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

doi:10.11924/j.issn.1000-6850.casb20191000740

中国农学通报 ›› 2020, Vol. 36 ›› Issue (30): 143-140.doi: 10.11924/j.issn.1000-6850.casb20191000740

所属专题：生物技术；畜牧兽医

• 畜牧·动物医学·蚕·蜂 • 上一篇下一篇

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

韩涛涛¹(), 黎露¹, 唐青海¹(), 吴广艳¹, 孙国印², 李晓楠², 常秋玲², 杨海¹, 唐斯萍¹, 王芳宇¹

¹衡阳师范学院生命科学与环境学院,衡阳师范学院畜禽养殖生物工程技术研究所,湖南衡阳 421008
²南阳市天华制药有限公司,河南南阳 473061

收稿日期:2019-10-25 修回日期:2019-11-15 出版日期:2020-10-25 发布日期:2020-10-16
通讯作者: 唐青海
作者简介:韩涛涛,男,1996年出生,陕西商洛人,本科,从事动物传染病病原学研究。通信地址：421000 湖南省衡阳市雁峰区黄白路衡阳师范学院生命科学与环境学院,Tel：13548504557,E-mail： 326891396@qq.com。
基金资助:
国家级大学生创新创业训练计划项目“猪腹泻性病原卵黄抗体的制备”(201710546001);湖南省教育厅优秀青年项目“猪重要腹泻性病原卵黄抗体制剂的研制和应用”(17B041);校企合作项目“仔猪腹泻性疾病卵黄抗体制剂的研制”(ZXXQ201701)

Different Adjuvants: Effects on S Protein Immunogenicity of Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

Han Taotao¹(), Li Lu¹, Tang Qinghai¹(), Wu Guangyan¹, Sun Guoyin², Li Xiaonan², Chang Qiuling², Yang Hai¹, Tang Siping¹, Wang Fangyu¹

¹College of Life Sciences and Environment, Institute of Bio-technology for Livestock, Hengyang Normal University, Hengyang Hunan 421008
²Nanyang Tianhua Pharmaceutical Co., LTD., Nanyang Henan 473061

Received:2019-10-25 Revised:2019-11-15 Online:2020-10-25 Published:2020-10-16
Contact: Tang Qinghai

摘要/Abstract

摘要：

本研究旨在探究不同佐剂对猪传染性胃肠炎病毒(TGEV)S蛋白和猪流行性腹泻病毒(PEDV)S蛋白免疫原性的影响。IPGT诱导重组表达菌株Rosetta(DE3)-pET28a-TGEV-S2/3和BL21(DE3)-pET28a-PEDV-S1/2/3,制备重组蛋白TGEV-S2、TGEV-S3、PEDV-S1、PEDV-S2和PEDV-S3,并进行SDS-PAGE和Western blot鉴定,将重组蛋白等量混匀分别与3种佐剂ISA 71VG、ISA 201VG和ISA 15AVG进行乳化制备免疫原免疫蛋鸡;Western blot检测蛋鸡血清抗体效价,免疫过氧化物酶单层细胞染色法(IPMA)检测抗体与病原的反应活性;监测鸡群平均日增重。结果显示,重组蛋白TGEV-S2、TGEV-S3、PEDV-S1、PEDV-S2和PEDV-S3的分子量依次为25 ku、28 ku、26 ku、24 ku和21 ku。Western blot分析显示,5种蛋白均具有良好的免疫原性;疫苗二次免疫后7天抗体效价达到高峰,ISA 201VG佐剂组、ISA 71VG佐剂和ISA 15AVG佐剂组组最高抗体效价依次为64000倍、32000倍和8000倍,对照组未检测到特异性抗体;ISA 71VG和ISA 201VG组对产蛋率下降相对较大,ISA15AVG 佐剂疫苗免疫组对产蛋率影响相对较小,免疫后7天后试验组产蛋率恢复正常。ISA 71VG佐剂组蛋鸡平均日增重显著降低,ISA 201VG和ISA 15AVG佐剂组蛋鸡平均日增重与对照组无显著差异;ISA 201VG佐剂组血清抗体与病毒反应活性良好,中和效价可达1:256。ISA 201VG佐剂配伍的疫苗免疫效果优于其他两种佐剂疫苗。

关键词: 猪传染性胃肠炎病毒S蛋白, 猪流行性腹泻病毒S蛋白, 佐剂, 抗体, 免疫原性

Abstract:

