Preparation of small molecule antibody Fab specific to S1 protein of canine coronavirus

doi:10.3969/j.issn.1004-1524.20221618

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (11): 2568-2583.DOI: 10.3969/j.issn.1004-1524.20221618

• Animal Science • Previous Articles Next Articles

Preparation of small molecule antibody Fab specific to S1 protein of canine coronavirus

XUE Jiaoxiong¹(), ZHAO Tingfang¹, ZHANG Qian²^,³, TANG Qinghai¹^,^*(), GAO Cuicui¹, ZHAO Cheng¹, ZHANG Yan¹, QUAN Feiyang¹, LIU Ting¹, YANG Can¹, YANG Hai¹, WANG Wenxiu³^,^*()

1. Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region, College of Life Sciences, Hengyang Normal University, Hengyang 421008, Hunan, China
2. Binzhou Zhanhua District Science and Technology Innovation and Development Research Center, Binzhou 256600, Shandong, China
3. Shandong Binzhou Animal Science & Veterinary Medicine Academy, Binzhou 256600, Shandong, China

Received:2022-11-16 Online:2023-11-25 Published:2023-12-04

Abstract

Abstract:

To express S1 protein of canine coronavirus (CCV) and prepare its specific small molecule antibody Fab, so as to provide a new material for the prevention and treatment of CCV. The full length and truncated fragments of CCV S1 gene were amplified by PCR and cloned into prokaryotic expression vector pET28a or pGEX4T-1, respectively, and transferred into BL21 (DE3) or Rosetta (DE3) receptive cells to construct recombinant expression strains. The target protein expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The recombinant proteins were identified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blot. The purified proteins were emulsified with ISA71AVG or ISA201VG respectively to prepare the immunogen. Laying hens were immunized, and yolk antibodies against CCV S1 protein were extracted by polyethylene glycol (PEG) method. Western blot was used to detect antibody titers, and egg yolk antibody was hydrolyzed by pepsin to prepare Fab. The results showed that the whole length of S1 protein was not expressed, and the molecular weights of CCV S1-a and CCV S1-b were 79 ku and 68 ku, respectively. On the 35th day after immunization, the titer of yolk antibody in the 2 adjuvant groups was 1:128 000. The optimal digestion conditions for Fab were as follows: mass ratio of egg yolk antibody to pepsin 20:1, pH value 4.1, 37 ℃ and 8 h. Complete IgY could react specifically with CCV, and the small molecule antibody Fab prepared by pepsin treatment could effectively block the infectivity of CCV. In conclusion, the 2 truncated fragments of CCV S1 protein were successfully expressed in this experiment, and the yolk antibody and its small molecule antibody Fab were prepared, which laid a foundation for further research on diagnosis and prevention of canine coronavirus disease.

Key words: canine coronavirus, prokaryotic expression, adjuvant, egg yolk antibody, small molecule antibody Fab

CLC Number:

S855.3

XUE Jiaoxiong, ZHAO Tingfang, ZHANG Qian, TANG Qinghai, GAO Cuicui, ZHAO Cheng, ZHANG Yan, QUAN Feiyang, LIU Ting, YANG Can, YANG Hai, WANG Wenxiu. Preparation of small molecule antibody Fab specific to S1 protein of canine coronavirus[J]. Acta Agriculturae Zhejiangensis, 2023, 35(11): 2568-2583.

Figures/Tables 16

Table 1 Primer sequences

引物名称 Primer name	序列 Sequence (5'→3')
CCV S1-a-F	ACTGGATCCATGATTGTTTTAATTTTATG
CCV S1-a-R	ACTCTCGAGAGAGTTACAATAAGTAACTTTTTTAATAG
CCV S1-b-F	ACTGGATCCACTGATGCTTTAGTTCAAGTTGA
CCV S1-b-R	ACTCTCGAGAGAAACTGGAGTAGTATAAACTTGAA

Table 2 Adjuvant compatibility list

组别(数量)Group (Quantity)	抗原Antigen	佐剂Adjuvant volume	总体积Total volume/mL
CCV-71(n=3)	CCV S1-ab 1.2 mL	ISA71AVG 2.8 mL	4
CCV-201(n=3)	CCV S1-ab 1.2 mL+0.8 mL ddH₂O	ISA201VG 2.0 mL	4
PBS对照组PBS control group (n=3)	PBS 4 mL	0	4

Fig.1 PCR amplification products of full-length and truncated fragments of CCV S1 gene M, DL5 000 DNA marker; 1, CCV S1 gene PCR product; 2, CCV S1-a gene PCR product; 3, CCV S1-b gene PCR product.

