Immunogenicity of Porcine Epidemic Diarrhea Virus Tandem Epitope Subunit Vaccine
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摘要: 将纤突蛋白S的COE区域 (E1)、S1D区域 (E2)、C末端区域 (E3) 以及膜蛋白M的M3区域 (E4) 设计成串联表位亚单位 (EC)，以报道的COE和S1亚单位作为对照，构建了不同亚单位疫苗的杆状病毒载体表达系统。杆状病毒载体表达系统生产的目标蛋白采用镍柱亲和层析进行纯化。在BALB/c小鼠上，不同亚单位疫苗的免疫原性初步评价结果表明，EC、COE和S1序列分别成功插入杆状病毒基因组，EC、COE和S1蛋白均能在Sf9细胞中分泌表达，但是纯化条件依蛋白不同而有差异。与COE和S1亚单位疫苗相比，EC亚单位疫苗能激发小鼠产生更多的特异性免疫球蛋白IgG、干扰素-γ和肿瘤坏死因子-α。结果表明，各个亚单位疫苗均能激发小鼠的体液免疫与细胞免疫，与COE和S1亚单位疫苗相比，EC亚单位疫苗能更好地激发小鼠的体液免疫与细胞免疫。
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关键词:
- 猪流行性腹泻病毒 /
- 串联表位 /
- 亚单位疫苗 /
- 杆状病毒载体表达系统
Abstract: Porcine epidemic diarrhea virus can infect pigs at different ages and cause porcine epidemic diarrhea, leading to heavy economic losses to the pig industry around the world. There is currently no effective treatment for porcine epidemic diarrhea, and vaccination seems to be its key preventive approach. In order to develop an effective porcine epidemic diarrhea virus subunit vaccine, a tandem epitope subunit (EC) was assembled from the COE region (E1), S1D region (E2), and C-terminal region (E3) of spike protein, and the M3 region (E4) of membrane protein, and a baculovirus expression system was constructed for the production of subunit vaccine EC. The reported candidate subunits COE and S1 were used as positive controls. Three target proteins of EC, COE and S1 were produced by baculovirus expression vector systems in insect cell line Sf9, and purified with a nickel affinity chromatography column, respectively. The immunogenicity of the different subunit vaccines was evaluated on BALB/c mouse. The results showed that the EC, COE and S1 gene sequences were successfully inserted into the baculovirus genome. All the three proteins could be expressed and secreted into culture supernatant. Compared with the subunit vaccines of COE and S1, subunit vaccine EC could stimulate mouse to produce larger amount of specific immunoglobulin G, interferon-γ and tumor necrosis factor-α than controls. The above results indicate that each subunit vaccine can stimulate the humoral and cellular immunity of mouse, and the immunogenicity of subunit vaccine EC is much stronger. -
图 1 携带EC、COE、S1序列的重组杆状病毒菌落验证
Figure 1. Validation of recombinant baculovirus colonies born with EC, COE, S1 sequences
图 2 Western Blot 检测COE (a)、EC (b) 和S1 (c) 蛋白在昆虫细胞系Sf9中表达
Figure 2. Expressions of the COE (a), EC (b) and S1 (c) proteins in insect cell line Sf9 detected by Western Blot
1— Culture supernatant of sf9 cells infected with recombinant baculovirus; 2— Lysate supernatant of sf9 cells infected with recombinant baculovirus; 3—Lysate precipitation of sf9 cells infected with recombinant baculovirus
图 3 SDS-PAGE检测镍柱亲和层析纯化的EC (a)、COE (b) 和S1 (c) 蛋白
Figure 3. SDS-PAGE determinations of EC (a), COE (b) and S1 (c) proteins purified with Ni affinity chromatography column
Y—Pre-column sample; L—Flow-through sample; 10, 15, 50, 100, 500: 10, 15, 50, 100, 500 mmol/L imidazole eluent, respectively
图 4 ELISA检测两次免疫EC、COE或S1亚单位疫苗小鼠血清IgG (a)、IFN-γ (b) 和TNF-α (c) 水平
Figure 4. ELISA determination of serum IgG (a), IFN-γ (b) and TNF-α (c) levels of mouse vaccinated with EC, COE or S1 subunit vaccine twice
表 1 引物信息
Table 1. Primers information
Primer names Primer sequences (5’→3’) signal-F GGCGCGGGATCCATGCGCTCACTGATCTAC signal-R GCAGAGTCACACGGGTCACGTCCTGAGGCA E1-F CGTGACCCGTGTGACTCTGCCCTCATTCAA E1-R CATGAAGGAGACGTCAGTGCTACCGCCTCCACCGACGTCAGTGAT E1-R1 GTGATGGTGATGGTGATGGACGTCAGTGATGCCTTCCAGAGGC E2-F TAGCACTGACGTCTCCTTCATGACCCTGGA E2-R GAAAGCCTCGTAGGGCTGCAGGCTACCGCCTCCACCGATAGACATGCT E4-F GTAGCCTGCAGCCCTACGAGGCTTTCGGTGGAGGCGGTAGCGTGCAGGTC E4-R GTGATGGTGATGGTGATGCACCAGGTGCAGGACCTTCTCGGAGTCAGT S1-R GTGATGGTGATGGTGATGGTGGTAGAAGAAACCTGGCAGTTCA P-EC-F GTGCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAG P-E1-F GTCCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAGGA P-S1-F ACCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAG P-EC-R TCAGTGAGCGCATGGATCCCGCGCCCGATGGTGGGACG 
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