CN107619438B - 新型环二核苷酸受体及其激动剂或抑制剂筛选的方法和试剂盒 - Google Patents
新型环二核苷酸受体及其激动剂或抑制剂筛选的方法和试剂盒 Download PDFInfo
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Abstract
本发明提供了能表达新型的感受外源性的环二核苷酸的受体(STING亚型M)的重组载体,同时提供特异性的鉴定和检测这个受体表达的方法和试剂盒;进一步提供以新基因STING亚型M为靶点的激活剂或抑制剂药物筛选模型和试剂盒及其应用。本申请的筛选模型和试剂盒可以简化筛选的步骤,提高筛选效率,并扩大可筛选药物的范围。
Description
技术领域
本发明属于生物工程领域以及药物筛选领域,具体涉及一种感受外源性的环二核苷酸的受体、其编码基因、载体、鉴定方法、表达量测量、稳定细胞系及在药物筛选中的应用和相关试剂盒。
背景技术
环二核苷酸(包括细菌所产生的c-di-AMP和c-di-GMP以及哺乳动物细胞所产生的cGAMP)能够通过激活STING-TBK1-IRF3信号通路从而刺激机体产生强烈的免疫应答。近年来的研究证明外源性的环二核苷酸能够显著增强疫苗的免疫反应,同时研究显示外源性的环二核苷酸在抗肿瘤免疫治疗中也有非常好的疗效。外源性的环二核苷酸能在体外和体内激活CD4和CD8细胞的肿瘤特异性的攻击效应,从而抑制肿瘤的生长。因此,环二核苷酸被认为是新的一代的疫苗佐剂以及肿瘤治疗药物。2015年3月底,诺华制药(Novartis)和Aduro公司达成7.5亿美元的交易,共同开发新的针对STING的靶向药物。
环二核苷酸的受体被认为是位于细胞内的内质网(ER)上的蛋白STING亚型1,这个受体蛋白是由Tmem173基因所编码产生。但由于细胞外环二核苷酸不能直接穿透细胞膜,而内质网又在细胞质内,外源性的环二核苷酸是如何通过细胞膜结构来激活在内质网上的受体蛋白STING亚型1一直未知。因此现有的理论体系不能解释外源性的环二核苷酸是如何激活机体的免疫反应的,这导致在筛选环二核苷酸抑制剂和激活剂时为了使待筛选药物接触到细胞内质网上的STING亚型1,需要对细胞进行细胞膜通透步骤或者用脂质体来包裹待筛选药物进行转染,这大大降低了筛选的效率及可筛选药物的范围(例如待筛选药物需要可以被脂质体所包裹),同时也限制了进一步优化外源性的环二核苷酸作为疫苗佐剂以及抗肿瘤免疫治疗的应用。
本发明旨在提供能表达新型的感受外源性的环二核苷酸的受体(称为STING亚型M)的重组载体,同时提供特异性的鉴定和检测这个受体表达的方法和试剂盒;进一步提供以新基因STING亚型M为靶点的药物筛选模型和试剂盒及其应用。
发明内容
通过对于全转录组数据的分析,发明人发现Tmem173基因可编码除了在细胞内质网上的传统型STING(STING亚型1)之外,还可以编码表达在细胞表面的其他STING亚型(发明人命名为人STING亚型M和小鼠STING亚型M,以下统称STING亚型M)。通过5’RACE的方法,发明人确认了STING亚型M在人和小鼠体内以及广泛肿瘤细胞系中的存在。体外研究发现,这些STING亚型M可以直接感受细胞外的环二核苷酸从而作为细胞外环二核苷酸的受体。细胞外的环二核苷酸可以激活STING亚型M从而激活下游的一型干扰素的启动子进而促使一型干扰素的表达上调。因此,作为外源性的环二核苷酸的受体,STING亚型M是疫苗开发以及肿瘤免疫治疗的重要药物靶点。
发明人发现Tmem173基因编码的STING亚型M位于细胞膜上以及内质网中,小鼠STING亚型M氨基酸序列如SEQ ID NO:1所示,人的STING亚型M氨基酸序列如SEQ ID NO:2所示。
另一方面,本发明提供一种用于表达STING亚型M的重组载体,例如可以是pCMV6-Entry,其含有上述的DNA序列。
