猪流行性腹泻病毒S蛋白纳米抗体的筛选与鉴定

doi:10.11843/j.issn.0366-6964.2021.09.022

Abstract

Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease, which mainly harms piglets less than 1 week of age, and the infection mortality rate of piglets is up to 100%, and it is a major disease that harms the world's pig industry. We aimed to prepare a specific nanobody against porcine epidemic diarrhea virus (PEDV) S protein and to identify its binding activity. The prokaryotic expression and purification of PEDV S1 protein were performed, and the purified recombinant PEDV S1 protein was used to immunize Bactrian camel. The peripheral blood lymphocytes were isolated, the lymphocyte RNA was extracted, and the cDNA was obtained by reverse transcription. The VHH fragment was amplified by nested PCR, then was constructed into pCANTAB-5E vector and transformed into TG1 competent cells, the VHH phage antibody display library was obtained. The constructed phage antibody display library was rescued and enriched for three rounds. Phage display technology was used to screen the PEDV S protein nanobodies from the library. The specificity and binding force of the selected specific nanobodies were verified by ELISA. The binding activity of the nanobody to PEDV was verified by Western blot and indirect immunofluorescence. Results were as follows:PEDV S1 protein was successfully expressed and purified and the titer of specific antibodies in the serum of Bactrian camels reached 1:256 000 after four immunizations of the PEDV S1 protein. The capacity of the library was 2.1×10⁷, and the positive rate was 85%. After three rounds of screening and enrichment of the display library, six nanobodies with different amino acid sequences were screened out. ELISA results showed that all of them have good binding ability and specificity for PEDV S1 recombinant protein. Subsequently, it was verified that Nb3 can bind to PEDV virus, indicating that it has good activity. The specific nanobodies against PEDV S1 protein were successfully screened. The screened nanobodies are expected to be used for the diagnosis and treatment of PED, and provide antibody materials for the study of the pathogenic mechanism of PEDV.

Key words: porcine epidemic diarrhea virus, structural protein S, nanobody, phage display technology

CLC Number:

WANG Tianyu, LI Zhiwei, YANG Ting, DONG Linfang, MA Zhiqian, BIANBA Ciren, XIAO Shuqi, LI Shuang. Screening and Identification of Nanobodies against Porcine Epidemic Diarrhea Virus S Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2589-2598.

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Screening and Identification of Nanobodies against Porcine Epidemic Diarrhea Virus S Protein

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