CN114107247B - 鸡ripk3基因多克隆抗体及其制备方法与应用 - Google Patents
鸡ripk3基因多克隆抗体及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种鸡RIPK3基因多克隆抗体及其制备方法与应用,属于生物技术领域。该多克隆抗体是以RIPK3基因为抗原,通过动物免疫反应,制备的鸡RIPK3基因多克隆抗体。经实验验证发现该多克隆抗体能特异性结合鸡组织中的RIPK3蛋白。该多克隆抗体的制备简便,成本低,灵敏度高,作用范围广,为后期RIPK3蛋白的检测和病毒感染类疾病病理机制的研究奠定基础。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种鸡RIPK3基因多克隆抗体及其制备方法与应用。
背景技术
肾型传染性支气管炎病毒(Nephropathogenic infectious bronchitis virus,NIBV)是属于γ冠状病毒家族的单链正义RNA病毒,传染性强,且传播速度快,表现出强烈的肾脏组织嗜性,是诱发内脏痛风爆发的主要病原体之一。内脏型痛风不仅常见于家禽,也是困扰世界各种鸟类的常见疾病之一,有较高的致病率和死亡率,给世界家禽养殖业造成了巨大的经济损失。凡是能引起肾脏损伤和尿酸盐排泄障碍的因素都能引起痛风,据研究表明,能引起痛风的因素有20多种。目前,研究和报道最多的是NIBV。NIBV感染鸡可呈现白稀粪便,肾脏肿大苍白,呈花斑状,肾小管和输尿管乳白色,内有尿酸盐结晶等症状。根据组织病理学,NIBV在肾上皮细胞表面复制,导致肾小管上皮细胞颗粒变性、空泡化和脱落,以及间质中大量炎性细胞的浸润。然而,目前关于NIBV感染导致肾损伤的机制,我们知之甚少。
RIPK3是一种苏氨酸/丝氨酸蛋白激酶,与RIPK1、RIPK2有30%的共同序列同源性。N-末端激酶结构域同源性最高。RIPK3是坏死性凋亡的主要启动子,当RIPK3被磷酸化时,RIPK3和RIPK1通过RIP相互作用结构区域(RHIM),促进坏死复合体的形成。在坏死复合体中,RIPK3利用其C末端激酶样结构域招募并磷酸化混合谱系激酶结构域样蛋白(MLKL),磷酸化的MLKL移位到质膜上,并破坏细胞膜的完整性,扰乱细胞的渗透平衡,最终导致细胞破裂。在病毒感染的细胞中,这会导致损伤相关分子模式(DAMPs)和病原体相关分子模式(PAMPs)的释放。而DAMPs和PAMPs会被邻近的吞噬细胞识别,激活免疫系统,使坏死下垂成为一种强烈的免疫原性形式细胞死亡。与最初的假设相反,在没有RIPK1的情况下,坏死性凋亡也可以通过RIPK3进行。RIPK3在宿主防御细菌和病毒感染方面也是至关重要的。最有力的证据来自于单疱疹病毒(HSV)通过编码抗凋亡病毒蛋白抑制宿主细胞凋亡,此时宿主细胞通过RIPK3驱动坏死性凋亡来抑制病毒复制。此外,小鼠巨细胞病毒(CMV)M45基因编码的病毒蛋白,可以通过RHIM结构域与RIPK3相互作用而直接抑制病毒诱导的宿主细胞坏死性凋亡,从而促进其在宿主细胞内的复制。这些研究都表明RIPK3驱动的坏死性凋亡能作为一种限制病毒复制的途径,在体内清除病毒感染细胞过程中起着重要作用。尽管RIPK3主要被描述为坏死性下垂的调节因子,但它也在细胞凋亡和下睑下垂以及炎症信号中发挥作用。当RIPK3的激酶活性被抑制或者当该蛋白过度表达时,RIPK3会触发依赖于caspase-8的细胞凋亡。此外,在甲型流感模型中Ripk3−/−、Ripk3−/−Fadd−/−和Mlk1−/−Fadd−/−小鼠比野生型和MLKL−/−小鼠更容易感染甲型流感病毒,这也表明当坏死性下垂被抑制时,RIPK3还可以在细胞凋亡中发挥作用。焦亡(炎性细胞死亡),其特征是NLRP3炎症小体的组装,它导致caspace1的加工与激活,最终导致促炎细胞因子IL-1β、IL-18的释放。而炎症体可以以依赖RIPK3的方式被激活。由此可见,RIPK3驱动的细胞死亡在病毒感染中是必不可少的。目前还没有关于RIPK3在NIBV感染中作用的研究。
发明内容
本发明的目的是提供一种鸡RIPK3基因多克隆抗体及其制备方法与应用,以解决上述现有技术存在的问题,该多克隆抗体的制备简便,成本低,灵敏度高,作用范围广,为后期RIPK3蛋白的检测和病毒感染类疾病病理机制的研究奠定基础。