本发明还提供了一种能特异性鉴别以及测量STING亚型1和M mRNA水平的方法和试剂盒。本试剂盒检测的基本原理是利用特异性的寡核苷酸引物,在逆转录酶、耐热DNA聚合酶、RNA酶抑制剂、高品质的脱氧核糖核苷三磷酸(dNTPs)以及Mg2+等RT-PCR反应缓冲液,通过RT-PCR扩增实现靶核苷酸的扩增,从而实现分别快速、高效、特异、定量检测STING亚型1和M mRNA水平的目的。
本发明中的试剂盒包括分别装有RNA提取试剂,RT-PCR扩增反应液,混合酶,阴性质控品,阳性质控品,STING亚型1和M mRNA阳性标准品的加盖密封的多个试剂瓶或管,和分隔并集中包装这些试剂瓶或管的包装盒。其中,所述混合酶包含Taq DNA聚合酶和逆转录酶(RT酶);所述RT-PCR扩增反应液包含有以下寡核苷酸引物或与下述序列同源性大于85%的序列或使用下述所有序列中的任意一种或一种以上的组合:
RT-PCR扩增反应液还包含有DNTPs、PCR Buffer和RNA酶抑制剂(RNas In)。RT-PCR扩增出来的产物可以通过电泳进行半定量(图1)或者Ion Torrent进行靶向测序而准确定量。
本发明还提供了一种能特异性鉴别以及测量STING亚型1和M蛋白含量的方法和试剂盒。本试剂盒检测的基本原理是蛋白质印记技术实现分别快速、高效、特异、定量检测STING亚型1和M蛋白表达水平检测的目的。
本发明中的试剂盒包括样品处理所需的溶液,蛋白质印记技术所需的预制胶,转膜所需要的PVDF膜,抗STING亚型1和M的抗体,二抗,STING亚型1和M蛋白阳性标准品,以及显色所需的试剂。优选的,通过使用试剂盒所提供的样品处理溶液来处理样品并进行蛋白印记实验,通过目标蛋白的条带(图2)以及STING亚型1和M标准品的对比可对STING亚型1和M蛋白表达水平进行半定量计算。
还一方面,本发明还提供了制备包含STING亚型1或M的慢病毒的载体。优选的,可用于在体内或体外稳定的表达STING亚型1或M。
此外,本发明还提供了稳定表达STING亚型M的细胞系,优选的,所述细胞系还包含报告基因,用于指示STING亚型M被激活或被抑制,更加优选的,所述报告基因位于一型干扰素或NF kappB反应原件启动子下游。本发明还提出了利用这些细胞系来进行筛选STING亚型M激活剂和/或抑制剂的方法和试剂盒。
本发明中的试剂盒包括稳定表达STING亚型M的细胞系,优选的,所述细胞系还包含报告基因,用于指示STING亚型M被激活或被抑制,更加优选的,所述报告基因位于一型干扰素或NF kappB反应原件启动子下游。优选的,试剂盒还可以包括报告基因所作用的底物,以及细胞裂解液。优选的,通过使用图4中所描述的利用本试剂盒筛选方法,可以达到高效筛选STING亚型M的激活剂以及抑制剂的目的。
可见,本发明首次发现了STING亚型M是感受细胞外环二核苷酸的受体,并实现其在真核细胞中的稳定表达。因为细胞外环二核苷酸在肿瘤免疫治疗以及疫苗开发中显示了良好的效果,证明了细胞外环二核苷酸的受体,也即发明所描述的STING亚型M,是肿瘤免疫治疗的重要靶点。针对细胞外环二核苷酸的受体(STING亚型M)进行进一步的药物筛选开发和优化对于促进疫苗开发及肿瘤免疫治疗的发展有重要作用。另外,这个受体本身表达的水平也将会是对于利用细胞外环二核苷酸以及其他STING激动剂来进行治疗的重要药敏性标志物。本发明所提供的特异性鉴别以及测量STING亚型M的mRNA以及蛋白水平的方法及试剂盒将会对这个靶标的检测起到重要作用。利用本发明进行高通量小分子药物和抗体药物的筛选能够有力的推动基于STING亚型M的疫苗开发和肿瘤免疫治疗的发展。
到目前为止,STING的激活剂只有有限的几个自然界存在的环二核苷酸,另外,而其抑制剂并没有被发现。本发明为发现STING的激活剂及抑制剂提供了一个新颖、高效、可靠、简便的平台,适于高通量药物筛选,对于寻找环二核苷酸的受体的激活剂及抑制剂具有重要意义。
