弓形虫 棒状体分泌抗原2(ROP2) 真核表达载体pcDNA3 基因重组 

分 类 号：R512.62[医药卫生—内科学]