Screening and Identification of Nanobodies Against Porcine epidemic diarrhea virus N Protein

doi:10.7668/hbnxb.20195229

Abstract

Abstract:

The highly contagious gastrointestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV)has led to significant economic losses in China's pig industry.It aimed to establish a basis for PEDV antibody detection methods and functional research of the N protein through the screening of specific nanobodies(Nbs)using phage display technology.Peripheral blood lymphocytes were isolated from a camel immunized with the N protein,and total RNA was extracted and reverse transcribed into cDNA.The variable domain of the heavy chain of heavy chain antibodies(VHH)was amplified by PCR,subcloned into the pCANTAB5E-ccdb vector,and electroporated into ER2738 competent cells to construct the VHH phage antibody display library.Subsequently,the library was subjected to four rounds of panning against the PEDV N protein,and positive phage clones were cloned into the pET-30a vector.The binding affinity and specificity of the Nbs were determined by indirect ELISA and Western Blot.The results showed that after the fifth immunization,the antibody titer reached 1∶25 600.The constructed phage display library had a capacity of 4.72×10⁸ and an abundance of 4.3×10¹⁰ cfu/mL,with a 93.75% positive rate.After four rounds of screening,16 Nb clones with different amino acid sequences were obtained,and Nb45 was validated to possess excellent specificity and binding ability to the PEDV N protein.This study successfully screened and obtained specific N protein-targeting Nbs,providing biological materials for the establishment of PEDV detection methods and foundational research.

Key words: Porcine epidemic diarrhea virus, N protein, Phage display, Nanobody, Characterization

YIN Dongdong, DING Xiang, LAN Mengdie, JI Kaiyuan, WANG Jieru, YIN Lei, SHEN Xuehuai, DAI Yin, ZHAO Ruihong, HOU Hongyan, HU Xiaomiao, PAN Xiaocheng. Screening and Identification of Nanobodies Against Porcine epidemic diarrhea virus N Protein[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 224-230. doi: 10.7668/hbnxb.20195229.

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Figures/Tables 7

Tab.1 Primer sequences for VHH fragments

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')
Call001-F	GTCCTGGCTGCTCTTCTACAAGG
Call001-R	GGTACGTGCTGTTGAACTGTTCC
VHH-F	TTTCTATTACTA GGCCCAGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG
VHH-R	AAACCGTT GGCCATAATGGCCTGGAGCTGGGGTCTTCGCTGTGGT

Fig.1 Titer of anti-N antibody in alpaca

Fig.2 Construction of a VHH phage display library A.The first round of PCR:M.DL2000 DNA Marker,1.Negative control,2—4.IgG;B.The second round of PCR:M.DL2000 DNA Marker,1.Negative control,2—4.VHH;C.The library positivity rate was identified by PCR:M.DL2000 DNA Marker,1—48.Different monoclonals.

Tab.2 Enrichment degree of four-round phage elutation

筛选轮数 Elutriation rounds	拯救噬菌体/ (cfu/mL) Input phage	洗脱噬菌体/ (cfu/mL) Output phage	回收率 Output/ Input
第1轮 First round	1.28×10¹³	4.50×10⁴	3.52×10^-9
第2轮 Second round	5.68×10¹³	5.30×10⁶	9.33×10^-8
第3轮 Third round	7.04×10¹³	2.90×10⁷	4.12×10^-7
第4轮 Fourth round	3.94×10¹²	1.46×10⁸	3.71×10^-5

Fig.3 Screening of specific nanobody of PEDV N protein A.Identification of specific phages;B.Alignment of Nb amino acid sequences.

Fig.4 Expression of Nbs A. Expression of Nb41 and Nb44: 1,5. The supernatant of Nb41, Nb44 was induced at 16 ℃, 2,6. Inclusion bodies of Nb41, Nb44 were induced at 16 ℃, 3,7. Supernatant of Nb41, Nb44 was induced at 37 ℃, 4,8. Inclusion bodies of Nb41, Nb44 were induced at 37 ℃; B. Expression of Nb45 and Nb46: 1,5. The supernatant of Nb45, Nb46 was induced at 16 ℃, 2,6. Inclusion bodies of Nb45, Nb46 were induced at 16 ℃, 3,7. Supernatant of Nb45, Nb46 was induced at 37 ℃, 4,8. Inclusion bodies of Nb45, Nb46 were induced at 37 ℃; C. Expression of Nb47 and Nb96: 1,5. The supernatant of Nb47,Nb96 was induced at 16 ℃, 2,6. Inclusion bodies of Nb47,Nb96 were induced at 16 ℃, 3,7. Supernatant of Nb47,Nb96 was induced at 37 ℃, 4,8. Inclusion bodies of Nb47,Nb96 were induced at 37 ℃; D. Purification of Nb45: 1. Lysate, 2. Draining liquid, 3—4. Washing liquid, 5—8. Eluent.

Fig.5 Binding capacity and specificity of Nb45 A.Specificity detection of Nb45;B.Binding capacity of Nb45.

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