Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (17): 3494-3506.doi: 10.3864/j.issn.0578-1752.2024.17.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Prokaryotic Expression, Antibody Preparation and Application of Major Non-Structural Proteins of Porcine Rotavirus

BIAN XianYu1,2(), LI SuFen2, WANG JianXin2, HAN Nan2, LU HongTing1,2, CHENG Xi2, ZHOU JinZhu2, TAO Ran2, ZHU XueJiao2, DONG HaiLong1(), ZHANG XueHan1,2(), LI Bin1,2   

  1. 1 College of Animal Science, Tibet Agriculture and Animal Husbandry University, Linzhi 860000, Xizang
    2 Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture and Rural Affairs, Nanjing 210014
  • Received:2024-03-14 Accepted:2024-06-07 Online:2024-09-01 Published:2024-09-04
  • Contact: DONG HaiLong, ZHANG XueHan

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Abstract:

【Background】Rotavirus (RV) is one of the main causes of acute viral gastroenteritis in young children and young animals worldwide, and it is of great significance in public health. Porcine rotavirus disease, caused by Porcine Rotavirus (PoRV), is an acute intestinal infectious disease that often results in gastrointestinal dysfunction in piglets, leading to severe symptoms, such as vomiting, diarrhea, and dehydration. Outbreaks of PoRV can result in significant economic losses in the pig industry. At present, there is no specific drug treatment for PoRV infection, so vaccination is the most economical way to control the infection. However, PoRV genotypes are various and easily mutable, and cross-protection between different genotypes is poor. Therefore, it is urgent to strengthen the epidemiological surveillance and pathogenic mechanism research of PoRV to explore new prevention and control strategies.【Objective】The prokaryotic system of Escherichia coli was employed to express the non-structural proteins (NSP), such as NSP2, NSP4, and NSP5 of PoRV. Subsequently, rabbits were immunized to produce polyclonal antibodies (pAbs) specific to these proteins, which could offer novel insights to the detection and prevention of PoRV.【Method】The NSP2, NSP4, and NSP5 genes of PoRV were codon-optimized and cloned into the pCold-sumo vector. The positive recombinant plasmids, with correct sequencing, were transformed into E. coli BL21 (DE3), and the recombinant proteins were obtained with IPTG induction. Protein expression was identified using SDS-PAGE and Western blot assay. The recombinant proteins were then purified and quantified using affinity chromatography. New Zealand white rabbits were immunized with NSP2, NSP4, and NSP5 recombinant proteins by a subcutaneous multi-point injection method to prepare pAbs. The titers of pAbs were determined using indirect ELISA technology. The reactivity of pAbs with PoRV was verified by indirect immunofluorescence (IFA) and Western blot assay, and their applications in PoRV infection were also explored.【Result】SDS-PAGE analysis indicated that the recombinant proteins of NSP2, NSP4, and NSP5 were expressed well in a soluble form. Indirect ELISA analysis showed that the titers of pAbs against the three proteins reached 1﹕81 000, indicating good immunogenicity of the expressed proteins. The results from IFA and Western blot assay demonstrated that the prepared pAbs could specifically react with the dominant prevalent genotypes of PoRV, but had no reaction with other common diarrhea pathogens. Western blot assay results also showed that the pAbs could be used for dynamic expression analysis of NSPs during PoRV infection and validating transfection of the three recombinant eukaryotic plasmids.【Conclusion】Here, the NSP2, NSP4 and NSP5 of PoRV were successfully expressed in E. coli, and their pAbs with high titer and good specificity were obtained, which laid the foundation for the study of PoRV pathogenesis and the development of prevention and control strategies.

