犬冠状病毒S1蛋白特异性小分子抗体Fab的制备

doi:10.3969/j.issn.1004-1524.20221618

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (11): 2568-2583.DOI: 10.3969/j.issn.1004-1524.20221618

犬冠状病毒S1蛋白特异性小分子抗体Fab的制备

薛姣雄¹(), 赵婷芳¹, 张倩²^,³, 唐青海¹^,^*(), 高翠翠¹, 赵铖¹, 张妍¹, 全飞杨¹, 刘婷¹, 杨灿¹, 杨海¹, 王文秀³^,^*()

1.衡阳师范学院生命科学学院,南岳山区生物资源保护与利用湖南省重点实验室,湖南衡阳 421008
2.滨州市沾化区科技创新发展研究中心, 山东滨州 256600
3.山东省滨州畜牧兽医研究院, 山东滨州 256600

收稿日期:2022-11-16 出版日期:2023-11-25 发布日期:2023-12-04
作者简介:薛姣雄(2001—),女,湖南桃江人,本科生,主要从事微生物学与免疫学研究。E-mail:1654181665@qq.com
通讯作者: * 唐青海,E-mail: qinghaitang109@126.com;王文秀,E-mail:wwx1997@126.com
基金资助:
国家级大学创新创业训练计划(校科字〔2021〕1-9/cxcy2022001);2022年中央引导地方科技发展资金项目(2022ZYC091);湖南省自然科学基金面上项目(2021JJ30060);湖南省教育厅科学研究项目重点项目(21A0442)

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

摘要/Abstract

摘要：

为表达犬冠状病毒(canine coronavirus, CCV)S1蛋白,制备其特异性小分子抗体Fab,为CCV的防治提供新材料。采用PCR扩增CCV S1基因全长及其截短片段,分别克隆到原核表达载体pET28a或pGEX4T-1中,转到BL21(DE3)或Rosetta(DE3)感受态细胞构建重组表达菌株;用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot鉴定分析重组蛋白;纯化蛋白分别与ISA71AVG或ISA201VG佐剂配伍乳化后制备免疫原,免疫蛋鸡,用聚乙二醇(PEG)沉淀法提取得到CCV S1蛋白的卵黄抗体,通过Western blot检测抗体滴度,用胃蛋白酶水解卵黄抗体制备小分子抗体Fab。结果显示,S1蛋白全长无表达,CCV S1-a和CCV S1-b分子量分别为79 ku和68 ku。在免疫后第35天2种佐剂组的卵黄抗体滴度可达1:128 000。制备小分子抗体Fab最佳酶切条件为卵黄抗体与胃蛋白酶混合质量比20:1、pH值4.1、37 ℃酶切8 h。完整的IgY可以与CCV呈特异性反应,胃蛋白酶处理后制备的小分子抗体Fab可以有效阻断CCV的感染性。综上,本研究成功表达了CCV S1的2个截短片段蛋白,制备了卵黄抗体及其小分子抗体Fab,为进一步开展犬冠状病毒病的诊断和防治技术研究奠定了基础。

关键词: 犬冠状病毒, 原核表达, 佐剂, 卵黄抗体, 小分子抗体Fab

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

中图分类号:

S855.3

薛姣雄, 赵婷芳, 张倩, 唐青海, 高翠翠, 赵铖, 张妍, 全飞杨, 刘婷, 杨灿, 杨海, 王文秀. 犬冠状病毒S1蛋白特异性小分子抗体Fab的制备[J]. 浙江农业学报, 2023, 35(11): 2568-2583.

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.

图/表 16

表1 引物序列

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

表2 佐剂配伍表

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

图1 CCV S1基因全长和截短片段的PCR扩增产物 M,DL5 000 DNA分子量标准;1,CCV S1基因PCR产物;2,CCV S1-a基因PCR产物;3,CCV S1-b基因PCR产物。

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.