Fig.2 Double digestion of plasmids in recombinant strain BL21(DE3)-pGEX4T-CCV S1 and Rosetta (DE3)-pGEX4T-CCV S1 A, Identification of recombinant strain BL21(DE3)-pGEX4T-CCV S1; B, Identification of recombinant strain Rosetta (DE3)-pGEX4T-CCV S1. M, DL5 000 DNA marker; 1, 3, 5, 7 were recombinant plasmid pGEX4T-CCV S1; 2, 4, 6, 8 were recombinant plasmid pGEX4T-CCV S1 digested by BamHⅠ and XhoⅠ; 9 and 11, Recombinant plasmid pGEX4T-CCV S1; 10 and 12, Recombinant plasmid pGEX4T-CCV S1 digested by BamHⅠ and XhoⅠ; 13 and 15, Empty plasmid pGEX4T-1 in control group; 14 and 16, Empty plasmid pGEX4T-1 in control group digested by BamHⅠ and XhoⅠ.

Fig.3 Double digestion of plasmid in recombinant strain Rosetta (DE3)-pET28a-CCV S1-a, Rosetta (DE3)-pGEX4T-CCV S1-a and Rosetta (DE3)-pGEX4T-CCV S1-b A, Identification of recombinant Rosetta (DE3)-pET28a-CCV S1-a and Rosetta (DE3)-pGEX4T-CCV S1-a; B, Identification of recombinant Rosetta (DE3)-pGEX4T-CCV S1-b. M, DL5 000 DNA marker; 1 and 3, Recombinant plasmid pET28a-CCV S1-a; 2 and 4, Recombinant plasmid pET28a-CCV S1-a digested by BamHⅠ and XhoⅠ; 5 and 7, Recombinant plasmid pGEX4T-CCV S1-a; 6 and 8, Recombinant plasmid pGEX4T-CCV S1-a digested by BamHⅠ and XhoⅠ; 9-12, Recombinant plasmid pGEX4T-CCV S1-b digested by BamHⅠ and XhoⅠ; 13-15, Recombinant plasmid pGEX4T-CCV S1-b.

Fig.4 SDS-PAGE analysis of recombinant strain Rosetta (DE3)-pGEX4T-CCV S1-a and Rosetta (DE3)-pGEX4T-CCV S1-b A, SDS-PAGE detection of recombinant Rosetta (DE3)-pGEX4T-CCV S1-a; B, SDS-PAGE detection of recombinant Rosetta (DE3)-pGEX4T-CCV S1-b. M, Protein marker; 1, Induced precipitation of pGEX4T-CCV S1-a; 2, Induced supernatant of pGEX4T-CCV S1-a in the first ultrasonic crushing; 3, Induced supernatant of pGEX4T-CCV S1-a in the second ultrasonic crushing; 4, pGEX4T-CCV S1-a precipitation without induced expression; 5, pGEX4T-CCV S1-a supernatant without induced expression; 6, Induced precipitation of pGEX4T-1; 7, Induced supernatant of pGEX4T-1; 8, Uninduced precipitation of pGEX4T-1; 9, Uninduced supernatant of pGEX4T-1; 10, Induced precipitation of pGEX4T-CCV S1-b; 11, Uninduced precipitation of pGEX4T-CCV S1-b; 12, Induced precipitation of pGEX4T-1; 13, Uninduced precipitation of pGEX4T-1; 14, Induced supernatant of pGEX4T-CCV S1-b in the first ultrasonic crushing; 15, Induced supernatant of pGEX4T-CCV S1-b in second ultrasonic crushing; 16, Induced supernatant of pGEX4T-1; 17, Uninduced supernatant of pGEX4T-1.