Key words: Porcine Rotavirus (PoRV), nonstructural protein, prokaryotic expression, polyclonal antibody

Table 1

Rotavirus strains used in this study"

毒株名称 Strain name 基因型 Genotype 宿主 Host GenBank登录号 Accession number
AHFY2022 G9P[23] 猪 Porcine OQ979280.1-OQ979290.1
NJ2012 G9P[7] 猪 Porcine MT874983.1-MT874993.1
JSNJ2019 G1P[7] 猪 Porcine PP100160.1-PP100180.1
JSJR2023 G4P[23] 猪 Porcine PP100149.1-PP100159.1
JSNJ2024 G5P[11] 猪 Porcine 未登录Not logged in
SA11 G3P[2] 猿猴 Simian LC178564.1-LC178574.1
NCDV G6P[1] 牛 Bovine JF693026.1-JF693036.1
SDLY G9P[1] 牛 Bovine 未登录Not logged in
N3 G10P[11] 牛 Bovine 未登录Not logged in

Fig. 1

Construction of recombinant plasmid A. NSP2 recombinant plasmid; B. NSP4 recombinant plasmid; C. NSP5 recombinant plasmid"

Table 2

Groups of immunized rabbits"

编号
Serial number		 免疫原
Immunogen		 剂量
Dose (μg)
001 NSP2重组蛋白
NSP2 recombinant protein		 200
002
003 NSP4蛋白
NSP4 recombinant protein		 200
004
005 NSP5蛋白
NSP5 recombinant protein		 200
006

Fig. 2

Comparison of amino acid sequences of major non- structural proteins of rotaviruses A. NSP2 amino acid sequence; B. NSP4 amino acid sequence; C. NSP5 amino acid sequence"

Fig. 3

The phylogenetic tree was constructed based on the amino acid sequences of the main nonstructural proteins of rotavirus A. NSP2 amino acid sequence; B. NSP4 amino acid sequence; C. NSP5 amino acid sequence"

Fig. 4

Enzyme digestion identification of recombinant plasmids A. pCold-sumo-NSP2 recombinant plasmid; B. pCold-sumo-NSP4 recombinant plasmid; C. pCold-sumo-NSP5 recombinant plasmid. M. DL10000 DNA Marker; 1. Double enzyme digestion of recombinant plasmid; 2. Double enzyme digestion of pCold-sumo plasmid"

Fig. 5

SDS-PAGE assay analysis of three recombinant protein expression A. Recombinant bacteria of BL21(DE3)/pCold-sumo-NSP2; B. Recombinant bacteria of BL21(DE3)/pCold-sumo-NSP4; C. Recombinant bacteria of BL21(DE3)/pCold-sumo-NSP5. M. Prestained middle molecular weight protein marker; 1. Whole bacterial lysates before induction; 2. Whole bacterial lysate after induction; 3. The supernatant from whole bacteria lysis after induction; 4. The pellet from whole bacteria lysis after induction"

Fig. 6

Ni-NTA affinity chromatography column purification and Western blot identification of recombinant proteins A. Purified recombinant protein of NSP2; B. Purified recombinant protein of NSP4; C. Purified recombinant protein of NSP5. M. Prestained middle molecular weight protein marker; 1-7. The elution with imidazole; 8: Western blot assay analysis of reactivity"

Fig. 7

Detection of polyclonal antibody titer by ELISA assay"

Fig. 8

Identification of NSP2, NSP4 and NSP5 polyclonal antibody reactivity to PoRV by IFA assay"

Fig. 9

Western blot identification of NSP2, NSP4 and NSP5 polyclonal antibody reactivity to PoRV A. Detection of rabbit NSP4 polyclonal antibody reactivity; B. Detection of rabbit NSP5 polyclonal antibody reactivity detection. M. Prestained middle molecular weight protein standard; 1.MA104 cells infected with JSJR2023 (G4P[23]); 2. MA104 cells infected with AHFY2022 (G9P[23]); 3. MA104 cells"

Fig. 10

Identification specificity of NSP2, NSP4 and NSP5 polyclonal antibody to PoRV by IFA"

Fig. 11

Detection of overexpression of NSP2, NSP4 and NSP5 in cells transfected with recombinant plasmid by Western blot assay M. Prestained middle molecular weight protein standard; 1. HEK293A cells transfected with 1 μg of recombinant plasmid; 2. HEK293A cells transfected with 2 μg of recombinant plasmid; 3. HEK293A cells transfected with 5 μg of recombinant plasmid; NC. Untranfected HEK293A cells"

Fig. 12

Detection of the expression of NSP4 and NSP5 after virus infection by Western blot assay M. Prestained middle molecular weight protein standard; 1. MA104 cells infected with RV for 4 hours; 2. MA104 cells infected with RV for 8 hours; 3. MA104 cells infected with RV for 12 hours; 4. Uninfected MA104 cells"

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