【摘要】 目的构建嗜肺军团菌主要免疫原基因的真核表达重组质粒pcDNA3.1-ip,并观察其在真核细胞中的表达。方法以嗜肺军团菌1型DNA为模板,PCR扩增获得嗜肺军团菌ip基因,将其定向插入载体pcDNA3.1(+),构建真核表达重组质粒pcDNA3.1-ip。经限制性核酸内切酶Hind和BamH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-ip转染NIH3T3细胞,采用免疫荧光法和Western blot分别鉴定pcD-NA3.1-ip的瞬时和稳定表达。结果成功构建了嗜肺军团菌ip基因的真核表达重组质粒。在细胞膜与细胞内观察到较强的绿色荧光,表明pcDNA3.1-ip成功转入NIH3T3细胞,并在NIH3T3细胞中得到了瞬时表达;用嗜肺军团菌兔血清抗体检测pcDNA3.1-ip稳定转染的NIH3T3细胞,在相对分子质量为29×103处检测到阳性杂交信号,表明pcDNA3.1-ip在细胞内可稳定表达IP蛋白。结论本研究构建的嗜肺军团菌主要免疫原基因真核表达质粒pcDNA3.1-ip能够成功表达IP蛋白,表达的蛋白具有良好的免疫原性与免疫反应性。更多还原

【Abstract】 Objective To construct the recombinant plasmid of Lgeionella pneumophila immunogenic protein gene (ip) and detect the immunoprotein expression in NIH3T3 cells. Methods The immunogenic protein gene was amplified from DNA of Legionella pneumophila by polymerase chain reaction (PCR), then inserted into pcDNA3.1(+) vector. The recombinant plasmid was named as pcDNA3.1-ip and analyzed with restriction-endonuclease Hind Ⅲ and BamHⅠ digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-ip with lipofection strategy. The transient and stable expression products of immunogenic protein gene were detected by immunofluorescence and Western blot. Results We have successfully constructed the recombinant plasmid of Lgeionella pneumophila immunogenic protein gene. It was found that there was dark green fluorescence on the cell membrane and inside the cell. Our results showed that NIH3T3 cell was transfected by pcDNA3.1-ip successfully. The rabbit serum antibody of Lgeionella pneumophila detected the NIH3T3 cell transfected stably by pcDNA3.1-ip to express the immunogenic protein with the relative molecular mass 29×103. Conculsion We have successfully expressed immunogenic protein of Lgeionella pneumophila. The expressed product showed the good immunogenicity and immunoreactivity.更多还原