图2 重组表达菌株BL21(DE)-pGEX4T-CCV S1、Rosetta(DE3)-pGEX4T-CCV S1的质粒双酶切鉴定 A,重组菌株BL21(DE3)-pGEX4T-CCV S1的鉴定;B,重组菌株Rosetta(DE3)-pGEX4T-CCV S1的鉴定。M,DL 5 000 DNA分子量标准;1、3、5、7均为重组质粒pGEX4T-CCV S1;2、4、6、8均为重组质粒pGEX4T-CCV S1经BamHⅠ和XhoⅠ双酶切产物;9、11为重组质粒pGEX4T-CCV S1;10、12为重组质粒pGEX4T-CCV S1经BamHⅠ和XhoⅠ双酶切产物;13和15为对照组pGEX4T-1空质粒;14和16为对照组pGEX4T-1空质粒经BamHⅠ和XhoⅠ双酶切产物。

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.

图3 重组表达菌株Rosetta(DE3)-pET28a-CCV S1-a、Rosetta(DE3)-pGEX4T-CCV S1-a和Rosetta(DE3)-pGEX4T-CCV S1-b的质粒双酶切鉴定 A,重组菌株Rosetta(DE3)-pET28a-CCV S1-a和Rosetta(DE3)-pGEX4T-CCV S1-a的鉴定;B,重组菌株Rosetta(DE3)-pGEX4T-CCV S1-b的鉴定。M,DL5 000 DNA分子量标准;1、3,重组质粒pET28a-CCV S1-a;2、4,重组质粒pET28a-CCV S1-a经BamHⅠ和XhoⅠ双酶切产物;5、7,重组质粒pGEX4T-CCV S1-a;6、8,重组质粒pGEX4T-CCV S1-a经BamHⅠ和XhoⅠ双酶切产物;9、10、11、12,重组质粒pGEX4T-CCV S1-b经BamHⅠ和XhoⅠ双酶切产物;13、14、15,重组质粒pGEX4T-CCV S1-b。

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.

图4 重组表达菌株Rosetta(DE3)-pGEX4T-CCV S1-a和Rosetta(DE3)-pGEX4T-CCV S1-b的SDS-PAGE检测 A,重组菌株Rosetta(DE3)-pGEX4T-CCV S1-a的SDS-PAGE检测;B,重组菌株Rosetta(DE)-pGEX4T-CCV S1-b的SDS-PAGE检测。M,蛋白分子量标准;1,诱导表达的pGEX4T-CCV S1-a沉淀;2,诱导表达的pGEX4T-CCV S1-a第1次超声破碎的上清液;3,诱导表达的pGEX4T-CCV S1-a第2次超声破碎的上清液;4,未诱导表达的pGEX4T-CCV S1-a沉淀;5,未诱导表达的pGEX4T-CCV S1-a上清液;6,诱导表达的pGEX4T-1沉淀;7,诱导表达的pGEX4T-1上清液;8,未诱导表达的pGEX4T-1沉淀;9,未诱导表达的pGEX4T-1上清液;10,诱导表达的pGEX4T-CCV S1-b沉淀;11,未诱导表达的pGEX4T-CCV S1-b沉淀;12,诱导表达的pGEX4T-1沉淀;13,未诱导表达的pGEX4T-1沉淀;14,诱导表达的pGEX4T-CCV S1-b第1次超声破碎的上清液;15,诱导表达的pGEX4T-CCV S1-b第2次超声破碎的上清液;16,诱导表达的pGEX4T-1上清液;17,未诱导表达的pGEX4T-1上清液。

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.

图5 重组蛋白Rosetta(DE3)-pGEX4T-CCV S1-a和Rosetta(DE3)-pGEX4T-CCV S1-b鉴定 A,Rosetta(DE3)-pGEX4T-CCV S1-a 蛋白的Western bolt鉴定;B,Rosetta(DE3)-pGEX4T-CCV S1-b蛋白的Western bolt鉴定。M,蛋白分子量标准;1,诱导表达的 pGEX4T-CCV S1-a沉淀;2,诱导表达的pGEX4T-CCV S1-a第1次超声的上清液;3,诱导表达的pGEX4T-CCV S1-a第2次超声的上清液;4,未诱导表达的pGEX4T-CCV S1-a沉淀;5,未诱导表达的pGEX4T-CCV S1-a上清液;6,诱导表达的pGEX4T-1沉淀;7,诱导表达的pGEX4T-1上清液;8,未诱导表达的pGEX4T-1沉淀;9,未诱导表达的pGEX4T-1上清液;10,诱导表达的pGEX4T-CCV S1-b沉淀;11,未诱导表达的pGEX4T-CCV S1-b沉淀;12,诱导表达的pGEX4T-1沉淀;13,未诱导表达的pGEX4T-1沉淀;14,诱导表达的pGEX4T-CCV S1-b上清液;15,未诱导表达的pGEX4T-CCV S1-b上清液;16,诱导表达的pGEX4T-1上清液;17,未诱导表达的pGEX4T-1上清液。用虚线框标识重组目的蛋白条带。

