Abstract:

【Objective】 The gosling gout disease caused by the novel goose astrovirus (nGAstV) has brought significant economic losses to the goose industry. In this study, a nanobody phage display library for nGAstV was constructed to obtain the specific nanobodies (Nbs) that recognize the ORF2 protein of nGAstV, which could pave a way for the establishment of antibody-based detection methods and the study on the structure and function of nGAstV ORF2 protein. 【Method】 The proliferated nGAstV in LMH cells was purified by sucrose gradient centrifugation. nGAstV was identified by RT-PCR and the virus titer was determined by cytopathic effect. The two-year-old alpacas were immunized with purified nGAstV inactivated by 0.6% formaldehyde solution. For the first immunization, inactivated nGAstV was emulsified with an equal volume of Freund's complete adjuvant. For the second to fifth immunization, inactivated nGAstV was emulsified with an equal volume of Freund's incomplete adjuvant. The immunization was performed every two weeks with a dose of 50 μg. And the titer of IgG against nGAstV in alpaca serum collected at 14 days post the fifth immunization was determined by indirect ELISA. When the IgG titer reached the standard for constructing a library, the alpaca peripheral blood lymphocytes (PBL) were isolated. The total RNA of PBL was extracted and reverse-transcribed into cDNA. The variable region gene of the heavy chain was amplified by nested PCR. It was constructed into pComb phage vector and combined with phage display technology to construct nGAstV Nb phage display library. The capacity of the library was calculated and its diversity was analyzed. nGAstV was used as a target antigen for three rounds of enrichment and panning to obtain recombinant phage Nb positive clones. The positive clones were then cloned into pcDNA3.1-Fc eukaryotic expression vectors followed by sequencing analysis. The plasmids with different sequences were transfected into HEK-293F cells, and the expression level was identified by SDS-PAGE. The nGAstV was used as a target antigen to test the specificity and binding activity of the expressed Nb by ELISA and Western blot. The affinity of Nb was verified using indirect ELISA using nGAstV ORF2 protein as the target antigen and to screen Nbs with better biological activity. 【Result】 The results of RT-PCR showed that nGAstV was ready to be proliferated in LMH cells. The titer of nGAstV virus was 4.38 Log₁₀ TCID₅₀/mL by calculated by the Reed-Muench method. The titer of nGAstV antibodies in alpaca serum reached over 1:64 000 after five immunizations. The VHH gene was amplified by nested PCR and a phage display library of nGAstV Nb with a library capacity of 3.8×10⁸ CFU/mL was successfully constructed. Phylogenetic tree analysis showed that the phage Nb library had an excellent diversity. 39 phage positive clones were acquired, which reacted with nGAstV post three rounds of enrichment and panning, including 25 Nbs with different sequences. The results of SDS-PAGE identification showed that a total of 10 Nbs were successfully expressed. Among them, 8 Nbs that reacted explicitly with nGAstV ORF2 protein were further confirmed by ELISA and western blot, among which one Nb showed the best biological activity. 【Conclusion】 In this study, Nbs that reacted specifically with nGAstV ORF2 protein were screened for the first time, which provided materials for basic research and developing nGAstV detection methods.

Key words: novel Goose Astrovirus, ORF2 Protein, nanobody, phage display