猴D型逆转录病毒p27基因的原核表达及流式免疫荧光微球检测技术的建立

发布时间：2018-12-14 20:13

【摘要】：目的构建猴D型逆转录病毒的p27蛋白,并建立流式免疫荧光微球检测技术用于猴D型逆转录病毒的检测。方法通过PCR扩增p27基因片段,与p GEX-4T-1表达载体进行连接,转化至BL21(DE3)进行表达。通过SDS-PAGE分析蛋白表达的形式及最佳诱导时间。采取GST树脂纯化目标蛋白,包被磁珠,建立流式免疫荧光微球检测技术,用于临床样本的检测。结果重组蛋白以可溶的上清液进行表达,诱导的最佳时间为4 h。包被磁珠后,成功建立流式免疫荧光微球检测技术,对阳性血清稀释81倍仍可检测为阳性,对猴类其他病原的阳性血清无交叉反应,特异性强,与ELISA法同时对24份临床样本进行检测,其中3份样品的流式免疫荧光微球检测结果为阳性,ELISA检测结果为2份阳性,1份阴性,两种方法的检测结果符合率为96%。结论成功表达猴D型逆转录病毒p27蛋白,并运用该蛋白建立了灵敏度高,特异性强,样本需求少的流式免疫荧光微球检测技术,为后续推广应用多重流式免疫荧光微球检测技术奠定了基础。
[Abstract]:Objective to construct the p27 protein of monkey type D retrovirus and to establish a flow immunofluorescence microsphere assay for detection of monkey type D retrovirus. Methods p27 gene fragment was amplified by PCR, ligated with p GEX-4T-1 expression vector and transformed into BL21 (DE3) for expression. The expression of protein and the optimal induction time were analyzed by SDS-PAGE. The target protein was purified by GST resin and coated with magnetic beads. Flow immunofluorescence microspheres were used for the detection of clinical samples. Results the recombinant protein was expressed in soluble supernatant for 4 h. After coated with magnetic beads, the flow immunofluorescence microspheres were successfully established. The positive sera diluted 81 times could still be detected as positive, and the positive sera of other pathogens of monkeys had no cross reaction and the specificity was strong. 24 clinical samples were detected simultaneously with ELISA method. The results of flow immunofluorescence microspheres were positive in 3 samples, 2 positive and 1 negative in ELISA. The coincidence rate between the two methods was 96.1%. Conclusion the p27 protein of monkey type D retrovirus was successfully expressed, and a flow immunofluorescence microsphere detection technique with high sensitivity, strong specificity and less sample requirement was established. It lays a foundation for the further application of multiplex immunofluorescence microspheres detection technology.
【作者单位】：广东省实验动物监测所广东省实验动物重点实验室;
【基金】：广东省科技计划项目(编号:2016A040403059,2014A040401035,2014B070706006) 国家科技支撑计划项目(编号:2015BAI07B01)
【分类号】：S852.65

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