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.

图6 重组表达菌株Rosetta(DE3)-pGEX4T-CCV S1-a和Rosetta(DE3)-pGEX4T-CCV S1-b的SDS-PAGE和Western blot鉴定 A,SDS-PAGE检测;B,Western blot鉴定。M,蛋白分子量标准;1,诱导表达的pGEX4T-CCV S1-a沉淀;2,诱导表达的pGEX4T-CCV S1-b沉淀;3,诱导表达的pGEX4T-1沉淀。

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.

图7 CCV S1-ab蛋白卵黄抗体透析前后对比 M,蛋白分子量标准;1、3、5,透析前卵黄抗体;2、4、6,透析后卵黄抗体。

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.

图8 CCV S1-ab蛋白卵黄抗体免疫活性的Western blot鉴定 M,蛋白分子量标准;1~6依次为稀释4 000、8 000、16 000、32 000、64 000、128 000倍的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.

图9 卵黄抗体与CCV反应活性的免疫过氧化物酶单层细胞试验鉴定 A,所制备的特异性IgY与CCV反应;B,阴性IgY与CCV反应。

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.

图10 卵黄抗体与胃蛋白酶不同混合质量比和不同酶切时间SDS-PAGE检测 A,不同质量浓度胃蛋白酶溶液稀释成100 μL体系;B~F,依次为IgY与胃蛋白酶混合质量比为5:1、10:1、20:1、40:1、80:1酶切SDS-PAGE检测结果。M,蛋白分子量标准;1~5依次为6.4、3.2、1.6、0.8、0.4 mg·mL-1胃蛋白酶溶液稀释成100 μL体系;6,IgY原液;7~14依次为酶切10 min、0.5 h、1 h、2 h、4 h、8 h、12 h、24 h。

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.

图11 不同pH值乙酸钠缓冲液溶解卵黄抗体检测结果 M,蛋白分子量标准;1~13依次为pH值为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乙酸钠缓冲液溶解的卵黄抗体。

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.

图12 不同pH条件下的酶切产物SDS-PAGE检测结果(卵黄抗体与胃蛋白酶混合质量比20:1,37 ℃酶切24 h) M,蛋白分子量标准;1~13依次为pH值为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的酶切产物。

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.

图13 不同酶切pH值与不同酶切时间的产物SDS-PAGE检测 A~I,pH值分别为2.0、2.5、3.0、3.5、3.8、4.1、4.4、4.7、5.0时不同酶切时间的产物。M,蛋白分子量标准;1~8依次为酶切5 min、10 min、20 min、0.5 h、1 h、2 h、4 h、8 h;9,pH值2.0的IgY原液;10,对应pH值的IgY原液;11~18依次为酶切5 min、10 min、20 min、0.5 h、1 h、2 h、4 h、8 h;19,对应pH值的IgY原液;20~27依次为酶切10 min、30 min、1 h、2 h、4 h、8 h、12 h、24 h。

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.

图14 小分子抗体Fab对病毒感染性的阻断活性IPMA鉴定 A,特异性小分子Fab孵育的CCV感染CRFK细胞;B,阴性小分子Fab孵育的CCV感染CRFK细胞。

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.

