CN117517657B - Lnx1基因或蛋白在调控禽类先天免疫应答反应中的应用 - Google Patents
Lnx1基因或蛋白在调控禽类先天免疫应答反应中的应用 Download PDFInfo
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Abstract
本发明属于基因工程领域,公开了LNX1基因或蛋白在调控禽类先天免疫应答反应中的应用。本发明首次揭示了LNX1基因或蛋白在调节先天免疫通路中的用途,并阐明了E3泛素连接酶LNX1调控先天免疫通路受体接头蛋白TIFA的机理,为沙门氏菌等革兰氏阴性菌的预防、控制和治疗提供了有力的科学依据和工具。本发明提供的靶向LNX1基因的寡核苷酸,能够显著提高鸡巨噬细胞吞噬能力,为沙门氏菌等革兰氏阴性菌防控相关的科学研究和产业化应用提供科学手段。
Description
技术领域
本发明属于基因工程领域,具体地说,涉及LNX1基因或蛋白在调控禽类先天免疫应答反应中的应用。
背景技术
沙门氏菌是一类能侵染多种宿主的革兰氏阴性菌,相对厌氧,不产孢子,属于肠杆菌科的直棒状细菌,是细胞内兼性病原体,大小在2到3微米之间,有菌毛,一般无荚膜和芽孢,有 2000 多种血清型。沙门氏菌可在各种条件下存活。适应宿主生物条件的能力和由此产生的致病性取决于沙门氏菌属细菌的血清型(Fàbrega et al.,2013; Cianflone etal.,2008; McSorley et al.,2014)。
禽沙门氏菌以垂直和水平两种方式进行传播,能够引起禽类患急性或慢性疾病,经消化道感染健康家禽,致病致死率高,是危害禽类养殖业最严重的疾病之一。由鸡白痢沙门氏杆菌引起的称为鸡白痢,各品种、各年龄的鸡均可发生,三周龄以内的雏鸡发病严重,死亡率高,成年鸡多为慢性,严重影响产蛋性能(张胜国等,2023)。由伤寒沙门氏杆菌引起的称为鸡伤寒,多发于成年鸡,以肝、脾肿大,肝呈黄绿色或古铜色为主要特征(刘勇,2021)。
在家禽生产中,肠炎沙门氏菌(Salmonella Enteritidis, SE)不仅能引起家禽发病、严重时甚至死亡,而且还可以造成家禽产品污染、携带肠炎沙门氏菌进而危害人类健康,大部分人类感染沙门氏菌都与消费禽源性食品如鸡蛋、鸡肉等有关。多次爆发由沙门氏菌感染禽引发的公共卫生危机,给家禽产业带来了巨大的经济损失,已成为困扰家禽产业的一大难题(汪敏,2009;徐成刚等,2015)。
发明内容
本发明的目的是提供LNX1基因或蛋白在调控禽类先天免疫应答反应中的应用。
为了实现本发明目的,第一方面,本发明提供LNX1基因或蛋白在调控禽类先天免疫应答反应中的应用(含非疾病诊断和治疗目的)。
本发明中,来自鸡的LNX1基因在NCBI上的参考序列编号为XM_040700234.2。
所述禽类先天免疫应答反应是指鸡感知革兰氏阴性菌感染并启动免疫应答反应;
优选地,本发明所述革兰氏阴性菌为沙门氏菌(Salmonella)、志贺氏菌等,更优选肠炎沙门氏菌(Salmonella Enteritidis)。
具体地,当LNX1基因或蛋白表达水平升高,则禽类先天免疫应答反应降低;当LNX1基因或蛋白表达水平降低,则禽类先天免疫应答反应升高且鸡单核巨噬细胞系(HD11)吞噬能力增强。
第二方面,本发明提供LNX1基因作为靶基因在提高禽类先天免疫应答能力中的应用(含非疾病诊断和治疗目的)。
优选地,本发明所述禽类为鸡。
第三方面,本发明提供LNX1基因或蛋白抑制剂在制备提高禽类先天免疫应答能力的制剂中的应用(含非疾病诊断和治疗目的)。
