含有Th细胞表位的合成PCV2 ORF2 Cap多肽抗原的表达及免疫原性分析

doi:10.11843/j.issn.0366-6964.2015.12.020

摘要/Abstract

摘要：

拟测定在PCV2 ORF2 Cap P1多肽抗原中添加从PCV2 ORF1、ORF3中筛选的Th细胞表位多肽在促进其免疫原性中的作用，以及合成表达的PCV2 ORF2 Cap P1多肽抗原作为疫苗抗原的可行性。采用生物信息学及分子生物学方法，筛选获得1条泛宿主新型Th细胞表位多肽和3条PCV2特异的含有Th细胞抗原表位的多肽序列，然后将这4条Th细胞多肽氨基酸序列与筛选自ORF2 Cap1上的1条B细胞表位多肽序列进行串联组合，密码子优化，插入酶切位点、终止密码子，然后进行密码子适应指数和分布频率分析，预测可以实现高效表达后再进行化学合成。合成的P1多肽抗原连接到pET-30a表达载体，并转化至BL21(DE3)pLysS感受态细胞中，构建表达工程菌BL21(DE3)pLysS-pET-30a-P1。经IPTG诱导表达后P1可实现高效表达，SDS-PAGE鉴定表达量，Western Blot鉴定其生物活性，然后分别免疫小鼠和猪，测定小鼠免疫后的抗体水平以及猪免疫后P1合成多肽抗原对外周血淋巴细胞的刺激增殖情况。结果表明，设计合成表达的PCV2 P1多肽抗原序列，密码子适应性指数为0.89，能够实现高水平表达，目的片段大小约为28.29 ku，具有良好的免疫原性；MTT试验结果表明，P1多肽抗原免疫后机体内IFN-γ和IL-4的表达量明显增加，且与对照组差异显著（P＜0.05）。这说明P1能够诱导机体产生细胞免疫和体液免疫反应，为其作为疫苗用抗原的进一步研究奠定了理论基础。

Abstract:

To determine the role of Th cell epitopes，which were screened from PCV2 ORF1，and ORF3，in improving the PCV2 ORF2 Cap P1 immunogenicity when they were added to the sequence，and the feasibility of synthetic expression of PCV2 ORF2 Cap P1 as a vaccine antigen，bioinformatics and molecular biology methods were employed to screen the peptide sequences.A pan-host of T helper cell epitope peptides and three Th cell epitopes peptide sequences of PCV2-specific were identified.These 4 Th cells peptides sequences were combined with a B-cell epitope peptide sequence screened from PCV2 ORF2 Cap1 gene，and then codon was optimized and inserted the restriction sites and stop codons.After analyzing the codon adaptation index and the frequency distribution，predicted it can achieve efficient expression，further chemical synthesis or peptides were carried out.The synthetic polypeptide antigen P1 was connected to the pET-30a expression vector，and transformed into BL21(DE3) pLysS competent cells，the expression in the engineered bacteria BL21(DE3) pLysS-pET-30a-P1 was constructed.The P1 antigen was able to be expressed after IPTG inducting.The expression level was identified by SDS-PAGE，and its biological activity was identified by Western Blot，followed by mice and pigs immunization with P1.Antibody levels in mice and the peripheral blood lymphocyte proliferation in pigs were tested.The results showed that the codon adaptation index of PCV2 P1 sequence was 0.89，it can achieve to high levels of expression，and the peptide fragment is about 28.29 kDa.MTT results showed that P1 peptide antigen had good immunogenicity.In immunized pigs，the expression of IFN-γ and IL-4 can increase obviously，and were significantly different to the control group(P<0.05).These results indicated that P1 could induce a strong humoral and celluar immune response，and it will provide a theoretical basis on PCV2 P1 peptide antigen as a vaccine antigen for further studies.

中图分类号:

S852.4

陈善真，赵焱，李中圣，罗均，陈克宏，王贵平，李其昌. 含有Th细胞表位的合成PCV2 ORF2 Cap多肽抗原的表达及免疫原性分析[J]. 畜牧兽医学报, 2015, 46(12): 2273-2281.

CHEN Shan-zhen，ZHAO Yan，LI Zhong-sheng，LUO Jun，CHEN Ke-hong，WANG Gui-ping，LI Qi-chang. Expression and Immunogenicity Analysis of PCV2 Cap Recombinant Protein Antigen which Contains Helper T cell Epitope Peptides[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2273-2281.

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https://www.xmsyxb.com/CN/Y2015/V46/I12/2273

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