附图说明
图1显示从小鼠脑组织和脾淋巴细胞中以及人的Hela细胞和外周血单个核细胞(PBMCs)对于STING亚型M特异性片段进行RT-PCR的电泳结果,其中泳道1为100bp Marker,上图中泳道2和3分别为在脑组织和脾淋巴细胞中对于小鼠STING亚型1和M进行RT-PCR的电泳结果。桑格尔测序证明所示条带分别为STING亚型1和M。下图中泳道2和3分别为在Hela细胞和PBMCs中对于人的STING亚型1和M进行RT-PCR的电泳结果。桑格尔测序证明所示条带分别为STING亚型1和M。
图2是对两只小鼠脾淋巴细胞以及人的PBMC用抗STING抗体进行蛋白质印记检测的结果。所示条带分别为STING亚型1和M。
图3显示通过转染一型干扰素报告质粒进入稳定表达STING亚型1和M的细胞系中可以看出STING亚型M是感受细胞外环二核苷酸的受体并激活一型干扰素生成。STING亚型1和M都可以感受细胞内的环二核苷酸并激活一型干扰素生成。
图4显示使用本发明高通量筛选药物的方式,并和传统方式进行对比。
具体实施方式
实施例一 人和小鼠STING亚型M基因表达质粒的构建和鉴定以及人和小鼠STING亚型M基因表达量的鉴定。
我们通过高通量RNA测序的方法发现Tmem173除了编码传统的STING亚型1之外,还编码其他的亚型,我们命名为STING亚型M。为了确认人和小鼠STING亚型M的存在,我们设计了引物特异性的扩增人和小鼠STING亚型M。简言之,使用RNA提取试剂从野生型小鼠脾细胞或人PBMC制备总RNA,然后用逆转录试剂盒(Invitrogen,18080-051),遵循制造商的说明把RNA逆转录为cDNA。扩增人和小鼠STING亚型M基因的引物如下所示:
对于小鼠的STING亚型M只需使用引物mIsoform M-F/R在小鼠脾细胞中进行1轮PCR扩增。对于人的亚型M需进行巢式PCR以扩增特异性序列。人的PBMCs cDNA作为具有外引物(hIsoform M-F-1和hIsoforms M-commonR)的第一轮PCR模板。再将第一轮的PCR产物作为模板进行第二轮PCR(使用引物hIsoforms M-F-2和hIsoforms M-commonR)。所得到的人和小鼠的STING亚型M的特异性的序列和全转录组测序所得到的人和小鼠的STING亚型M完全一致。因此我们确认了人和小鼠STING亚型M在人和小鼠的原代免疫细胞中的存在。
为了构建人和小鼠STING亚型M的重组表达载体,根据STING亚型1和STING亚型M的差异,我们采用了在表达STING亚型1的pCMV6表达载体中进行定点诱变将STING亚型1改造为人STING亚型M和小鼠STING亚型M。定点诱变使用的是Q5位点定向诱变试剂盒(NewEngland Biolabs,产品E0554S)并遵循制造商的说明。具有特异突变位点的引物是为每个突变载体设计的。用于诱变的PCR程序在94℃5分钟,然后37次循环94℃1分钟,55℃30秒,72℃3分钟,然后在72℃最终延伸2分钟。PCR产物通过琼脂糖凝胶电泳。目标条带使用QuickClean II胶回收试剂盒(金斯瑞公司,产品L00418)并用kinase-Ligase-DPNI(KLD)酶混合物连接10分钟,然后转化成DH5α感受态细胞。16小时后,细菌单克隆被扩增,然后纯化其中的表达质粒和进行测序来确认目标序列的正确性。
为了构建人和小鼠STING亚型M的慢病毒表达载体,以上表达人STING亚型M和小鼠STING亚型M的表达载体通过含有Sgf I限制酶切割位点的正向引物和含有MluI限制酶切割位点的反向引物进行PCR扩增(94℃5分钟,然后循环:94℃1分钟,55℃1分钟,72℃ 3分钟,在40个循环后,然后在72℃最后延伸10分钟。)。目标条带使用QuickClean II胶回收试剂盒(金斯瑞公司,产品L00418)并使用T4DNA连接酶将目标片段插入带有SgfI/Mlu I酶切位点的pLenti载体(Origene,产品RC208418L2V)。然后将慢病毒载体纯化并测序。
