抗犬细小病毒纳米抗体的制备及其中和活性鉴定

doi:10.16431/j.cnki.1671-7236.2022.09.028

Abstract

Abstract: 【Objective】 The aim of the experiment was to establish a canine nanobody (Nb) library against Canine parvovirus(CPV) and screen out the Nb with neutralizing activity.【Method】 Heavy chain variable region (VH) amplification was performed using cDNA from spleen tissue of high free Beagle dogs, then connected VH to pBSD vector to establish pBSD-Nb library.The high-affinity nanobodies were screened out from pBSD-Nb library by bacterial display technology combined with flow cytometry detection, and then positive clones were obtained and sequenced.The upstream and downstream primers of Nb genes were designed by Primer Premier 5.0 software, then Nb gene fragments were amplified by PCR.The target protein was expressed by pET-27b and purified by denaturation and renaturation.The purified protein were analyzed by SDS-PAGE.Nb was coated with 96 well plate, and the affinity of Nb to CPV was detected by ELISA method.The inhibition of Nb on CPV infection of F81 cells was detected by in vitro virus neutralization test.【Result】 PCR amplification results showed that a obvious band at 384 bp was consistent with the expected size, which proved that the pBSD-Nb library was successfully constructed.After the bacteria were shown, the results of flow cytometry showed that 9 clones of Nb with high positive peak deviation were screened and named Nb1, Nb2, Nb3, Nb4, Nb5, Nb6, Nb7, Nb8 and Nb9, respectively.9 clones of Nb gene fragments were amplified by PCR, and a band of 384 bp was obtained.SDS-PAGE results showed that there was a band at about 14 ku, indicating that the purified protein was successfully obtained.ELISA results showed that 6 of the 9 Nb strains had strong affinity for CPV.In vitro virus neutralization test showed that 1 strain of Nb could neutralize the virus and inhibit F81 cytopathic changes at an effective concentration of 0.05 mg/mL.【Conclusion】 In this study, the pBSD-Nb library was successfully established, 6 clones of Nb with high affinity for CPV were screened, and 1 clone of Nb with neutralizing activity was successfully obtained, providing a new solution for the development of CPV the rapeutic antibody.

Key words: Canine parvovirus (CPV); nanobody; bacterial display; antibody activity

CLC Number:

S852.65

REN Zeheng, PI Xuelei, XIA Anran, SUN Yue, HU Changhui, REN Guiping. Preparation and Identification of Neutralization Activity of Nanobody Against Canine Parvovirus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3539-3548.

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Preparation and Identification of Neutralization Activity of Nanobody Against Canine Parvovirus

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