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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
引用本文: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. DOI: 10.11924/j.issn.1000-6850.casb20191000740
作者姓名: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
作者单位: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
基金项目: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)
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  
收稿时间:2019-10-25

Different Adjuvants: Effects on S Protein Immunogenicity of Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus
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. DOI: 10.11924/j.issn.1000-6850.casb20191000740
Authors:Han Taotao  Li Lu  Tang Qinghai  Wu Guangyan  Sun Guoyin  Li Xiaonan  Chang Qiuling  Yang Hai  Tang Siping  Wang Fangyu
Affiliation:1.College of Life Sciences and Environment, Institute of Bio-technology for Livestock, Hengyang Normal University, Hengyang Hunan 421008;2.Nanyang Tianhua Pharmaceutical Co., LTD., Nanyang Henan 473061
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.
Keywords:swine transmissible gastroenteritis virus S protein  porcine epidemic diarrhea virus S protein  adjuvant  antibodies  immunogenicity  
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