实施例二 稳定转染细胞株的构建(以HEK293T细胞为例)
首先使用Lenti-vpak包装盒(Origene,产品TR30022)将含有以上人STING亚型M和小鼠STING亚型M的慢病毒表达载体进行包装并产生慢病毒颗粒。
将HEK293T细胞保持在37℃培养,并培养在含有10%(v/v)胎牛血清以及10单位/ml青霉素-链霉素溶液的Dulbecco’s Modified Eagle Medium(DMEM)完全培养液当中。将含有慢病毒颗粒的细胞上清加入HEK293T培养液中并进行离心感染。转染48h后,离心HEK293T细胞重悬于含有300μg/ml G418的完全生长培养液中。两周后出现耐药菌落。将GFP+细胞在FACSAria II细胞分选仪(BD生物科学)进行分选。所取得的GFP+细胞在用有限稀释法进行单克隆细胞的挑选。
在取得稳定表达人和小鼠STING亚型M的细胞系后,将稳定表达重组蛋白的HEK293T细胞系接种在24孔培养板中(0.2x106个细胞/孔)过夜。然后将一型干扰素的报告基因进行瞬时转染。具体来说在150μl无血清DMEM培养基中稀释3μg带有一型干扰素的启动子序列的报告基因质粒并与含有9μl TurboFectin Transfection Reagent(美国OriGene公司)的150μl无血清DMEM培养基混合。室温孵育20min。将稳定细胞系的上清吸去,并将合并后的DNA-脂质体复合物溶液中按照每孔300μl加入稳定细胞系。将24孔板置于二氧化碳培养箱内37℃培养。转染6小时后换液,用完全生长培养基代替无血清培养基。24小时后,吸去上清。一组加入含有c-di-AMP(终浓度为30μM,(InvivoGen,产品vac-cda)完全培养基。另一组只加完全培养基。在配体刺激后16小时使用加入荧光素酶的底物测定荧光素酶活性。结果发现:加入c-di-AMP的组比只对照转染组的荧光素酶活性显著升高,证明人和小鼠STING亚型M可以成功的表达并行使其功能。
实施例三 特异性鉴定及检测人和小鼠STING亚型M的方法及试剂盒。
在mRNA的水平上检测及鉴定人和小鼠STING亚型M的方法:使用上述试剂盒提取目标组织或细RNA,并使用试剂盒中的RT酶把RNA逆转录为cDNA。对于小鼠的STING亚型M只需使用引物mIsoform M-F/R进行1轮PCR扩增。对于人的亚型M需进行巢式PCR以扩增特异性序列。人的cDNA作为具有外引物(hIsoform M-F-1和hIsoforms M-commonR)的第一轮PCR模板。再将第一轮的PCR产物作为模板进行第二轮PCR(使用引物hIsoforms M-F-2和hIsoforms M-commonR)。在琼脂糖凝胶电泳上可看到,小鼠STING亚型1和M的条带(经测序验证)(图1)。根据条带的亮度和管家基因的亮度的比较可进行相对定量。同样的引物可用于二代靶向测序,例如Ion Torrent或者Illumina平台的测序,可根据reads的量进行精确定量。
使用常规的蛋白质印迹方法在蛋白质水平上检测及鉴定人和小鼠STING亚型M。简要来说,对于目标组织或细胞制备加入试剂盒所提供的蛋白提取溶液制备蛋白提取物(可使用组织高速匀浆仪,研钵和研磨匀浆器以及超声破碎等方法)。在煮沸5分钟后,进行SDS-PAGE实验并使用半干法将蛋白质转移到硝酸纤维素膜(Bio-Rad,产品162-0115)上。使用含有5%(w/v)BSA的1X TBST在室温下2小时对硝酸纤维素膜进行封闭。然后将硝酸纤维素膜转移至含有一抗(Cell Signaling,产品13647,1∶1000稀释)以及5%(w/v)BSA的1×TBST在4℃下在摇摆孵育过夜。在含有5%(w/v)BSA的1xTBST的溶液中清洗3次,每次5分钟。然后将膜与含有二抗(抗兔-HRP或抗兔-AP抗体,1∶5000稀释)以及5%(w/v)BSA的1×TBST室温下孵育2小时。然后在含有5%(w/v)BSA的1xTBST的溶液中清洗3次,每次5分钟。之后进行显色和拍照。如图2所示,通过目标蛋白的条带对STING亚型1和M蛋白表达水平进行半定量计算。
