抗非洲猪瘟病毒NP419L蛋白纳米抗体的筛选鉴定及其在抗体检测中的初步应用

doi:10.11843/j.issn.0366-6964.2023.06.029

Abstract

Abstract: African swine fever (ASF) is a highly infectious disease of pigs, which seriously threatens the development of the global pig industry. The purpose of this study is to screen specific nanobodies against the NP419L protein of ASF virus (ASFV) and evaluate its application of anti-ASFV antibody detection. We prepared the recombinant NP419L protein by E.coli expression system and Ni-NTA affinity chromatography purification. The purified recombinant protein was used to immunize the Bactrian camels. After the fifth immunization, peripheral blood lymphocytes were collected and total RNA was extracted. A phage display library against NP419L protein was constructed by nested PCR, enzyme digestion, ligation and electroporation. Nanobodies against NP419L were obtained through three rounds of screening using phage display screening technology. The eukaryotic expression vectors of nanobodies fused with horseradish peroxidase (HRP) proteins were constructed and transfected into HEK293T cells. The secretory expression of the fusion protein and its binding to NP419L protein were identified by ELISA and indirect immunofluorescence (IFA), and preliminary evaluation of the obtained nanobody fused with HRP proteins in ASF detection using competitive ELISA. The results showed that the NP419L recombinant protein with the expected size of 48 ku was successfully prepared with high purity; a phage display library with a positive rate of 89.5% and a library capacity of 6×10⁸was constructed; six strains of anti-NP419L protein nanobodies were obtained by screening; the fusion proteins of these six nanobodies with HRP were secretively expressed, the results of ELISA showed that the six fusion proteins specifically bind to the recombinant NP419L protein, one fusion protein was found to bind to NP419L protein expressed with eukaryotic system by IFA, and this fusion protein can be used as a probe for the detection of ASFV antibodies. In the study, six nanobodies against ASFV NP419L protein were successfully screened. The preliminary characterization of its binding protein and its application of antibody detection were also carried out, which provided the basis for the subsequent development of ASF diagnostic technology and the functional study of NP419L protein.

Key words: African swine fever virus, NP419L protein, nanobody

CLC Number:

S852.659.1

WANG Ying, ZHU Jiahong, ZHAO Jiakai, JI Pinpin, CHEN Xu, ZHANG Lu, LIU Baoyuan, SUN Yani, ZHAO Qin. Screening and Identification of Nanobodies against NP419L Protein of African Swine Fever Virus and Its Preliminary Application of Antibody Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2509-2520.

References 34

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Screening and Identification of Nanobodies against NP419L Protein of African Swine Fever Virus and Its Preliminary Application of Antibody Detection

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