参考文献 36

[1]	刘亮, 金玉荣, 任婧. 一例犬冠状病毒病的诊治与体会[J]. 广西畜牧兽医, 2021, 37(3): 125-126.
	LIU L, JIN Y R, REN J. Diagnosis and treatment of a case of canine coronavirus disease[J]. Guangxi Journal of Animal Husbandry & Veterinary Medicine, 2021, 37(3): 125-126. (in Chinese)
[2]	张婷, 蒋郁明, 曾俊霞, 等. 犬冠状病毒的检测与防治措施[J]. 中国畜禽种业, 2022, 18(3): 140-142.
	ZHANG T, JIANG Y M, ZENG J X, et al. Detection and control measures of canine coronavirus[J]. The Chinese Livestock and Poultry Breeding, 2022, 18(3): 140-142. (in Chinese)
[3]	CAVANAGH D. Nidovirales: a new order comprising Coronaviridae and Arteriviridae[J]. Arch Virol, 1997, 142(3):629-633.
[4]	纪海旺, 孙晓荣, 孙科. 犬冠状病毒研究进展[J]. 中国动物检疫, 2020, 37(10): 87-91.
	JI H W, SUN X R, SUN K. Research progress of canine coronavirus[J]. China Animal Health Inspection, 2020, 37(10): 87-91. (in Chinese)
[5]	VLASOVA A N, DIAZ A, DAMTIE D, et al. Novel canine coronavirus isolated from a hospitalized patient with pneumonia in East Malaysia[J]. Clinical Infectious Diseases, 2022, 74(3): 446-454.
[6]	LU S, CHEN Y Z, QIN K, et al. Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China[J]. Science China Life Sciences, 2016, 59(6): 615-621.
[7]	CHEN S, LIU D F, TIAN J, et al. Molecular characterization of HLJ-073, a recombinant canine coronavirus strain from China with an ORF3abc deletion[J]. Archives of Virology, 2019, 164(8): 2159-2164.
[8]	韦欣欣, 李泽辉, 靳晓慧, 等. 猪氨基肽酶N作为受体介导猪冠状病毒感染的研究进展[J]. 河南农业科学, 2021, 50(8): 1-8.
	WEI X X, LI Z H, JIN X H, et al. Review of aminopeptidase N as the receptor of porcine coronavirus infection[J]. Journal of Henan Agricultural Sciences, 2021, 50(8): 1-8. (in Chinese with English abstract)
[9]	单颖, 许伟成, 施杏芬, 等. 猪代尔塔冠状病毒荧光定量PCR检测方法的建立及其S基因分子特征分析[J]. 浙江农业学报, 2018, 30(2): 220-227.
	SHAN Y, XU W C, SHI X F, et al. Establishment of one-step Taqman quantitative PCR detection method and molecular S gene characterization analysis of porcine deltacoronavirus[J]. Acta Agriculturae Zhejiangensis, 2018, 30(2): 220-227. (in Chinese with English abstract)
[10]	乔军, 夏咸柱, 胡桂学, 等. 犬冠状病毒S蛋白主要抗原区基因片段的表达及免疫原性[J]. 中国病毒学, 2004, 19(6): 616-619.
	QIAO J, XIA X Z, HU G X, et al. Expression of the major antigenic region fragment of spike gene of canine coronavirus and immunogenicity of expressed products[J]. Virologica Sinica, 2004, 19(6): 616-619. (in Chinese with English abstract)
[11]	王静. 犬冠状病毒的分离鉴定及两种检测方法的建立[D]. 北京: 中国农业科学院, 2018.
	WANG J. Isolation and identification of canine coronavirus and establishment of two detection methods[D]. Beijing: Chinese Academy of Agricultural Sciences, 2018. (in Chinese with English abstract)
[12]	柳青云, 孙康迪, 方咏青, 等. 三种免疫增效剂对犬冠状病毒疫苗免疫效果影响的研究[J]. 今日畜牧兽医, 2019, 35(12): 15.
	LIU Q Y, SUN K D, FANG Y Q, et al. Study on the influence of three immune synergists on the immune effect of canine coronavirus vaccine[J]. Today Animal Husbandry and Veterinary Medicine, 2019, 35(12): 15. (in Chinese)
[13]	刘玉秀, 黄柏成, 习向锋, 等. 犬冠状病毒的进化和致病性研究进展[J]. 中国兽医杂志, 2020, 56(2): 59-63.