本发明提供一种所述的鸡RIPK3基因多克隆抗体的制备方法,包括以RIPK3基因为抗原,通过动物免疫反应,制备鸡RIPK3基因多克隆抗体。
优选的是,具体包括以下步骤:
(1)获取所述RIPK3基因的序列,并构建重组表达载体;
(2)利用所述重组表达载体诱导原核表达RIPK3重组蛋白;
(3)用所述的重组表达蛋白免疫动物,从免疫动物助攻分别纯化血清,获取鸡RIPK3基因多克隆抗体。
优选的是,获取所述RIPK3基因序列的引物为:
上游引物(SEQ ID NO:1):5’-CCGGAATTCGAAGGTCAAACAAACCGACT-3’;
下游引物(SEQ ID NO:2):5’-CCCAAGCTTTGGATCCTTACGCTGACA-3’。
本发明还提供一种所述的鸡RIPK3基因多克隆抗体在制备检测鸡组织中RIPK3表达水平的试剂盒中的应用。
本发明还提供一种检测鸡组织中RIPK3表达水平的试剂盒,包括所述的鸡RIPK3基因多克隆抗体。
本发明还提供一种所述的鸡RIPK3基因多克隆抗体在制备检测肾型传染性支气管炎病毒感染和其他病毒感染类疾病的产品中的应用。
优选的是,所述产品包括检测试剂、检测试剂盒或药物。
本发明公开了以下技术效果:
本发明通过生物信息学分析筛选出RIPK3基因优质的核苷酸编码序列,并以此为抗原免疫新西兰大白兔,再通过分离纯化血清获取鸡RIPK3多克隆抗体。经过对该多克隆抗体的灵敏性和特异性检测试验发现,RIPK3的血清效价为1:102400,RIPK3蛋白的表达主要集中于动物各种器官的细胞质部分,并且该多克隆抗体能特异性结合动物组织中的RIPK3蛋白。说明该多克隆抗体具有灵敏度高、特异性好、应用范围广等优势,这为后期RIPK3蛋白的检测和病毒感染类疾病病理机制的研究奠定基础。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为与肾型传染性支气管病毒致痛风鸡肾脏损伤发生发展相关的RIPK3基因多克隆抗体研制方法的技术路线图;
图2为表达重组质粒的构建的结果;A:RIPK3基因的聚合酶链式反应扩增产物(561bp),泳道1、2:RIPK3基因的PCR扩增产物;泳道3:阴性对照;M为DNA Maker (2000);B:双酶切鉴定,泳道1、2:EcorI和HindIII对重组质粒PET-32a-RIPK3双酶切鉴定结果,M为DNAMaker (15000);C:菌液PCR,泳道1、2、3:PCR扩增产物(516bp),泳道4:阴性对照。
图3为重组RIPK3蛋白的诱导表达和纯化结果;A:IPTG诱导的重组RIPK3蛋白表达,泳道1:未诱导的PET-32a-RIPK3总蛋白;泳道2、3:诱导4小时的PET-32a-RIPK3总蛋白;泳道4、5:诱导6小时的PET-32a-RIPK3总蛋白;泳道6、7:诱导8小时的PET-32a-RIPK3总蛋白;B:37℃诱导,重组RIPK3蛋白表达形式的鉴定;泳道1:未诱导的PET32a-RIPK3蛋白的上清液;泳道2:未诱导的PET-32a-RIPK3蛋白的沉淀;泳道3:诱导的PET32a-RIPK3蛋白的上清液;泳道4:诱导的PET-32a-RIPK3蛋白的沉淀;C:重组蛋白纯化洗脱缓冲液的最适咪唑浓度检测,泳道1-5:不同咪唑浓度(100mM、200mM、300mM、400Mm、500mM)的洗脱缓冲液洗脱目的蛋白;D:透析复性的目标蛋白;泳道1-4:透析复性后的重组RIPK3总蛋白(40kDa);标准蛋白Marker(10kDa-180kDa);
图4为间接酶联免疫吸附试验测定制备的抗血清的效价;X轴表示抗RIPK3血清的稀释梯度;Y轴表示多克隆抗体在650nm波长处的吸光度值;阳性血清表示抗RIPK3血清,阴性血清为免疫前血清在相同稀释梯度下的吸光值;
图5为Western Blot 检测RIPK3基因的蛋白水平;A、B为western blot 分析结果;C为RIPK3基因实时荧光定量结果;
图6为间接免疫荧光检测RIPK3基因的蛋白水平;A为间接免疫荧光检测RIPK3基因的蛋白水平的荧光图;B为间接免疫荧光检测RIPK3基因的蛋白水平的荧光强度统计结果。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