第四方面,本发明提供LNX1基因或蛋白抑制剂在制备预防和/或防止禽类感染革兰氏阴性菌的制剂中的应用。
所述抑制剂可选自小分子抑制剂、寡核苷酸、抗体、多肽或融合蛋白等。
优选地,所述寡核苷酸为miRNA、sgRNA、siRNA、shRNA、dsRNA、cDNA或反义RNA/DNA,更优选靶向LNX1基因的shRNA。
更优选地,所述靶向LNX1基因的shRNA的核苷酸序列如SEQ ID NO:1所示。
第五方面,本发明提供一种寡核苷酸,所述寡核苷酸的序列如SEQ ID NO:1所示。
第六方面,本发明提供一种用于预防和/或治疗革兰氏阴性菌感染并提高禽类先天免疫应答能力的药物或组合物,其有效成分为LNX1基因或蛋白抑制剂。
优选地,所述禽类为鸡。
优选地,所述革兰氏阴性菌为沙门氏菌、志贺氏菌等,更优选肠炎沙门氏菌。
优选地,所述药物或组合物的有效成分为SEQ ID NO:1所示的寡核苷酸。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(一)本发明公开了LNX1基因或蛋白在调节先天免疫通路中的用途,从这一角度阐明了E3泛素连接酶LNX1调控先天免疫通路受体接头蛋白TIFA的机理,为沙门氏菌等革兰氏阴性菌的预防、控制和治疗提供了有力的科学依据和工具。
(二)本发明提供的靶向LNX1基因的寡核苷酸,能够显著提高鸡巨噬细胞吞噬能力,为沙门氏菌等革兰氏阴性菌防控相关的科学研究和产业化应用提供科学手段。
附图说明
图1为本发明实施例1中稳定干扰LNX1基因表达的鸡单核巨噬细胞的镜下荧光观察照片。
图2为本发明实施例1中荧光定量PCR检测shLNX1的干扰效率。shLNX1可以有效降低LNX1基因的表达,shLNX1为实验组,shNC为对照组。表示P<0.001。
图3为本发明实施例1中流式细胞术检测鸡巨噬细胞的吞噬能力。LNX1的表达水平被敲降后,鸡巨噬细胞的吞噬能力极显著升高,shLNX1为实验组,shNC为对照组。表示P<0.01。
图4为本发明实施例2中双萤光素酶报告实验检测NF-κB报告载体萤光强度。HD11细胞经ADP-Heptose(二磷酸腺苷庚糖)处理后,当LNX1基因表达被干扰时,转录因子NF-κB的活性显著上升,shLNX1为实验组,shNC为对照组。表示P<0.01。
图5为本发明实施例3中免疫共沉淀结合Western blot试验鉴定LNX1蛋白与TIFA蛋白的互作关系。
具体实施方式
本发明以鸡单核巨噬细胞系(HD11)为实验素材,利用流式细胞术(FCM)与亲和纯化-质谱技术(AP-MS),开展了蛋白互作调控鸡先天免疫通路的研究,发现了蛋白LNX1可以调控鸡单核巨噬细胞的吞噬能力,与感知沙门氏菌等革兰氏阴性菌感染的ALPK1-TIFA-NK-κB先天免疫信号通路中的受体适配蛋白TIFA存在相互作用并且可调控其蛋白泛素化修饰水平,当LNX1基因或蛋白表达水平升高,则NK-κB转录因子活性显著降低,反之,当LNX1基因或蛋白表达水平降低,则NK-κB转录因子活性显著升高且鸡单核巨噬细胞吞噬能力增强。
LNX1是一种 E3 泛素连接酶,可以催化底物蛋白发生泛素化并降解底物。 LNX1蛋白包含一个具有 E3 泛素酶活性的 RING 结构域和4个决定底物特异性的 PDZ 结构域。LNX1 基因的每个PDZ 结构域都有其独特的底物结合偏好。
本发明采用如下技术方案:
本发明提供一种LNX1基因或蛋白在调节鸡单核巨噬细胞(HD11)的吞噬能力和先天免疫通路活性中的应用。首先使用靶向干扰LNX1的shRNA质粒进行第二代慢病毒包装,收集病毒上清经纯化浓缩后感染HD11,获得了稳定干扰LNX1的HD11细胞系;在利用流式细胞术(FCM)对HD11的吞噬能力进行评价时,shRNA干扰LNX1蛋白表达导致HD11阳性率显著高于对照组约15%。