	LIU Y X, HUANG B C, XI X F, et al. Research progress on evolution and pathogenicity of canine coronavirus[J]. Chinese Journal of Veterinary Medicine, 2020, 56(2): 59-63. (in Chinese)
[14]	余春, 夏咸柱, 池元斌, 等. 犬冠状病毒压力灭活苗的制备与应用研究[J]. 中国兽医科技, 2000, 30(5): 5-7.
	YU C, XIA X Z, CHI Y B, et al. Preparation and application of canine coronavirus pressure inactivated vaccine[J]. Chinese Veterinary Science, 2000, 30(5): 5-7. (in Chinese)
[15]	孙秋艳, 沈美艳, 李舫, 等. 浒苔多糖对犬冠状病毒灭活苗的免疫增强作用[J]. 中国兽医学报, 2017, 37(9): 1664-1669.
	SUN Q Y, SHEN M Y, LI F, et al. Immuno-enhancement of enteromorpha polysaccharide on canine coronavirus inactivated vaccine[J]. Chinese Journal of Veterinary Science, 2017, 37(9): 1664-1669. (in Chinese with English abstract)
[16]	ZHANG X Y, CALVERT R A, SUTTON B J, et al. IgY: a key isotype in antibody evolution[J]. Biological Reviews, 2017, 92(4): 2144-2156.
[17]	贺维朝, 张会艳, 王浩, 等. 卵黄抗体提取方法及其在畜禽细菌性肠道疾病防治中的应用[J]. 中国畜牧兽医, 2021, 48(2): 640-649.
	HE W Z, ZHANG H Y, WANG H, et al. Extraction methods of yolk antibody and its application in prevention and treatment of bacterial intestinal diseases in livestock and poultry[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(2): 640-649. (in Chinese with English abstract)
[18]	WU R, YAKHKESHI S, ZHANG X Y. Scientometric analysis and perspective of IgY technology study[J]. Poultry Science, 2022, 101(4): 101713.
[19]	FAN W H, SUN S S, ZHANG N, et al. Nasal delivery of thermostable and broadly neutralizing antibodies protects mice against SARS-CoV-2 infection[J]. Signal Transduction and Targeted Therapy, 2022, 7: 55.
[20]	钟欣欣, 曹立民, 林洪, 等. 酪氨酸酶特异性卵黄抗体Fab’片段的制备及其对酶活力的抑制性能[J]. 食品科学, 2018, 39(2): 119-123.
	ZHONG X X, CAO L M, LIN H, et al. Production and inhibitory effect of specific egg yolk antibody Fab' fragment against tyrosinase[J]. Food Science, 2018, 39(2): 119-123. (in Chinese with English abstract)
[21]	刘美君, 高向东, 徐晨. Fab类抗体的研究进展[J]. 国际药学研究杂志, 2014, 41(3): 318-324.
	LIU M J, GAO X D, XU C. Research advances on Fabs antibodies[J]. Journal of International Pharmaceutical Research, 2014, 41(3): 318-324. (in Chinese with English abstract)
[22]	ZHANG C, CODINA N, TANG J Z, et al. Comparison of the pH-and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation[J]. Computational and Structural Biotechnology Journal, 2021, 19: 2726-2741.
[23]	DUAN H L, HE Q Y, ZHOU B, et al. Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy[J]. Journal of Venomous Animals and Toxins Including Tropical Diseases, 2016, 22(1): 1-6.
[24]	乔军. 犬冠状病毒基因疫苗与重组腺病毒的构建、表达及免疫原性研究[D]. 长春: 吉林大学, 2005.
	QIAO J. Construction, expression and immunogenicity of canine coronavirus gene vaccine and recombinant adenovirus[D]. Changchun: Jilin University, 2005. (in Chinese with English abstract)
[25]	郝雲峰. 犬冠状病毒S蛋白抗原多表位基因的重组表达及rSP-ELISA方法的建立[D]. 北京: 中国农业科学院, 2021.
	HAO Y F. Recombinant expression of multi-epitope gene of canine coronavirus S protein antigen and establishment of rSP-ELISA method[D]. Beijing: Chinese Academy of Agricultural Sciences, 2021. (in Chinese with English abstract)
