动物造模,猴D型逆转录病毒 z-bio 2022-11-08 504

　　OBJECTIVE: To construct the p27 protein of simian D-type retrovirus, and establish a flow immunofluorescence microsphere detection technology for the detection of simian D-type retrovirus.

　　Methods: The p27 gene fragment was amplified by PCR, ligated with pGEX-4T-1 expression vector, and transformed into BL21 (DE3) for expression. The form of protein expression and the optimal induction time were analyzed by SDS-PAGE. The target protein was purified by GST resin, coated with magnetic beads, and a flow-type immunofluorescence microsphere detection technology was established for the detection of clinical samples.

　　RESULTS: The recombinant protein was expressed in soluble supernatant, and the optimal induction time was 4 h. After coating the magnetic beads, the flow-type immunofluorescence microsphere detection technology was successfully established. The positive serum can still be detected as positive when diluted 81 times. It has no cross-reaction with the positive serum of other monkey pathogens, and has strong specificity. Twenty-four clinical samples were tested, of which 3 samples were positive for flow immunofluorescence microspheres, 2 were positive for ELISA, and 1 was negative. The coincidence rate of the two methods was 96%.

　　Conclusion: The monkey D-type retrovirus p27 protein was successfully expressed, and the protein was used to establish a flow-based immunofluorescence microsphere detection technology with high sensitivity, strong specificity, and less sample requirements, which was used for the subsequent promotion and application of multiple flow-based immunofluorescence microspheres. Detection technology laid the foundation.