在此基础上,本发明提供一种LNX1基因或蛋白作为药物作用靶点在提高鸡先天免疫应答能力中的应用。
进一步地,本发明提供了一种LNX1基因或蛋白抑制剂在制备增强抗菌能力的药物中的应用;以及LNX1基因或蛋白抑制剂在制备预防和/或治疗沙门氏菌等革兰氏阴性菌的药物中的应用。
本发明所述“应用”,即可表示治疗目的的应用,也可表示非治疗目的的应用,如科学研究等。
本发明所述“药物”或“抑制剂”可选自小分子抑制剂、寡核苷酸、抗体、多肽或融合蛋白。所述寡核苷酸优选为miRNA、siRNA或shRNA,更优选为靶向LNX1基因的shRNA。
本发明提供一组寡核苷酸,所述寡核苷酸的序列如SEQ ID NO:1所示。上述寡核苷酸可用于预防和/或治疗沙门氏菌等革兰氏阴性菌感染并提高鸡只先天免疫能力。
在此基础上,本发明提供一种试剂盒,所述试剂盒包含如SEQ ID NO:1所示的寡核苷酸序列。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning: a Laboratory Manual, 2001),或按照制造厂商说明书建议的条件。
实施例1 shRNA干扰LNX1基因表达,增强鸡单核巨噬细胞吞噬能力
1、shRNA设计与实验分组
针对鸡源LNX1基因序列(Gene ID: 422752)设计shRNA序列,序列SEQ ID NO:1与SEQ ID NO:2由北京擎科生物有限公司合成,shRNA序列如表1所示,使用质粒骨架为pLV3ltr-ZsGreen-Puro-U6(购自北京擎科生物科技股份有限公司)。shNC为阴性对照。
表1
实验分两组:实验组(shLNX1)、对照组(shNC)。
2、HEK293T细胞(人胚肾细胞,购自中国科学院细胞库)培养
(1)细胞复苏:从液氮中取出冻存细胞,立即置于37℃水浴中并快速摇晃,待细胞溶液完全溶解之后,将细胞溶液加入到 9mL 完全培养基中混匀,3000rpm离心 5 分钟,去上清,用新的完全培养基充分混匀细胞沉淀,加入到细胞培养皿中,混匀,放入37℃、5%CO2培养箱中培养。
(2)细胞换液:显微镜下观察细胞密度及形态,一般24h换液,提前温浴细胞培养基,倒掉旧培养基,加入新培养基即可。
(3)细胞传代:显微镜下观察,细胞汇合度达到70%-80%时要进行传代,倒掉旧的培养基,用PBS轻柔清洗细胞,加入1.5mL 0.25%的胰酶,消化至细胞开始有悬浮时立即加入4.5mL完全培养基终止消化,制备单细胞悬液。将细胞悬液加入到15mL离心管中,3000rpm离心5min,去上清,用6mL完全培养基充分悬浮细胞沉淀,加入到细胞培养皿中,混匀,放入37℃、5%CO2培养箱中培养。
3、shRNA质粒慢病毒包装
采用脂质体转染法将质粒转入到HEK293T细胞中。
细胞转染按Lipofectamine® 3000试剂(Life Technologies)说明书进行:
(1)用Opti-MEM®培养基稀释Lipofectamine® 3000试剂并充分混匀,以shRNA质粒:包装质粒 psPAX2:包膜质粒pMD2.G=1:2:1的比例配置质粒预混液并使用Opti-MEM®培养基稀释;
(2)向质粒预混液中添加P3000™试剂,在每管已稀释的Lipofectamine® 3000试剂中加入稀释的质粒预混液(1:1体积比),室温孵育5min后将质粒-脂质体复合物至细胞培养皿中;
