研究目的
Investigating the therapeutic effects of single-domain antibodies on classical swine fever virus (CSFV) and their application in rapid identification and research on CSFV and host interactions.
研究成果
The study successfully obtained two sdAb fragments with high specificity and affinity for the E2 protein of CSFV. These sdAbs were effectively used to construct molecular probes for imaging CSFV in cells and capturing the virus and its protein, demonstrating their potential for rapid identification of CSFV and research on virus-host interactions.
研究不足
The study did not explore the neutralizing activity of the selected sdAbs against CSFV, limiting their application in therapeutic contexts. The specificity and affinity of sdAbs were tested against a limited set of viral proteins, potentially overlooking cross-reactivity with other pathogens.
1：Experimental Design and Method Selection:
The study involved the selection and characterization of CSFV-specific single-domain antibodies (sdAbs) from an immunized camel, their expression in E. coli, and conjugation with quantum dots (QD)/AF488 and immunomagnetic nanobeads (IMNBs) for imaging and capturing CSFV virions and recombinant E2 protein.
2：Sample Selection and Data Sources:
Peripheral blood lymphocyte cells (PBLC) from an immunized camel were used to construct a sdAb library. CSFV-infected swine testis (ST) cells and recombinant E2 protein were used for testing.
3：List of Experimental Equipment and Materials:
Equipment included a Biacore 3000 for SPR analysis, a laser scanning confocal microscope for imaging, and various reagents for ELISA, WB, and RT-PCR. Materials included recombinant E2 protein, QD605nm, AF488, and carboxyl-magnetic beads.
4：Experimental Procedures and Operational Workflow:
The workflow included immunization of a camel, library construction, phage display, sdAb expression and purification, conjugation with QD/AF488 and IMNBs, and application in imaging and capturing CSFV.
5：Data Analysis Methods:
Data were analyzed using SPR for binding kinetics, ELISA and WB for specificity and binding activity, and RT-PCR for viral RNA detection.
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