中国比较医学杂志2017，Vol.27Issue(9)：17-23,7.DOI:10.3969.j.issn.1671-7856.2017.09.004
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
Prokaryotic expression of p27 gene of simian type?D retrovirus and establishment of flow microsphere immunofluorescence assay
摘要
Abstract
Objective The p27 gene of the simian type?D retrovirus was prokaryotically expressed to establish the flow microsphere immunofluorescence assay for detection of simian type?D retrovirus in nonhuman primates. Methods The gene of p27 was amplified by PCR and linked to the pGEX?4T?1 expression vector digested with the restriction enzymes of EcoR I and Xho I, and the recombinant pGEX?4T?1?p27 plasmid was transfected into the BL21 (DE3) cells for expression of target protein. The form of expressed protein and the optimal time for induction were analyzed by SDS?PAGE. The target protein was purified with GST resin and coupled with magnetic beads to establish the flow microsphere immunofluorescence assay for detection of simian type?D retrovirus in clinical specimens. Results The recombinant protein was expressed in the soluble supernatant, and the optimal time for induction was 4 h. After coupling the protein with the magnetic beads, the flow microsphere immunofluorescence assay was successfully established. Using this method, 81?fold diluted serum could still be detected as positive, and no cross?reaction was found in the positive serum of other pathogens of nonhuman prima?tes, indicating the strong specificity of this method. A total of 24 clinical specimens were tested by this flow microsphere im?munofluorescence assay as well as ELISA. Among all the 24 specimens, 3 samples were positive detected by the flow mi?crosphere immunofluorescence assay, of which 2 were positive and 1 was negative by ELISA. The coincidence rate of these two methods was 96％. Conclusions The prokaryotic expression of p27 protein of the simian type?D retrovirus is success?ful and the flow microsphere immunofluorescence assay with high sensitivity, strong specificity and tiny samples needed is established, which lays the foundation for further application of the multi?flow microsphere immunofluorescence technique.关键词
猴D型逆转录病毒/p27基因/原核表达/流式免疫荧光微球检测技术Key words
Simian type⁃D retrovirus/p27 gene/Prokaryotic expression/Flow microsphere immunofluorescence assay分类
医药卫生引用本文复制引用
刘助红,王静,李秀珍,尹雪琴,张钰,郭鹏举,黄韧..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[J].中国比较医学杂志,2017,27(9):17-23,7.基金项目
广东省科技计划项目(编号:2016A040403059, 2014A040401035, 2014B070706006 ) （编号:2016A040403059, 2014A040401035, 2014B070706006 ）
国家科技支撑计划项目(编号:2015BAI07B01). （编号:2015BAI07B01）