实施例四 人和小鼠STING亚型M是感受细胞外环二核苷酸的受体并激活一型干扰素生成
利用慢病毒转导小鼠的STNG亚型1和M以及人的STING亚型1和M进入不同的STING缺失的细胞系中构建稳定表达每一种STING亚型的细胞系。同时这些细胞系稳定的表达一型干扰素启动子介导的报告萤光素酶(luciferase)。这些细胞系在加入培养基或培养基加上30μM的细胞外环二核苷酸c-di-AMP或通过使用lipofectamine 2000转染c-di-AMP进入细胞内16小时之后,可以直接检测细胞内或上清中的一型干扰素含量,或者细胞被裂解并与萤光素酶的底物混合。荧光强度被读取。同一种细胞系加与不加c-di-AMP的荧光强度进行比值来反应加上c-di-AMP后荧光强度上升的倍数。试验表明小鼠的STING亚型M和人的STING亚型M是感受细胞外的c-di-AMP的受体。而小鼠和人的STING亚型1和M都可以感受细胞内的c-di-AMP(图3)。
实施例五 稳定细胞株在筛选人STING亚型M和小鼠STING亚型M激活剂中的应用
步骤1,把上述试剂盒提供的稳定表达STING亚型M以及一型干扰素的报告基因的报告细胞系铺入96孔板,并加入待筛选药物。
对于贴壁生长的报告细胞系(例如HEK293T细胞系)需预先经胰蛋白酶/EDTA在37℃处理10分钟,加入有10%胎牛血清的RPMI1640中和胰蛋白酶。对于悬浮的报告细胞系(例如THP-1细胞系),可直接进行下一步的离心步骤。在500g离心5分钟后,将细胞重悬在10%胎牛血清、青霉素100U/mL和链霉素100μg/mL的DMEM完全培养液中,细胞密度为1×105/ml。将细胞铺于96孔板中(200微升每孔)。在37℃、含5%CO2培养孵箱培养,备用。16小时后,吸去上清,加入200μl含有待筛选药物的DMEM完全培养液中.同时设立阳性对照组(30μM的c-di-AMP,InvivoGen,产品vac-cda)及阴性对照组(只加培养液,不加任何药物)。
由于STING亚型M激活后会进一步激活一型干扰素的启动子,本发明在一型干扰素的启动子下游加入了荧光素报告基因。STING亚型M的激活剂会导致荧光素酶报告基因表达的上升。也会激活一型干扰素的表达。通过直接检测细胞内或上清中的一型干扰素含量,或者加入荧光素酶底物并读取底物所产生的荧光量,就可以测量荧光素酶的表达量从而反应药物对一型干扰素启动子的激活程度。
步骤2,采用光度计对加入待筛选药物后报告基因表达进行实时动态监测。
在加入药物16小数后,吸去上清。每个孔加入100μl的细胞裂解液并进行震荡20分钟。将20微升细胞裂解液和80μl荧光素酶底物混合,使用光度计进行测量荧光素酶底物所发出的荧光强度。
步骤3,利用该系统可以准确高效筛选增加荧光素酶表达的化合物,所筛选出的药物即为STING亚型M的激活剂。
如图4所示,这个方法与传统的针对细胞内环二核苷酸的受体STING相比,省去了把报告细胞系打孔或者把药物包裹在脂质体中的步骤,大大简化了药物筛选的流程并扩大了可筛选药物的范围(例如药物不需要能被脂质体所包裹)。
实施例六 稳定细胞株在筛选STING亚型M抑制剂中的应用
本发明所公开的是一种基于本发明所建立的报告细胞系统来高通量筛选STING亚型M的抑制剂的方法。这个发明是基于细胞外抗STING蛋白C端的抗体(Abcam,产品ab189430)可以阻断人STING亚型M和小鼠STING亚型M对于外源性配体的识别。