Fig.5 Detection of recombinant protein Rosetta (DE3)-pGEX4T-CCV S1-a and Rosetta (DE3)-pGEX4T-CCV S1-b by Western blot A, Western bolt identification of Rosetta (DE3)-pGEX4T-CCV S1-a protein; B, Western bolt identification of Rosetta (DE3)-pGEX4T-CCV S1-b protein. M, Protein marker; 1, Induced precipitation of pGEX4T-CCV S1-a; 2, Induced supernatant of pGEX4T-CCV S1-a in the first ultrasonic crushing; 3, Induced supernatant of pGEX4T-CCV S1-a in the second ultrasonic crushing; 4, pGEX4T-CCV S1-a precipitation without induced expression; 5, pGEX4T-CCV S1-a supernatant without induced expression; 6, Induced precipitation of pGEX4T-1; 7, Induced supernatant of pGEX4T-1; 8, Uninduced precipitation of pGEX4T-1; 9, Uninduced supernatant of pGEX4T-1; 10, Induced precipitation of pGEX4T-CCV S1-b; 11, Uninduced precipitation of pGEX4T-CCV S1-b; 12, Induced precipitation of pGEX4T-1; 13, Uninduced precipitation of pGEX4T-1; 14, Induced supernatant of pGEX4T-CCV S1-b; 15, pGEX4T-CCV S1-b supernatant without induced expression; 16, Induced supernatant of pGEX4T-1; 17, Uninduced supernatant of pGEX4T-1. The recombinant target protein bands were marked with dashed box.

Fig.6 SDS-PAGE and Western bolt analysis of recombinant strain Rosetta (DE3)-pGEX4T-CCV S1-a and Rosetta (DE3)-pGEX4T-CCV S1-b A, SDS-PAGE test; B, Western blot identification. M, Protein marker; 1, Induced precipitation of pGEX4T-CCV S1-a; 2, Induced precipitation of pGEX4T-CCV S1-b; 3, Induced precipitation of pGEX4T-1.

Fig.7 Comparison of IgY against CCV S1-ab protein before and after dialysis M, Protein marker; 1, 3 and 5, Pre-dialysis IgY; 2, 4 and 6, Post-dialysis IgY.

Fig.8 Immunologic activity of IgY against CCV S1-ab protein by Western blot M, Protein marker; 1-6 were 4 000, 8 000, 16 000, 32 000, 64 000, 128 000 times dilution of IgY.

Fig.9 Identification of the reactivity of IgY to CCV by immunoperoxidase monolayer cell assay A, Prepared specific IgY reacted with CCV; B, Negative IgY reacted with CCV.

Fig.10 SDS-PAGE detection of IgY and pepsin with different mixed mass ratio and different digestion time A, Different concentrations of pepsin solution diluted to 100 μL; B-F, The mixing mass ratio of IgY and pepsin were 5:1, 10:1, 20:1, 40:1, 80:1, respectively.M, Protein marker; 1-5, 6.4 mg·mL-1, 3.2 mg·mL-1, 1.6 mg·mL-1, 0.8 mg·mL-1 and 0.4 mg·mL-1 pepsin solution were diluted into 100 μL; 6, IgY stock solution; 7-14, The results of digested for 10 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h, respectively.

Fig.11 Detection results of IgY dissolved in sodium acetate buffer at different pH values M, Protein marker; 1-13 were the IgY dissolved by sodium acetate buffer at pH 2.0, 2.5, 3.0, 3.5, 3.8, 4.1, 4.4, 4.7, 5.0, 5.5, 6.0, 6.5, 7.0, respectively.

Fig.12 SDS-PAGE detection of enzyme-digested products under different pH conditions (mixing mass ratio of IgY and pepsin was 20:1, digested for 24 h at 37 ℃) M, Protein marker; 1-13 were enzyme-digested products at pH 2.0, 2.5, 3.0, 3.5, 3.8, 4.1, 4.4, 4.7, 5.0, 5.5, 6.0, 6.5, 7.0,respectively.

Fig.13 SDS-PAGE detection of products with different enzyme digestion pH values and different enzyme digestion time A-I, Products digested for different time at pH 2.0, 2.5, 3.0, 3.5, 3.8, 4.1, 4.4, 4.7 and 5.0, respectively.M, Protein marker; 1-8 were digested for 5 min, 10 min, 20 min, 0.5 h, 1 h, 2 h, 4 h and 8 h, respectively; 9, pH 2.0 IgY stock solution; 10, The corresponding pH IgY stock solution; 11-18 were digested for 5 min, 10 min, 20 min, 0.5 h, 1 h, 2 h, 4 h and 8 h, respectively; 19, The corresponding pH IgY stock solution; 20-27 were digested for 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h, respectively.

Fig.14 IPMA identification of blocking activity of small molecule antibody Fab against viral infectivity A, CRFK cells were infected by CCV incubated with specific small molecule Fab; B, CRFK cells were infected by CCV incubated with negative small molecule Fab.

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Preparation of small molecule antibody Fab specific to S1 protein of canine coronavirus

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