(3)转染后6-8h轻柔换液为病毒收获培养基,转染后48h时收集上清,400g离心5min,并用0.45 µm滤头(Millipore, Millex®-HP)过滤,使用PEG-it™ (SBI, LV825A-1)浓缩病毒,并使用p24 ELISA(GenScript, L00938 )检测试剂盒测定病毒滴度。
4、慢病毒转染与流式细胞分选
(1)培养鸡单核巨噬细胞系(HD11, 由扬州大学陈国宏教授惠赠),培养方法与上述HEK293T培养方法相同;
(2)细胞密度为60-70%时,培养基中加入终浓度为8µg/ml的Polybrene(MCE,HY-112735)处理4-6h;
(3)将浓缩后的shRNA慢病毒加入细胞培养基,混合均匀,感染48h;
(4)感染结束后倒掉旧培养基,PBS冲洗后加0.25%的胰蛋白酶(Gibco)于37℃培养箱消化5min,轻晃培养皿,显微镜下见细胞游离时立即加入2倍体积完全培养基终止消化;
(5)将细胞悬浮液加入到15mL离心管中,1000rpm离心5min,去上清,用PBS重悬细胞并转入流式管中,根据GFP荧光信号进行流式分选,得到稳定转染的阳性细胞。
5、荧光定量PCR实验
RNA提取按照总RNA提取试剂盒(天根,DP419)说明书进行,反转录按照FastQuantcDNA第一链合成试剂盒(天根,KR106),shLNX1的干扰效率通过荧光定量PCR检测。qPCR反应液使用 One Step SYBR® PrimeScript™ RT-PCR Kit II (Takara)配置反应体系,通过 ABI Q7 Flex system (Applied Biosystems, USA) 分析LNX1的表达量,β-Actin为内参对照基因,使用2-ΔΔct方法计算。
6、鼠伤寒沙门氏菌感染与流式细胞分析
使用带有mCherry荧光标签的鼠伤寒沙门氏菌感染shLNX1组与shNC组,每组设置三个生物学重复,感染4h后换为含浓度为100µg/ml的庆大霉素细胞培养基继续培养12h以杀灭胞外菌,收集细胞并用PBS制成细胞悬液转入流式管中,上流式细胞仪进行分析细胞阳性率。
7、结果
参见图1,展示了慢病毒感染后稳定干扰LNX1基因表达的鸡单核巨噬细胞的镜下荧光观察照片。
参见图2,展示了荧光定量PCR检测shLNX1的干扰效率。shLNX1为实验组,shNC为对照组。以β-Actin为内参对照基因,使用2-ΔΔct方法计算LNX1与β-Actin的相对表达量。由图可见,shLNX1可以有效降低LNX1的表达水平。
参见图3,展示了流式细胞术检测鸡巨噬细胞的吞噬能力。shLNX1为实验组,shNC为对照组。由图可见,LNX1的表达水平被敲降后,鸡巨噬细胞的吞噬能力极显著升高。
实施例2 shRNA干扰LNX1基因表达,NK-κB转录因子活性显著升高
1、实验分组与布板
实验共分五组,实验组(shLNX1)、对照组(shNC)、Control组、Promoter组、Basic组,每组设置三个生物学重复;
将实施例1所获得的shLNX1和shNC稳定转染细胞系与野生型HD11细胞系按照实验设计接种适量细胞至24孔板中于37℃、5%CO2培养箱中培养。
2、shLNX1稳转HD11细胞系转染及ADP-Heptose处理
当接种细胞密度80%左右进行细胞转染,细胞转染按TransIT-X2® DynamicDelivery System试剂(Mirus,MIR6000)说明书进行:
(1)用Opti-MEM®培养基稀释所转萤光素酶报告质粒并充分混匀制成质粒预混液;
