嗜肺军团菌pilE基因体外表达及其免疫原性研究  

In vitro expression of Lgeionella pneumophila pilE gene and its immunogenicity

作　　者：杨志伟[1] 陈建平[1] 王涛[1] 田玉[1] 刘德松[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫学教研室,四川成都610041

出　　处：《南方医科大学学报》2007年第2期141-145,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(303000302);教育部博士学科点专项科研基金(20060610091)~~

摘　　要：目的构建嗜肺军团菌Ⅳ型菌毛pilE基因的真核表达重组质粒pcDNA3.1-pilE,研究其真核细胞中的表达及其免疫原性。方法以嗜肺军团菌1型DNA为模板,PCR扩增获得嗜肺军团菌pilE基因,将其定向插入载体pcDNA3.1(+),构建真核表达重组质粒pcDNA3.1--pilE。经限制性核酸内切酶酶切鉴定、PCR和核酸序列分析后,用脂质体法将重组质粒pcDNA3.1-pilE转染NIH3T3细胞,采用免疫荧光法和Western-blot分别鉴定pcDNA3.1-pilE的瞬时和稳定表达产物。将pcDNA3.1-pilE作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFN-γ产生水平、CTL杀伤活性等指标,评价疫苗的免疫原性。结果扩增出了429bp的PilE基因,在细胞膜与细胞内观察到较强的绿色荧光,在相对分子质量15000处检测到阳性杂交信号,表明pcDNA3.1-pilE在细胞内表达PilE蛋白。pcDNA3.1-pilE免疫组的免疫原性均高于对照组pcDNA3.1(+)组(P<0.01)。结论成功构建了嗜肺军团菌pilE基因的真核表达重组质粒,并在NIH3T3细胞中得到了表达,免疫小鼠后诱导了特异的体液免疫应答和细胞免疫应答。Objective To construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity. Methods PilE gene （LP） was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1 （＋） vector to construct the recombinant plasmid pcDNA 3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pile gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity ofpcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon （IFN）-γ production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated. Results The pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected ceils with Western blotting, suggesting successful protein expression of pile gene. pcDNA 3.1-pile resulted in much slxonger immune response in the immunized mice than pcDNA3.1 （＋） （P〈0.01）. Coneulsion The recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 ceils, and can induce specific humoral and cellular immune responses in mice.

关 键 词：嗜肺军团菌 Ⅳ型菌毛蛋白 免疫原性 体外表达 

分 类 号：R378[医药卫生—病原生物学]