This study aims to explore the effects of different adjuvants on the immunogenicity of Transmissible gastroenteritis virus (TGEV) S protein and Porcine epidemic diarrhea virus (PEDV) S protein. IPTG-induced recombinant expression strains Rosetta (DE3)-pET28a-TGEV-S2/3 and BL21 (DE3) -pET28a-PEDV-S1/2/3 were used to prepare recombinant proteins TGEV-S2, TGEV-S3, PEDV-S1, PEDV-S2 and PEDV-S3, and to conduct SDS-PAGE and western blot identification. The recombinant protein was mixed with three types of adjuvants, ISA 71VG, ISA 201VG and ISA 15AVG respectively, to prepare immunogen which was used to vaccinate layers. Western blot was used to detect serum antibody titer of layers, and immunoperoxidase monolayer cell staining assay (IPMA) was used to detect antibody and pathogen reactivity. The average daily weight gain of the flock was monitored. The results showed that the molecular weights of recombinant proteins TGEV-S2, TGEV-S3, PEDV-S1, PEDV-S2 and PEDV-S3 were 25 ku, 28 ku, 26 ku, 24 ku and 21 ku, respectively. Western blot analysis showed that all the five proteins had good immunogenicity. The highest antibody titer was 64000 times, 32000 times and 8000 times respectively in the ISA 201VG adjuvant group, ISA 71VG adjuvant group and ISA 15AVG adjuvant group, while no specific antibody was detected in the control group. The ISA 71VG and ISA 201VG group had a relatively large decrease in the egg production rate, while the ISA15AVG adjuvant group had a relatively small impact on the egg production rate, and the egg production rate in the experimental group returned to normal 7 days after immunization. The average daily weight gain of layers in the ISA 71VG adjuvant group was significantly decreased; while the average daily weight gain of layers in the ISA 201VG and ISA 15AVG adjuvant group was not significantly different from that in the control group. The serum antibody of ISA 201VG group had good reactivity with neutralization titer up to 1:256. The immune effect of the vaccine with ISA 201VG adjuvant was better than that of the other two adjuvants.

Key words: swine transmissible gastroenteritis virus S protein, porcine epidemic diarrhea virus S protein, adjuvant, antibodies, immunogenicity

中图分类号:

S855.3

韩涛涛, 黎露, 唐青海, 吴广艳, 孙国印, 李晓楠, 常秋玲, 杨海, 唐斯萍, 王芳宇. 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): 143-140.

Han Taotao, Li Lu, Tang Qinghai, Wu Guangyan, Sun Guoyin, Li Xiaonan, Chang Qiuling, Yang Hai, Tang Siping, Wang Fangyu. Different Adjuvants: Effects on S Protein Immunogenicity of Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus[J]. Chinese Agricultural Science Bulletin, 2020, 36(30): 143-140.

图/表 7

分组	抗原佐剂配伍	剂量	接种方式	免疫程序
A(n=30)	TGEV+PEDV- ISA 71VG	1 mL/羽/次	胸部肌肉注射	免疫2次,间隔21 d
B(n=30)	TGEV+PEDV- ISA 201VG	1 mL/羽/次	胸部肌肉注射	免疫2次,间隔21 d
C(n=30)	TGEV+PEDV- ISA 15AVG	1 mL/羽/次	胸部肌肉注射	免疫2次,间隔21 d
D(n=30)	0.85%生理盐水	1 mL/羽/次	胸部肌肉注射	免疫2次,间隔21 d

表1. 动物分组及免疫

图1. TGEV和PEDV S重组蛋白的SDS-PAGE检测(A)和对应的Western blot鉴定(B) M：Marker;1~5依次为：TGEV S2、TGEV S3、PEDV S1、PEDV S2和PEDV S3