步骤1,把上述试剂盒所提供的人STING亚型M和小鼠STING亚型M的稳定细胞系铺入96孔板。
对于贴壁生长的报告细胞系(例如HEK293T细胞系)需预先经胰蛋白酶/EDTA在37℃处理10分钟,加入有10%胎牛血清的RPMI1640中和胰蛋白酶。对于悬浮的报告细胞系(例如THP-1细胞系),可直接进行下一步的离心步骤。在500g离心5分钟后,将细胞重悬在10%胎牛血清、青霉素100U/mL和链霉素100μg/mL的DMEM完全培养液中,细胞密度为1×105/ml。将细胞铺于96孔板中(200微升每孔)。在37℃、含5%CO2培养孵箱培养,备用。
16小时后,吸去上清,加入100μl含有待筛选药物的DMEM完全培养液中.同时设立阳性对照组(50μg/ml的抗STING蛋白C端的抗体(Abcam,产品ab189430))及阴性对照组(只加培养液,不加任何药物或抗体)。2个小时后,加入c-di-AMP(终浓度为30μM,InvivoGen,产品vac-cda)。
步骤2,采用光度计对加入待筛选药物后报告基因表达进行实时动态监测。
由于STING亚型M激活后会进一步激活一型干扰素的启动子,本发明在一型干扰素的启动子下游加入了荧光素报告基因。STING亚型M的配体c-di-AMP会导致荧光素酶报告基因表达的上升以及一型干扰素本身表达的上升。如果所加药物可以抑制一型干扰素的启动子的激活以及一型干扰素本身的表达,表明该药物可以抑制人STING亚型M和小鼠STING亚型M的功能。
在加入c-di-AMP 16小时后,可以直接通过ELISA检测上清中一型干扰素的含量或细胞内一型干扰素的含量,又或者吸去上清。每个孔加入100μl的细胞裂解液并进行震荡20分钟。将20微升细胞裂解液和80μl荧光素酶底物混合,使用光度计进行测量荧光素酶底物所发出的荧光强度。
步骤3,利用该系统可以准确高效筛选抑制荧光素酶表达的化合物。所筛选出的药物即为STING亚型M的抑制剂。
如图4所示,这个方法与传统的针对细胞内环二核苷酸的受体STING相比,省去了把报告细胞系打孔或者把药物包裹在脂质体中的步骤,大大简化了药物筛选的流程并扩大了可筛选药物的范围(例如药物不需要能被脂质体所包裹)。
Claims (4)
1.包含序列如SEQ ID NO:1或SEQ ID NO:2所示核苷酸的细胞系在制备筛选胞外环二核苷酸受体的激活剂或抑制剂的试剂盒中的用途,其特征在于,所述序列如SEQ ID NO:1或SEQ ID NO:2所示核苷酸可以直接感受细胞外的环二核苷酸,所述序列如SEQ ID NO:1或SEQ ID NO:2所示核苷酸编码的蛋白质位于细胞膜上。
2.根据权利要求1所述的用途,其特征在于,细胞系中还包括报告基因,用于指示人STING亚型M或小鼠STING亚型M被激活;
小鼠STING亚型M的核苷酸序列如SEQ ID NO:1所示;人STING亚型M的核苷酸序列如SEQID NO:2所示;
所述序列如SEQ ID NO:1或SEQ ID NO:2所示核苷酸可以直接感受细胞外的环二核苷酸而作为细胞外环二核苷酸的受体。
3.根据权利要求2所述的用途,其特征在于,所述报告基因位于一型干扰素或NFKappaB 反应原件启动子下游。
4.筛选胞外环二核苷酸受体的激活剂或抑制剂的方法,其特征在于,采用权利要求3中所述的试剂盒,包括如下步骤:
步骤 1,培养包含序列如 SEQ ID NO:1 或 SEQ ID NO:2 所示核苷酸的细胞系;
步骤 2,加入待筛选药物;孵育;检测细胞内或细胞培养基中一型干扰素或 NF KappaB的含量,或采用光度计对加入待筛选药物后的细胞所产生的报告基因进行动态监测;
步骤 3,筛选出能够增加或抑制一型干扰素表达或报告基因表达的药物。
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