论文部分内容阅读
目的利用杆状病毒表达系统对RSVA型Long株F基因进行表达研究,为RSV重组亚单位疫苗的研制奠定基础。方法用反转录聚合酶链反应(RT-PCR)扩增F基因,将其克隆到pFastBacHT A载体中,阳性重组质粒转化DH10Bac感受态细胞,PCR鉴定获得阳性克隆,碱裂解法提取阳性质粒,转染Sf9昆虫细胞,获得含F基因的重组杆状病毒质粒,重组病毒感染Sf9细胞72h后,进行SDS-PAGE电泳、间接免疫荧光和Western-blot试验。免疫Balb/c小鼠,用ELISA测定抗体滴度,MTT试验检测T淋巴细胞增殖。结果目的蛋白F在昆虫细胞中有特异性表达,能被F蛋白单克隆抗体所识别,该蛋白诱导了高滴度的RSV特异性抗体。结论成功克隆RSVF基因,并在Sf9昆虫细胞中获得表达,该蛋白具有良好的免疫原性,为进一步研究开发RSV重组亚单位疫苗奠定了基础。
OBJECTIVE: To study the expression of F gene in RSVA Long strain by baculovirus expression system, and lay a foundation for the development of RSV recombinant subunit vaccine. Methods F gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), cloned into pFastBacHT A vector, positive recombinant plasmid was transformed into DH10Bac competent cells, positive clones were obtained by PCR, positive plasmids were extracted by alkaline lysis, The Sf9 cells were transfected with Sf9 insect cells to obtain recombinant baculovirus plasmids containing F gene. Sf9 cells were infected with recombinant virus for 72 hours, then subjected to SDS-PAGE, indirect immunofluorescence and Western-blot. Balb / c mice were immunized, antibody titers were determined by ELISA, and T lymphocyte proliferation was measured by the MTT assay. Results The target protein F was specifically expressed in insect cells and was recognized by the F protein monoclonal antibody, which induced a high titer of RSV specific antibody. Conclusion The RSVF gene was successfully cloned and expressed in Sf9 insect cells. This protein has good immunogenicity, which lays the foundation for further research on the development of RSV recombinant subunit vaccine.