CN113786480B - 非洲猪瘟病毒a137r和k205r基因的应用 - Google Patents
非洲猪瘟病毒a137r和k205r基因的应用 Download PDFInfo
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Abstract
本发明涉及一种非洲猪瘟病毒在制备非洲猪瘟病毒疫苗和调节细胞炎症反应中的应用。本发明提供的应用具体为非洲猪瘟病毒A137R和K205R在制备非洲猪瘟病毒疫苗和调节细胞炎症反应中的应用。非洲猪瘟病毒A137R和K205R基因能够显著抑制病毒感染诱导的IL‑8的表达。通过双荧光素酶报告基因系统检测发现，与对照组相比，表达A137R和K205R基因能够抑制仙台病毒(SEV)、水泡性口炎病毒(VSV)、伪狂犬病毒(PRV)、流感病毒(PR8毒株)诱导的IL‑8启动子活性。通过荧光定量PCR试验，发现A137R和K205R基因的表达能够显著抑制仙台病毒感染诱导的IL‑8的mRNA表达水平。综上，本发现鉴定了两种具有抑制宿主细胞抗病毒天然免疫应答功能的非洲猪瘟病毒蛋白，为制备非洲猪瘟病毒基因缺失疫苗提供了新的选择。
Description
技术领域
本发明属于生物医药领域，涉及两种病毒蛋白的功能鉴定及其在制备基因缺失疫苗中的应用，主要涉及非洲猪瘟病毒A137R和K205R蛋白的功能鉴定及其在制备非洲猪瘟病毒基因缺失疫苗中的应用。
背景技术
非洲猪瘟(ASF)于1921年在肯尼亚首次发现，随后蔓延至欧洲、南美洲和东南亚，在过去的一个世纪中，给全世界的养猪业造成了巨大的经济损失。ASF于2018年在辽宁省首次报道。ASF是发生在家猪和野猪身上的一种急性致死出血性病毒性疾病，死亡率高，接近100％。本病常导致淋巴结、肾脏、心脏等多个器官的出血性坏死，尤以异常脾肿大和出血为主。ASF的病原体是非洲猪瘟病毒(ASFV)，属于非洲猪瘟病毒属。ASFV是核胞浆大DNA病毒(NCLDV)，具有相当复杂的结构，包括含有DNA的中央核仁、核壳、内脂膜、二十面体衣壳和外脂膜。基因组长度的多样性是ASFV的重要特征之一，不仅在不同分离菌株之间不同，而且在同一菌株的不同培养代之间也有差异，主要原因是病毒基因组可以随机放弃或获得外源性序列，这可能是病毒免疫逃逸的原因之一。
ASFV基因组约170～194kb，编码150～170个蛋白，但大部分蛋白的功能尚不清楚。基于编码衣壳蛋白p72的B646L基因，ASFV可分为24个不同的基因型。在中国，基因型II型ASFV疫情暴发，摧毁了养猪业，导致猪肉价格迅速上涨，严重影响了人民的生活水平。因此，开发有效的疫苗和抗病毒药物尤为迫切。在过去的几十年里，人们尝试了许多不同的疫苗接种策略，包括灭活疫苗、DNA疫苗、亚单位疫苗和腺病毒载体疫苗，但所有这些疫苗都未能诱导对ASFV的保护性免疫。然而，一些基因缺失的疫苗删除了一些编码干扰素(IFN)抑制蛋白的基因(如MGF360 1R-3R、MGF505 12L-14L)已被证明可以减弱病毒的毒性并诱导对同源攻击的保护，但在猪的复制过程中仍有一些问题需要评估，包括转化为毒性更强的毒株，将病毒释放到环境中等。
白细胞介素8(IL-8)，也称为中性粒细胞激活因子(NAF)，是第一个被鉴定的趋化因子，最初被鉴定为白细胞趋化因子。IL-8主要由中性粒细胞、单核细胞、巨噬细胞、T细胞、上皮细胞和内皮细胞产生。CXCR1、CXCR2和DARC是IL-8的三种受体，它们通过与不同的受体结合而产生不同的生物学效应。IL-8的生物学作用为：(1)强烈趋化并激活中性粒细胞，此作用无种属特异性。中性粒细胞在血液的非特异免疫系统中起着十分重要的作用，它处于机体抵抗病原微生物，特别是在化脓性细菌侵入的第一线，IL-8可将其吸引到炎症部位并激活，通过非特异性吞噬杀灭病原体。还可通过中性粒细胞表达IgG的Fc受体，在IgG抗体的参与下通过细胞毒性作用而特异性的病原体机器他靶细胞。(2)趋化碱性粒细胞，并促进其释放活性物质，参与速发性的超敏反应。(3)趋化部分静止的T细胞，还可趋化IL-2活化的NK细胞。有研究发现，ASFV的一种毒性分离株Malawi Lil 20/1可以感染血管内皮细胞，导致表型变化，通过抑制细胞活化中重要分子的表面表达，例如粘附分子E-选择素和MHC I类以及炎症细胞因子IL-6和IL-8的转录，使感染的细胞启动血液凝固和白细胞募集的正常炎症反应失活，因此抑制相关免疫细胞活化可能是病毒的重要免疫逃避策略。当然，目前研究还发现，ASFV多基因家族蛋白，例如MGF360和MGF505家族，在非洲猪瘟病毒感染的初期能够抑制宿主分泌干扰素的能力，从而成功感染宿主细胞，由此可见，非洲猪瘟病毒对宿主的免疫逃逸策略是多种多样、相互协同作用的，探究非洲猪瘟病毒抑制天然免疫的机制有助于了解非洲猪瘟病毒的致病机制和疫苗的研发。
