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Journal of Henan Agricultural University. 2023, (02)

Screening and identification of African swine fever virus K205R protein-specific nanobodiesChinese Full TextEnglish Full Text (MT)

WU Shuya;ZHAO Huijun;REN Jiahui;DONG Haoxin;ZHANG Mingfang;LI Jiaji;DU Yongkun;WAN Bo;ZHANG Angke;ZHANG Gaiping;College of Veterinary Medicine, Henan Agricultural University;International Joint Research Center of National Animal Immunology, Henan Agricultural University;Key Laboratory of Animal Immunology, Ministry of Agriculture and Rural Affairs, Henan Academy of Agricultural Sciences;

Abstract: 【Objective】 Using phage display technology aims to screen specific nanobodies against African swine fever virus（ASFV） K205R protein.【Method】ASFV K205R protein was obtained by prokaryotic expression and purification, and immunized with Bactrian camels. Blood was collected on the seventh day after 4 immunizations, then isolated peripheral blood lymphocytes, extracted total RNA, and reversed transcribed into cDNA. The VHH genes were amplified using the cDNA as a template with nest PCR to construct a VHH phage library. Finally, ASFV K205R-specific nanobodies were screened by phage display technology. 【Result】 The VHH phage library was successfully constructed with a library capacity of 5×10~8. Then, after three rounds of panning by phage display technology, the specific VHH phages were significantly enriched. The amino acid sequence alignment found that five ASFV K205R protein-specific nanobodies were screened, named ASFV-K205R-Nb1, ASFV-K205R-Nb14, ASFV-K205R-Nb35, ASFV-K205R-Nb64 and ASFV-K205R-Nb82, respectively. 【Conclusion】 The five screened nanobodies have good specificity, among which ASFV-K205R-Nb1, ASFV-K205R-Nb35 and ASFV-K205R-Nb82 have better affinity, they provide a material basis for the establishment of a new type of ASFV serological diagnosis method with strong specificity, high sensitivity and low cost using nanobody as a sensitive probe.
Keywords：

African swine fever virus; K205R protein; VHH phage library; phage display; nanobody;

  • DOI：

    10.16445/j.cnki.1000-2340.20220819.001

  • Series：

    (D) Agriculture; (A) Mathematics/ Physics/ Mechanics/ Astronomy

  • Subject：

    Biology; Animal Husbandry and Veterinary

  • Classification Code：

    S852.651

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