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

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)-pET28aPEDV-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。

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)-pET28 a-TGEV-S2/3 and BL21(DE3)-pET28 a-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 71 VG, ISA 201 VG and ISA 15 AVG 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 201 VG adjuvant group,ISA 71 VG adjuvant group and ISA 15 AVG adjuvant group, while no specific antibody was detected in the control group. The ISA 71 VG and ISA 201 VG group had a relatively large decrease in the egg production rate,while the ISA15 AVG 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 71 VG adjuvant group was significantly decreased;while the average daily weight gain of layers in the ISA 201 VG and ISA 15 AVG adjuvant group was not significantly different from that in the control group. The serum antibody of ISA 201 VG group had good reactivity with neutralization titer up to1:256. The immune effect of the vaccine with ISA 201 VG adjuvant was better than that of the other two adjuvants.