奈瑟氏淋球菌外膜蛋白PIA基因的克隆、真核表达与鉴定  

Cloning,Expression,and Identification of the Major Outer Membrane Protein PIA of Neisseria gonorrhoeae in HELA Cell

作　　者：熊新华[1] 王华洪[2] 

机构地区：[1]湖南省华容县疾病预防控制中心,湖南华容414200 [2]湖南省岳阳市疾病预防控制中心

出　　处：《实用预防医学》2010年第3期581-583,共3页Practical Preventive Medicine

摘　　要：目的克隆奈瑟氏淋球菌外膜蛋白PIA基因,构建稳定的真核重组表达载体,并在HELA细胞中表达。方法根据淋球菌PIA基因序列,利用Primer Premier5.0生物学软件设计一对特异性扩增引物,以淋球菌国际标准株基因组DNA为模板,PCR扩增除掉信号肽后PIA基因开放读码框(ORF),将其插入真核表达载体pcDNA3.1(+),构建含目的基因的pcDNA3.1(+)/PIA重组质粒,PCR及酶切鉴定重组载体,脂质体转染HELA细胞,以间接免疫荧光法检测PIA基因在HELA细胞中的表达。结果成功构建了pcDNA3.1(+)/PIA重组质粒;pcDNA3.1(+)/PIA能在HELA细胞中高效表达PIA蛋白。结论重组质粒pcDNA3.1(+)/PIA构建成功并在HELA细胞中高效表达了PIA蛋白,为该蛋白的抗原性及功能学研究奠定了基础。Objective To clone the major membrane protein PIA gene of Neisseria gonorrhoeae and construct the eukaryotic expression vector expressed in HELA cell. Methods According to the sequence of PIA,design a pair of specific amplification primers by Primer Premier 5.0 Biology Software and amplify the ORF of PIA by PCR after removing the signal peptide. Clone the product of PCR to the pcDNA3.1（＋）eukaryotic vector,after identifying the recombinant eukaryotic plasmid by PCR and enzymes analysis,transfect the expression plasmid to the HELA cell by liposome and detect the expression of PIA in the cell by indirect immunofluorescence assay. Results The recombinant plasmid of pcDNA3.1（＋）/PIA was constructed successfully and the pcDNA3.1（＋）/PIA eukaryotic plasmid could highly express in HELA cell. Conclusions The recombinant plasmid of pcDNA3.1（＋）/PIA could highly express in HELA cell and it will be benefit to the function research of the PIA protein.

关 键 词：奈瑟氏淋球菌 PIA pcDNA3.1(+)/PIA 表达 

分 类 号：R378.16[医药卫生—病原生物学]