发明人基于基因工程和免疫学知识,设计本发明的技术方案实施路线,具体见图1所示。以下将对该技术方案的实施以实施例形式进行说明。
1、PET-32a-RIPK3重组表达载体的构建
1.1根据Genbank中报道的鸡源RIPK3基因核苷酸序列通过生物信息学分析筛选出优质的核苷酸编码序列再设计并合成特异性引物,与模板进行相应的结合从而扩增RIPK3基因,RIPK3基因PCR反应总体系为25μL,见表1。
表1 PCR反应总体系
PCR反应程序:94℃预变性5min,94℃变性30s,56℃退火35s,72℃延伸40s,39个循环,最后再72℃延伸10min。
由图2A易知,基于GenBank上公布的鸡RIPK3基因序列,设计了一对具有较强特异性的引物来扩增RIPK3基因的编码序列,在分子量约为516bp的位置可见清晰单一的特异性条带,引物见表2。
表2 PCR反应引物
1.2 对上述PCR扩增的目的基因和PET-32a(+)表达载体进行双酶切,将获得的目的片段与PET-32a(+)表达载体进行连接,构建出PET-32a-RIPK3重组表达载体。
由图2B-C易知,经由重组质粒的菌液PCR、双酶切鉴定分析证实在原有的扩增条件下,可以利用已知引物扩增出重组质粒中的目的片段,且双酶切重组质粒后,在相应大小的位置出现明显的目的条带,表明成功构建出了PET-32a-RIPK3表达载体。
2、抗RIPK3蛋白多克隆抗体血清的获取
2.1 将上述成功构建的PET-32a-RIPK3表达载体转入大肠杆菌BL21感受态细胞,并用终浓度为1mM的IPTG,于37℃,200rpm/min的摇床上摇8h,诱导培养大肠杆菌表达重组蛋白。将获得的重组蛋白进行12%的SDS-PAGE电泳,鉴定是否为所需目的蛋白。
由图3 A可知,在该条件下重组RIPK3蛋白大量表达。并且主要是在大肠杆菌培养液的沉淀中出现大量的RIPK3蛋白,见图3 B所示。
2.2 将表达的RIPK3重组蛋白进行镍亲和柱纯化,分别用500mM、400mM、300mM、200mM、100mM咪唑浓度的洗脱缓冲液进行洗脱条件优化,经12%的SDS-PAGE电泳确定最佳洗脱浓度为200mM咪唑,保存纯化后的重组蛋白经透析复性后备用于后期兔子免疫。
由图3 D所知,200mM浓度咪唑的洗脱液洗脱出的条带最为清晰单一,选用200mM咪唑浓度的洗脱缓冲液来洗脱蛋白,RIPK3重组蛋白过镍亲和柱纯化后得到分子量为40kDa的蛋白,这与预期大小相符合。
2.3将纯化的RIPK3重组蛋白与弗氏完全佐剂等体积混合,对新西兰大白兔进行皮下多点注射,每隔10天加强免疫一次(加强免疫要求RIPK3蛋白与弗氏不完全佐剂混合),免疫30天后取心脏血,离心获得免疫血清。
3、RIPK3基因多克隆抗体的质量验证方法
3.1 酶联免疫吸附试验
将纯化的RIPK3重组蛋白作为抗原稀释至2.5μg/mL,涂在96孔板上(100μL/孔)在4℃中孵化过夜。用PBS-T洗涤3次,在每孔中加入200μL的5%的脱脂奶粉封闭,37℃孵化2h,清洗后分别加入连续稀释(1:100-1:204800)的抗RIPK3血清和具有相同稀释梯度的免疫前血清(100μL/孔),37℃孵育1h清洗过后,加入按照1:3000稀释的辣根过氧化物酶标记的山羊抗兔IgG(120μL/孔)孵化1h,最后用PBS-T彻底清洗后加入可溶型单组分TMB底物溶液避光反应15min,用浓硫酸终止反应,在酶标仪中取650nm的吸光度值进行测量,进而计算RIPK3多克隆抗体的实际效价。
由图4易知,前期实验表明,选用了2.5μg/mL的抗原包被浓度可以获得最佳的抗体效价,根据OD阳性/OD阴性≥2.1的计算公式可知,抗RIPK3的血清效价都高达1:102400。
3.2 Western Blot 检测
提取正常组和肾型传染性支气管炎病毒感染后的海兰褐蛋鸡肾、气管组织总蛋白后,经由BCA法测定蛋白浓度后,将25μg的蛋白进行12%的SDS聚丙烯酰胺凝胶电泳,并以稳定表达的基因GAPDH为内参照,通过湿转将电泳条带转移到聚偏二氟乙烯膜(PVDF)上,在快速封闭液中室温封闭20 min,然后用抗RIPK3血清(1:500稀释)于摇床上4℃孵育过夜,用PBS-T洗涤4次(每次洗涤10 min),将膜与辣根过氧化物酶标记的山羊抗兔IgG(1:5000稀释)在室温下孵育40 min,最后清洗3次,用高敏型ECL化学发光检测试剂盒进行蛋白条带显色,进而检测组织中该基因的蛋白水平。