发明内容
本发明的目的是提供一种病毒蛋白的功能鉴定及其在制备基因缺失疫苗中的应用。
非洲猪瘟病毒A137R和K205R基因在制备非洲猪瘟病毒疫苗中的应用，非洲猪瘟病毒A137R和K205R基因核酸序列如SEQ ID No.1和SEQ ID No.2所示。
非洲猪瘟病毒A137R和K205R基因在抑制宿主细胞抗病毒天然免疫应答中的应用。
用非洲猪瘟病毒A137R和K205R基因翻译成的蛋白质在制备非洲猪瘟病毒疫苗中的应用，其中非洲猪瘟病毒A137R基因翻译成蛋白质的氨基酸序列为：
非洲猪瘟病毒K205R基因翻译成蛋白质的氨基酸序列为：
试验证明，非洲猪瘟病毒A137R和K205R基因能够显著抑制病毒感染诱导的IL-8的表达。通过双荧光素酶报告基因系统检测发现，与对照组相比，表达A137R和K205R基因能够抑制仙台病毒(SEV)、水泡性口炎病毒(VSV)、伪狂犬病毒(PRV)、流感病毒(PR8毒株)诱导的IL-8启动子活性。通过荧光定量PCR试验，发现A137R和K205R基因的表达能够显著抑制仙台病毒感染诱导的IL-8的mRNA表达水平。通过ELISA试验，发现A137R和K205R基因的表达能够显著抑制由仙台病毒感染诱导的IL-8的蛋白表达水平。综上，本发现鉴定了两种具有抑制宿主细胞抗病毒天然免疫应答功能的非洲猪瘟病毒蛋白，为制备非洲猪瘟病毒基因缺失疫苗提供了新的选择。
附图说明
图1为A137R和K205R基因对仙台病毒(SEV)、水泡性口炎病毒(VSV)、伪狂犬病毒(PRV)、流感病毒(PR8毒株)诱导的IL-8启动子活性的影响。**表示差异极显著(P<0.01)，***表示差异极极显著(P<0.001)；
图2-A为A137R和K205R基因对仙台病毒诱导的IL-8的mRNA表达量的影响。**表示差异极显著(P<0.01)，***表示差异极极显著(P<0.001)；
图2-B为A137R和K205R基因对仙台病毒诱导的IL-8蛋白表达水平的影响。**表示差异极显著(P<0.01)，***表示差异极极显著(P<0.001)。
具体实施方法
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法及装置，如无特殊说明，均为常规方法及装置。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购得。以下实施例中对突变体突变位点的确定，均由常规测序公司测序确定。为使本发明专利的目的、技术方案和优点更加清楚，下面结合具体实施例对本发明专利的具体实施方式进行详细说明。这些优选实施方式的示例在具体实施例中进行了例示。
在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明专利的技术方案，在实施例中仅仅示出了与根据本发明专利的方案密切相关的技术方案和/或处理步骤，而省略了关系不大的其他细节。
293T细胞(人胚肾细胞系)：美国标准生物品收藏中心(American Type CultureCollection，ATCC)，编号为CRL-3216。
实施例1.非洲猪瘟病毒A137R和K205R基因可显著抑制病毒感染诱导的IL-8启动子活性
本发明以非洲猪瘟病毒China/2018/AnhuiXCGQ(GenBank登录号：MK128995.1)A137R和K205R基因的核苷酸序列为模板，由苏州金唯智生物科技有限公司合成，并成功构建到pCAGGS载体上，分别命名为pCAGGS-A137R和pCAGGS-K205R，质粒构建后，送北京擎科生物技术有限公司进行测序，测序无误后，进行后续的细胞试验。