(2)按照推荐比例向质粒预混液中添加TransIT-X2试剂,充分混匀,室温孵育15min后将复合物加至细胞培养皿中;
(3)转染24h后,向培养皿中加入终浓度为1µM的ADP-Heptose(二磷酸腺苷庚糖,参考文献:Zhou, P., Y. She, et al Alpha-kinase 1 is a cytosolic innate immunereceptor for bacterial ADP-heptose. Nature, 2018. 561(7721): p. 122-126. 购自Invivogen)处理细胞6h,处理结束即检测萤光素酶化学发光。
3、双萤光素酶报告基因检测实验
双萤光素酶报告基因检测按照Dual-Luciferase® Reporter Assay System(Promega,E1910)说明书进行:
(1)裂解细胞:吸除培养基,向细胞培养板中加入细胞裂解液,充分裂解后,4°C,12000 rpm离心5 min,吸取上清用于后续测定;
(2)配制海肾萤光素酶检测缓冲液:按照每个样品100 µL的体积,计算所需的海肾萤光素酶检测缓冲液,按照海肾萤光素酶检测底物:海肾萤光素酶检测缓冲液=1:100的比例配制工作液;
(3)萤火虫萤光素酶的测定:向酶标板中加入100 µL的细胞裂解液,然后加入100µL萤火虫萤光素酶检测试剂,吹打混匀,置于酶标仪中,检测化学发光的数值即为萤火虫萤光素酶数值;
(4)海肾萤光素酶的测定:上述步骤完成后,加入100 µL海肾萤光素酶检测缓冲液,吹打混匀,按照上述方法混匀,置于酶标仪中测定海肾萤光素酶数值;
(5)计算:萤火虫萤光素酶数值和海肾萤光素酶数值的比值即为相关报告基因的活性。
4、结果
参见图4,展示了HD11细胞经ADP-Heptose处理后,当LNX1基因表达被干扰时,检测化学发光强度显示转录因子NF-κB的活性显著上升,说明LNX1通过影响转录因子NF-κB的活性从而调控下游免疫因子的表达。
实施例3 蛋白LNX1与蛋白TIFA的相互作用关系验证
1、LNX1和TIFA基因表达载体构建
根据pcDNA3.1-3×Flag C载体信息(参见硕士论文,王菲,SPOP通过降解MyD88负反馈调节先天免疫的分子机制,DOI: 10.27630/d.cnki.gznky.2020.000009)和NCBI上公布的鸡LNX1基因基因序列(GenBank:XM_040700234.2),基因CDS序列由北京擎科生物有限公司合成并连入载体。
根据pcDNA3.1-Myc载体信息和NCBI上公布的鸡TIFA基因序列(GenBank:XM_420645.8),基因CDS序列由北京擎科生物有限公司合成并连入载体。
2、DF1细胞培养
鸡胚成纤维细胞(DF1,购自中国科学院细胞库)培养方法与上述HEK293T培养方法相同。
3、细胞转染
待细胞培养至80%汇合度时,将基因表达载体LNX1-pcDNA3.1-3×Flag C和TIFA-pcDNA3.1-Myc采用脂质体转染法分别转入到DF1细胞中;
转染试剂选用Lipofectamine® 3000试剂(Life Technologies),操作与上述操作相同,转染24h后收集细胞用于下游实验。
4、蛋白质免疫共沉淀
(1)预处理beads:使用蛋白提取液(RIPA Buffer)清洗A/G蛋白亲和磁珠(ProteinA/G beads)和FLAG标签亲和磁珠(anti-FLAG M2 beads)四次,每次5min。洗剂过程于4℃摇床进行,过程需要缓慢震荡。
(2)细胞收集及裂解:向步骤3获得的细胞中加5倍细胞量的RIPA Buffer裂解细胞,4°C,12000 rpm离心30min。取上清于冰上备用。