由图5 A、B易知,抗RIPK3阳性血清作为一抗可以和肾组织中RIPK3蛋白特异性结合,且病理组RIPK3蛋白水平显著升高(p≤0.01)。再通过实时荧光定量分析RIPK3基因来验证其蛋白结果,由图5 C可知,病理组RIPK3基因水平显著升高,与蛋白趋势一致。
3.3间接免疫荧光检测
将海兰褐蛋鸡肾组织进行样品采集,并将其储存在4%多聚甲醛溶液中,方便后期进行相应的切片制备。抗原修复后,将5μm左右厚的脱蜡载玻片在山羊血清中封闭30 min。将载玻片分别与抗RIPK3蛋白抗体(1:200稀释)和免疫前血清(1:200稀释)在4℃孵育过夜,用PBS-T洗涤3次(每次5 min),将载玻片与1:300 稀释的Cy3 conjugated Goat Anti-Rabbit IgG(H+L)孵育液(Boster,Wuhan,China)室温避光孵育 50min。用PBS-T清洗3次(每次5min),滴加4',6-diamidino-2-phenylindole(DAPI;1:400稀释)后避光孵育10 min,最后清洗3次后,用抗荧光淬灭封片剂封片,置于尼康倒置荧光显微镜下进行镜检定位。
如图6 A所示,将PBS缓冲液、免疫前血清和抗RIPK3血清作为一抗与海兰褐蛋鸡肾组织进行孵育,可知与正常组相比,实验组表现出明亮的荧光信号,具有强阳性,说明该多克隆抗体可以特异性结合动物组织中的RIPK3蛋白,且RIPK3蛋白的表达主要集中于肾组织的细胞质部分。
如图6 B所示,对海兰褐蛋鸡正常组和病理组的荧光强度进行统计分析,表明在肾组织中RIPK3蛋白在正常组中的表达量以极显著的差异低于病理组(p≤0.01)。
以上实验数据应用 SPSS 25.0软件进行处理,计量方式以均数±标准差(M±SD)表示,方差分析进行组间比较,p<0.05表示差异显著性,p≤0.01表示差极显著。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 江西农业大学
<120> 鸡RIPK3基因多克隆抗体及其制备方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210>1
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggaattcg aaggtcaaac aaaccgact 29
<210> 2
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cccaagcttt ggatccttac gctgaca 27
Claims (5)
1.一种鸡RIPK3基因多克隆抗体的制备方法,其特征在于,包括以RIPK3基因为抗原,通过动物免疫反应,制备鸡RIPK3基因多克隆抗体,具体包括以下步骤:
(1)获取所述RIPK3基因的序列,并构建重组表达载体;
(2)利用所述重组表达载体诱导原核表达RIPK3重组蛋白;
(3)用所述的重组表达蛋白免疫动物,从免疫动物助攻分别纯化血清,获取鸡RIPK3基因多克隆抗体;
获取所述RIPK3基因序列的引物为:
上游引物:5’-CCGGAATTCGAAGGTCAAACAAACCGACT-3’;
下游引物:5’-CCCAAGCTTTGGATCCTTACGCTGACA-3’。
2.如权利要求1所述的方法制备的鸡RIPK3基因多克隆抗体在制备检测鸡组织中RIPK3表达水平的试剂盒中的应用。
3.一种检测鸡组织中RIPK3表达水平的试剂盒,其特征在于,包括权利要求1所述的方法制备的鸡RIPK3基因多克隆抗体。
4.一种如权利要求1所述的方法制备的鸡RIPK3基因多克隆抗体在制备检测肾型传染性支气管炎病毒感染的产品中的应用。
5.如权利要求4所述的应用,其特征在于,所述产品包括检测试剂、检测试剂盒或药物。
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