嗜肺军团菌pilE基因体外表达及其免疫原性研究

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429 bp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.

In vitro expression of Lgeionella pneumophila pilE gene and its immunogenicity

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 pcDNA3.1-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 of pcDNA3.1-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)-gamma 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 cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01). CONCLUSION: The recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.