(3)预清背景蛋白(Preclear):裂解液中加入预处理过的A/G蛋白磁珠 20μL,于4℃摇床缓慢震荡2h,以去除非特异性杂蛋白,降低背景,离心后收集上清于新离心管中。取1/50裂解液(全蛋白)保存至-80℃冰箱用作阳性对照(Input),剩余裂解液用作免疫沉淀(IP)即实验组。
(4)IP:向用作IP的裂解液中加入FLAG标签磁珠,于4℃摇床缓慢震荡过夜。
(5)清洗:离心收集琼脂糖珠,弃上清,用蛋白提取液于4℃摇床清洗四次,前两次每次5min,后两次每次30min。54K buffer清洗两次,第一次5min,第二次30min。
(6)纯化:加入60µL TBS溶液和5µL FLAG标签多肽,4℃摇床1h,离心抽取上清。
(7)加入上样缓冲液,96℃煮沸10min,立即置于冰上,短暂离心后吸取上清进行聚丙烯酰胺凝胶电泳。
5、Western blot
(1)蛋白经聚丙烯酰胺凝胶电泳后转至PVDF膜(Millipore)上,转膜条件为350mA恒流。
(2)PVDF膜封闭30min后,一抗孵育1h,TBST洗膜三次,每次5min,然后进行二抗孵育,TBST洗膜三次,每次5min。
(3)使用ImageQuant LAS 4000mini仪器自动曝光,采集Western杂交图像。
本实验所用Flag标签的一抗为DDDDK标签抗体(Anti-DDDDK tag antibody,AE005,爱博泰克),Myc标签的一抗为Myc标签抗体(Anti-Myc tag antibody,AE010,爱博泰克),二抗为HRP标记亲和纯化山羊抗兔IgG(H+L)[HRP Goat Anti-Rabbit IgG (H+L)](AS014,爱博泰克)。
6、结果
参见图5,展示了通过免疫共沉淀结合Western blot试验鉴定LNX1蛋白与TIFA蛋白的互作关系。互作验证实验共分3组,第1泳道为pcDNA3.1-Flag和TIFA基因表达载体共转染,第2泳道为pcDNA3.1-Myc和LNX1基因表达载体共转染,第3泳道为LNX1基因和TIFA基因表达载体共转染。24h后收集细胞,免疫共沉淀纯化FLAG标签融合蛋白,进行抗FLAG的Western blot检测,结果显示含有FLAG标签的LNX1蛋白被成功富集,说明LNX1基因表达载体在DF1细胞中成功表达。用抗Myc进行DF1t检测,结果显示含有Myc标签的TIFA蛋白存在于互作蛋白复合物中,说明LNX1与TIFA之间存在互作关系。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (2)
1.LNX1基因或蛋白抑制剂在制备提高禽类先天免疫应答能力的制剂中的应用,
其中,所述禽类为鸡,
来自鸡的LNX1基因在NCBI上的参考序列编号为XM_040700234.2,
所述禽类先天免疫应答是指鸡感知革兰氏阴性菌感染并启动免疫应答,
所述革兰氏阴性菌为沙门氏菌或志贺氏菌;
所述抑制剂为靶向LNX1基因的shRNA,其核苷酸序列如SEQ ID NO:1所示。
2.LNX1基因或蛋白抑制剂在制备预防和/或防止禽类感染革兰氏阴性菌的制剂中的应用,
其中,所述禽类为鸡,
来自鸡的LNX1基因在NCBI上的参考序列编号为XM_040700234.2,
所述革兰氏阴性菌为沙门氏菌或志贺氏菌;
所述抑制剂为靶向LNX1基因的shRNA,其核苷酸序列如SEQ ID NO:1所示。
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