复苏293T细胞，并培养于含有双抗(100units/mL青霉素和100units/mL链霉素)和10％的胎牛血清(Gibco)的DMEM(Gibco)培养基中。先将293T细胞均匀铺到24孔板中，置于37℃，5％CO2浓度的细胞培养箱中培养。待细胞长到汇和度为80-90％时，将500ngpCAGGS-A137R或pCAGGS-K205R分别与500ng的IL-8-Luc、50ng的内参pRL-TK质粒，依照VigoFect(威格拉斯生物技术有限公司)的说明书，共转染至293T细胞，转染4-6h后换成完全培养基继续培养，每组实验设置三个重复。转染24h后，将完全培养基换成病毒维持液(无血清DMEM中含2μg/ml的胰酶)，分别用仙台病毒、水泡性口炎病毒(VSV)、伪狂犬病毒(PRV)、禽流感病毒(PR8毒株)(100HAU/ml)感染细胞，病毒吸附1h，期间每15min倾斜摇晃细胞板一次。吸附1h后，吸去上清，用PBS清洗细胞3次，加入500uL病毒维持液。病毒感染20h后，按照双荧光素酶报告系统检测试剂盒(PromegaReport)的推荐方法收取蛋白样，离心后取上清到96孔板中，用GloMax进行荧光测定。用GraphPad Prism8对实验结果进行分析。结果发现，如图1所示，ASFV的A137R和K205R两个基因具有较为明显的抑制IL-8启动子活性。
实施例2.非洲猪瘟病毒A137R和K205R基因能抑制IL-8的转录
先将293T细胞铺到12孔板中，待细胞长到汇和度为80-90％时，按照VigoFect说明书将2μg的pCAGGS-A137R和pCAGGS-K205R分别瞬时转染293T细胞，4-6h后换成完全培养基。转染24h后用SeV(100HAU/ml)刺激细胞16h，然后用TRIzol(品牌：MNG，货号：740404.200)裂解细胞，并按照说明书提取RNA，反转录后用做荧光定量PCR(qPCR)的模板。按照qPCR(北京全式金生物技术有限公司，Green qPCR SuperMix)说明书的推荐体系进行荧光定量PCR实验。实验结果用GraphPad Prism8进行分析。结果表明，如图2-A所示，A137R和K205R蛋白都可抑制由SEV诱导的IL-8的转录。
实施例3.非洲猪瘟病毒A137R和K205R基因能抑制IL-8的蛋白表达水平
先将293T细胞铺到12孔板中，待细胞长到汇和度为80-90％时，按照VigoFect说明书将2μg的pCAGGS-A137R和pCAGGS-K205R分别瞬时转染293T细胞，4-6h后换成完全培养基。转染24h后用SeV(100HAU/ml)刺激细胞16h，然后收取上清，用Human IL-8/CXCL8 ELISAKit(品牌：ABclonal，货号：RK00011)，并按照说明书操作。实验结果用GraphPad Prism8进行分析。结果表明，如图2-B所示，A137R和K205R蛋白都可抑制由SeV诱导的IL-8的表达。IL-8作为趋化因子，能够激活多种免疫细胞进而抵抗病毒感染，在宿主抵抗病毒感染的过程中发挥着至关重要的作用。而A137R和K205R基因能够显著抑制IL-8的表达，进一步确定了A137R和K205R是与免疫抑制相关的基因，该发现为制备非洲猪瘟病毒基因缺失疫苗提供了新的选择。
综合上述三个实施例的结果，可证明非洲猪瘟病毒A137R和K205R基因具有抑制宿主细胞抗病毒天然免疫应答的功能，而且可作为构建非洲猪瘟病毒基因缺失疫苗的候选缺失基因。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
序列表
<110> 福建农林大学
<120> 非洲猪瘟病毒A137R和K205R基因的应用
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Claims (4)
1.非洲猪瘟病毒A137R基因在制备抑制IL-8转录和表达的试剂中的应用，非洲猪瘟病毒A137R基因核酸序列分别如SEQ ID No.1所示。
2.非洲猪瘟病毒K205R基因在制备抑制IL-8转录和表达的试剂中的应用，非洲猪瘟病毒K205R基因核酸序列分别如SEQ ID No.2所示。
3.非洲猪瘟病毒A137R基因翻译成的蛋白质在制备抑制IL-8转录和表达的试剂中的应用，非洲猪瘟病毒A137R基因核酸序列分别如SEQ ID No.1所示。
4.非洲猪瘟病毒K205R基因翻译成的蛋白质在制备抑制IL-8转录和表达的试剂中的应用，非洲猪瘟病毒K205R基因核酸序列分别如SEQ ID No.2所示。
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