图2. 不同佐剂配伍的TGEV-PEDV二联亚单位疫苗免疫蛋鸡血清中抗体滴度western blot检测

图3. 不同佐剂配伍的TGEV-PEDV二联亚单位疫苗接种鸡血清中抗体消长规律

图4. TGEV-PEDV二联免疫血清与TGEV反应活性检测

图5. TGEV-PEDV二联免疫血清与PEDV反应活性检测

图6. 整个实验期内所有蛋鸡的平均日增重

参考文献 23

[1]	Trudeau M P, Verma H, Urriola P E, et al. Survival of porcine epidemic diarrhea virus (PEDV) in thermally treated feed ingredients and on surfaces[J]. Porcine Health Manag, 2017,3:17. URL pmid: 28932412
[2]	Zhang Q, Hu R, Tang X, et al. Occurrence and investigation of enteric viral infections in pigs with diarrhea in China[J]. Archives of virology, 2013,158(08):1626-1631.
[3]	Annamalai T, Lin C M, Gao X, et al. Cross protective immune responses in nursing piglets infected with a US spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original US PEDV strain[J]. Vet Res, 2017,48(01):61. doi: 10.1186/s13567-017-0469-7 URL
[4]	Yu M, Wang L, Ma S, et al. Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus[J]. Viruses, 2017,9(10):49-51. doi: 10.3390/v9030049 URL
[5]	蒲翠敏, 冯旭芳, 张双翔, 等. PEDV、TGEV和PoRV三重PCR检测方法的建立及初步应用[J]. 动物医学进展, 2018,39(02):19-25.
[6]	Sun Y G, Li R, Xie S, et al. Identification of a novel linear B-cell epitope within the collagenase equivalent domain of porcine epidemic diarrhea virus spike glycoprotein[J]. Virus Res, 2019. doi: 10.1016/j.virusres.2020.198164 URL pmid: 32949657
[7]	Yuan X, Lin H, Fan H. Efficacy and immunogenicity of recombinant swinepox virus expressing the A epitope of the TGEV S protein[J]. Vaccine, 2015. URL pmid: 33012608
[8]	邓碧华, 朱俊, 马鹤毓, 等. 兽用疫苗佐剂的研究进展[J]. 畜牧兽医科学:电子版, 2017(12):5-6.
[9]	Booth J S, Nichani A K, Benjamin P, et al. Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells[J]. Veterinary Immunology & Immunopathology, 2007,115(1-2):24. doi: 10.1016/j.vetimm.2006.09.008 URL pmid: 17067685
[10]	翟路峰, 胡东波, 廖永洪, 等. 猪圆环病毒2型-肺炎支原体二联灭活疫苗佐剂的筛选[J]. 黑龙江畜牧兽医, 2017,298:147-150.
[11]	贾晓娟. 猪Gp96N增强PRRSV合成肽疫苗、亚单位疫苗及PCV2灭活疫苗免疫效应的分析[D]. 北京:中国农业大学, 2014.
[12]	Harandi A, Medaglini D, Shattock R. Vaccine Adjuvants:A priority for vaccine research[J]. Vaccine, 2010,28(12):2363-2366. URL pmid: 20064476
[13]	Zhang X, Calvert R A, Sutton B J, et al. IgY: a key isotype in antibody evolution[J]. Biol Rev Camb Philos Soc, 2017. doi: 10.1111/brv.12641 URL pmid: 32794644
[14]	Nafea N M, Sabbah M A, Al-Suhail R, et al. Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay[J]. Adv Biomed Res, 2015,4:100. URL pmid: 26015926
[15]	Vega C, Bok M, Saif L, et al. Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves[J]. Res Vet Sci, 2015,103:1-10. URL pmid: 26679788
[16]	Lee do 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]. Arch Virol, 2015,160(09):2197-2207. doi: 10.1007/s00705-015-2494-z URL
[17]	崔焕忠, 姜海龙, 张加力, 等. 抗猪传染性胃肠炎病毒与猪流行性腹泻病毒卵黄抗体制备及其临床应用研究[J]. 中国畜牧兽医, 2012,39(06):173-175.
[18]	王爱富. 抗猪流行性腹泻病毒S1蛋白卵黄抗体制剂的制备[D]. 佛山:佛山科学技术学院, 2017.
[19]	Yin Y, Zhu L, Liu P, et al. Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus[J]. Braz J Microbiol, 2019,50(01):279-286. doi: 10.1007/s42770-018-0022-5 URL
[20]	赖庆光. 不同佐剂对伪狂犬活疫苗免疫效果的比较研究[D]. 南昌:江西农业大学, 2011.
[21]	马涛, 王先炜, 李玉峰, 等. 不同佐剂对猪圆环病毒2型灭活抗原免疫效果的影响[J]. 中国兽医科学, 2011,41:593-600.
[22]	薛海燕, 杜枘宣, 韩芳, 等. 卵黄抗体/壳聚糖微囊的制备与体外释放性能的研究[J]. 食品工业科技, 2014,35:57-61.
[23]	于小娟. 不同佐剂配合ETEC灭活疫苗对蛋鸡免疫原性的影响[D]. 大连:大连理工大学, 2017.

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

Different Adjuvants: Effects on S Protein Immunogenicity of Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

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