Abstract: In order to obtain specific nanobodies against GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and explore their inhibitory effect on virus replication. The PRRSV-GP5 protein was expressed in a large amount by prokaryotic expression system and purified by nickel column affinity chromatography. The obtained recombinant GP5 protein was used to immunize alpacas, and blood was collected at the 0th, 14th, 28th, and 42nd days. After detecting the antibody titer in alpacas, the whole blood lymphocytes were isolated, and the total cell RNA was extracted. After reverse transcription, the heavy chain antibody variable region (VHH) fragment was amplified by nested PCR, and connected with pCANTAB-5E vector, and then transformed into TG1 receptor cells, The GP5-VHH phage antibody display library was constructed using phage display technology. After three rounds of screening and enrichment of specific phages, nanobodies with high affinity to PRRSV-GP5 protein were screened out, and their reactivity was identified by indirect ELISA. To investigate the effects of screened nanobodies targeting PRRSV-GP5 protein on virus replication and transcription in the cell,the obtained nanobody genes were cloned into eukaryotic expression vector pcDNA3.1 (-) and transfected into Marc-145 cells using Lipofectamine3000. They were incubated with PRRSV for 0, 24, 36, 48, 60, and 72 hours, respectively. The results showed that the PRRSV-GP5 protein was successfully expressed and purified in prokaryotic form, and the recombinant protein with a concentration of 2.16 mg·mL^-1 was obtained. After 14 days of three immunizations, the antibody titer in alpaca was as high as 1∶819 200. The GP5-VHH phage antibody display library was constructed, with a capacity of 2.93×10⁷ CFU·mL^-1, the insertion rate was 98%. After three consecutive rounds of panning and enrichment of phage antibody library, two nanobodies with different amino acid sequences were finally obtained. The results of indirect ELISA showed that the two nanobodies had good affinity with PRRSV-GP5 protein. Two nanobodies were transfected into Marc-145 cells, and they significantly hindered the replication and transcription of PRRSV at 36 and 60 hours,respectively.They demonstrated good ability to inhibit virus replication. In this study, the specific nanobodies against PRRSV-GP5 protein was screened for the first time and it has been verified that the screened nanobodies have the ability to inhibit PRRSV replication in cells. The research results could lay the experimental and material foundation for the development of new drugs against PRRSV.

Key words: porcine reproductive and respiratory syndrome virus, GP5 protein, nanobody, phage display technology, virus replication