非洲猪瘟病毒P72蛋白合成肽疫苗的构建及其免疫效力评估

【目的】构建非洲猪瘟病毒CAS19-01/2019株(GenBank登录号:MN172368.1)结构蛋白P72的合成肽疫苗,通过免疫小鼠评估合成肽疫苗的免疫效力。【方法】利用ProtParam、SOPMA等软件分析P72蛋白的理化性质与结构信息,通过ABCpred、SVMtrip、IEDB预测P72蛋白的T细胞与B细胞抗原表位,筛选出显著的表位多肽区域,合成多肽辅以弗氏佐剂腹腔注射免疫小鼠,检测免疫组小鼠产生的特异性抗体、T淋巴细胞亚群、脾脏淋巴细胞增殖、细胞因子白介素4(IL-4)、IL-2、γ干扰素(IFN-γ)、免疫球蛋白(IgG),从体液免疫与细胞免疫角度评估合成肽的免疫效力。【结果】综合分析得出P72蛋白是稳定性亲水蛋白,二级结构中α-螺旋、β-转角、延伸链、无规则卷曲分别占19.35%、5.42%、25.08%和50.15%。筛选出了P72蛋白的8个优势抗原表位,626－634、520－528、298－306、203－211位氨基酸处为T细胞抗原表位,587－606、232－251、110－129、39－58位氨基酸处为B细胞抗原表位。整合优势表位合成2个多肽P72-1与P72-2,首次免疫小鼠14 d时可检测到P72-1与P72-2的特异性抗体,首免后28 d达到最高值,其最高抗体效价分别为1∶25 600与1∶12 800;免疫后小鼠T淋巴细胞亚群CD4^＋/CD8^＋显著上升(P＜0.05);脾脏淋巴细胞增殖试验结果显示,免疫组淋巴细胞数量均极显著升高(P＜0.01);细胞因子IL-4、IL-2、IFN-γ含量均极显著增加(P＜0.01)。【结论】本研究成功研制2种合成肽疫苗,在免疫效力上P72-2高于P72-1,二者都能产生高水平的特异性抗体,刺激脾脏淋巴细胞增殖,诱导产生细胞因子IL-4、IL-2、IFN-γ,本研究为非洲猪瘟合成肽疫苗研制奠定技术基础。

Construction of Synthetic Peptide Vaccine of African Classical Swine Fever Virus P72 Protein and Evaluation of Its Immune Efficacy

【Objective】 The aim of this study was to construct a synthetic peptide vaccine based on protein P72 in African swine fever virus (ASFV) strain CAS19-01/2019(GenBank accession No.:MIN172368.1),and evaluate its immune efficiency by immunizing mice.【Method】 The physicochemical properties and structural information of P72 protein were analyzed by programs of ProtParam and SOPMA.The significant epitopes in T and B lymphocytes were screened by ABCpred,SVMtrip and IEDCB.The synthesized peptides were injected intramuscularly into mice with Freund's adjuvant.Antibodies against the synthesized peptides,T lymphocyte subsets,lymphocyte proliferation,cytokines contents of interleukin 4(IL-4),IL-2,interferon-γ (IFN-γ) and immunoglobulin (IgG) were detected using serum and other tissue from the immunized mice,and the immune efficacy of the synthetic peptide was evaluated from the perspectives of humoral immunity and cellular immunity.【Result】 Comprehensive analysis showed that P72 protein was a stable hydrophilic protein.In secondary structure,alpha helix,beta turn,extended strand and random coil accounted for 19.35%,5.42%,25.08% and 50.15%,respectively.Eight dominant epitopes of protein P72 were selected by comprehensive software analysis,including T cell epitopes 626-634,520-528,298-306,203-211 amino acids,and B cell epitopes 587-606,232-251,110-129,39-58 amino acids. Two peptides named P72-1 and P72-2 were synthesized by integrating the dominant epitopes together.The specific antibodies against the synthesized peptides P72-1 and P72-2 were determined in serum of mice after 14 days of the first immunization,reached the highest value on the 28th day after the first immunization,and highest antibody titers were 1:25 600 and 1:12 800,respectively.The value of CD4⁺/CD8⁺ of T lymphocyte subsets in immunized mice were significantly increased (P<0.05).The spleen lymphocyte proliferation test showed that the number of lymphocytes was enhanced in both groups (P<0.01).Cytokines contents of IL-4,IL-2 and IFN-γ were extremely significantly improved (P<0.01).【Conclusion】 In this study,two synthetic peptide vaccines were successfully developed.P72-2 was higher than P72-1 in immune efficacy,and both could produce high level of specific antibodies,stimulate the proliferation of lymphocytes,induce the production of cytokines IL-4,IL-2 and IFN-γ.This study laid a technical foundation for the development of African swine fever vaccine.