[26]	HAO Y F, LI S H, ZHANG G Z, et al. Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus[J]. Archives of Virology, 2021, 166(7): 1877-1883.
[27]	邓博, 马兵, 刘丹, 等. 免疫佐剂的作用机制及分类[J]. 国际生物医学工程杂志, 2020, 43(6): 470-475.
	DENG B, MA B, LIU D, et al. Mechanism and classification of immune adjuvants[J]. International Journal of Biomedical Engineering, 2020, 43(6): 470-475. (in Chinese with English abstract)
[28]	韩涛涛, 黎露, 唐青海, 等. 不同佐剂对猪传染性胃肠炎病毒S蛋白和猪流行性腹泻病毒S蛋白免疫原性的影响[J]. 中国农学通报, 2020, 36(30): 143-150.
	HAN T T, LI L, TANG Q H, et al. Effects of different adjuvants on immunogenicity of S protein of transmissible gastroenteritis virus and S protein of epidemic diarrhea virus in pigs[J]. Chinese Agricultural Science Bulletin, 2020, 36(30): 143-150. (in Chinese)
[29]	朱文姣, 常琛, 朱玉虎, 等. 猪流行性腹泻病毒coe基因的克隆表达及卵黄抗体的制备[J]. 河南农业大学学报, 2018, 52(4): 560-565.
	ZHU W J, CHANG C, ZHU Y H, et al. Clone and expression of coe gene and preparation of egg yolk antibody against coe of porcine epidemic diarrhea virus[J]. Journal of Henan Agricultural University, 2018, 52(4): 560-565. (in Chinese with English abstract)
[30]	GUJRAL N, YOO H, BAMDAD F, et al. A combination of egg yolk IgY and phosvitin inhibits the growth of enterotoxigenic Escherichia coli K88 and K99[J]. Current Pharmaceutical Biotechnology, 2017, 18(5): 400-409.
[31]	LEE D H, JEON Y S, PARK C K, et al. Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the porcine epidemic diarrhea virus spike protein in piglets[J]. Archives of Virology, 2015, 160(9): 2197-2207.
[32]	刘铭. 鸭细小病毒抗体ELISA检测方法的建立及卵黄抗体的制备[D]. 保定: 河北农业大学, 2020.
	LIU M. Establishment of ELISA for detection of duck parvovirus antibody and preparation of yolk antibody[D]. Baoding: Hebei Agricultural University, 2020. (in Chinese with English abstract)
[33]	AKITA E M, NAKAI S. Production and purification of Fab' fragments from chicken egg yolk immunoglobulin Y (IgY)[J]. Journal of Immunological Methods, 1993, 162(2): 155-164.
[34]	周欣. 鸡卵黄免疫球蛋白Fab片段的制备及其抗炎活性研究[D]. 武汉: 华中农业大学, 2020.
	ZHOU X. Preparation and anti-inflammatory activity of Fab fragment of chicken yolk immunoglobulin[D]. Wuhan: Huazhong Agricultural University, 2020. (in Chinese with English abstract)
[35]	张冯奕驰. 传染性法氏囊病卵黄抗体Fab’片段的制备条件优化及临床应用[D]. 南京: 南京农业大学, 2020.
	ZHANG F Y C. Optimization of preparation conditions and clinical application of Fab' fragment of yolk antibody against infectious bursal disease[D]. Nanjing: Nanjing Agricultural University, 2020. (in Chinese with English abstract)
[36]	邹强, 李敏惠, 杨淑霞, 等. 鸡卵黄抗体Fab’的制备及鉴定[J]. 医学研究生学报, 2010, 23(2): 133-136.
	ZOU Q, LI M H, YANG S X, et al. Preparation and identification of Fab' fragments from chicken egg yolk immunoglobulin Y[J]. Journal of Medical Postgraduates, 2010, 23(2): 133-136. (in Chinese with English abstract)

犬冠状病毒S1蛋白特异性小分子抗体Fab的制备

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

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