CN116813754A - 一种hbv特异性单克隆抗体及乙肝病毒检测试剂盒 - Google Patents
一种hbv特异性单克隆抗体及乙肝病毒检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种HBV特异性单克隆抗体及乙肝病毒检测试剂盒。本发明基于不同HBV的S蛋白的氨基酸序列,基于多序列比对,以及抗原表位在线软件筛选,最终选择较好的抗原表位肽,免疫小鼠制备了广谱单克隆抗体,所述抗体具有较好的结合特性,并且基于S蛋白的在病毒中的作用,通过中和实验证实了本发明制备的单克隆抗体也具有较好的中和病毒的效果,具有较好的应用前景。
Description
技术领域
本发明涉及生物领域,更具体的涉及一种HBV特异性单克隆抗体及乙肝病毒检测试剂盒。
背景技术
病毒性肝炎是由多种肝炎病毒引起的以肝脏病变为主的一种传染病。临床上以食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现。部分病人可有黄疸发热和肝大伴有肝功能损害。有些病人可慢性化,甚至发展成肝硬化,少数可发展为肝癌。病毒性肝炎的病原学分型,目前已被公认的有甲、乙、丙、丁、戊五种肝炎病毒,分别写作HAV、HBV、HCV、HDV、HEV,除乙型肝炎病毒为DNA病毒外,其余均为RNA病毒。
目前,在检测病毒性肝炎方面,主要是对肝炎病毒标志检测。甲型肝炎,急性肝炎患者,血清抗-HAVIgM阳性可确诊为HAV近期感染,抗-HAV-IgG阳性提示既往感染且已有免疫力。乙型肝炎,HBsAg与抗-HBs:HBsAg阳性示HBV目前处于感染阶段,抗-HBs为免疫保护性抗体阳性示已产生对HBV的免疫力。慢性HBsAg携带者的诊断依据为无任何临床症状和体征、肝功能正常,HBsAg持续阳性6个月以上者。HBeAg与抗-HBe:HBeAg阳性为HBV活跃复制及传染性强的指标,被检血清从HBeAg阳性转变为抗-HBe阳性表示疾病有缓解感染性减弱。③HBcAg与抗-HBc:HBcAg阳性提示存在完整的HBV颗粒直接反应,HBV活跃复制由于检测方法复杂临床少用。抗-HBc为HBV感染的标志,抗-HBc IgM阳性提示处于感染早期,体内有病毒复制。在慢性轻度乙型肝炎和HBsAg携带者中HBsAg、HBeAg和抗-HBc三项均阳性具有高度传染性指标难以阴转。丙型肝炎,由于血中抗原量太少无法测出,故只能检测抗体抗-HCV为HCV感染标记,不是保护性抗体。用套式反转录PCR法检测,血清HCV-RNA阳性示病毒活跃复制具有传染性。丁型肝炎,HDV为缺陷病毒,依赖HBsAg才能复制,可表现为HDV-HBV同时感染,HDAg仅在血中出现数天,随之出现IgM型抗-HD、慢性HDV感染抗-HD IgG持续升高,自血清中检出HDV-RNA则是更直接、更特异的诊断方法。戊型肝炎急性肝炎患者,血清中检出抗-HEVIgM抗体,恢复期血清中IgG抗体滴度很低,抗-HEV IgG在血清中持续时间短于1年,故抗-HEV IgM、抗-HEV IgG均可作为HEV近期感染指标。庚型肝炎,RT-PCR技术可检测HGVRNA,是HGV早期诊断和监测病毒血症的有效方法,抗-HGV的IgM和IgG抗体目前尚未成熟,存在检出率低且与RT-PCR结果不相符等特点。
当然,近些年来随着临床医学、免疫学和分子生物学的不断发展,众多高灵敏度的新型检测手段如化学发光、时间分辨荧光、表面增强拉曼光谱等技术开始在肝炎检测中得到应用。由于乙型肝炎病毒(Hepatitis B Virus,HBV)是引起乙型肝炎(简称乙肝)的病原体,属嗜肝DNA病毒科。乙肝病毒携带者中,50%~75%的人有活动性病毒复制的慢性乙肝,估计5年中从慢性乙肝进展为肝硬化的发生率为2%-20%;从代偿性肝硬化到肝脏失代偿为20%-23%;从代偿肝硬化到肝癌为6%-15%。慢性乙肝是进展为肝硬化、肝衰竭和肝细胞癌的首要危险因素。HBV的传染性很强,接种0.00004mL含病毒血液足以使人发生感染。
放射免疫法(RIA)也是常用的检测HBV的方法,其中RIA最常用的是固相放射免疫(SPRIA)法,它是利用放射性同位素标记在已知抗原或抗体上,使之与待测相应抗体或抗原结合。用计数器测定同位素的Cpm值,从而推算受检标本中的抗原或抗体的量。该法灵敏度较高.但试验条件要求高,一般难以在基层单位推广。
酶免法(EIA)常用的检测HBV的方法,其中最常用的是酶联免疫吸附试验(ELISA)。一般用聚苯乙烯吸附小孔为载体,吸附特异的抗原或抗体。使它与待测标本中相应的抗体或抗原结合。在酶的催化下显色,根据色泽的深浅用肉眼判断结果(定性);亦可用比色计(酶标仪)作出精确测定(定量)。此法目前已在各级单位推广使用。而乙肝病毒又常常产生突变,这给乙肝的确诊和治疗带来极大的麻烦,所以迫切需要一种既准确又快速的实验室检验方法来检测乙肝病毒。
发明内容
本发明提供了一种能够广谱性的检测HBV的单克隆抗体及其应用的试剂盒或者试纸条。
一方面,本发明提供一种广谱性的检测HBV的单克隆抗体HBV-S-2F6。
在本申请中,所述单克隆抗体包含重链可变区和轻链可变区,其中,
所述轻链可变区的氨基酸序列如SEQ ID NO:1所示,其氨基酸序列为
DIVITQRPALMAASPGEKVTITCRPINGEHNFSEVWYQQKSGISPKPWIYGPVPTWEGVPARFSGSGSGTSYSLTITSMEAEDAATYYCLFDPINDPPFGAGTKLELK
所述重链可变区的氨基酸序列如SEQ ID NO:2所示,其氨基酸序列为
EVQLEESATELARPGASVKLSCKASGYIFSIEVFPWIKQRPGQGLEWIGRCSMQQPTRTDPTNHFGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGWRWECNVWGLGTTLAVSS。
还提供了衍生自本文所述的抗体或抗体结合片段的抗体。通常,如本文所用,本文提供的衍生物基本上类似于本文所述的抗体或抗体结合片段。例如,它们可以在其氨基酸序列中包含一个或多个保守取代,或者可以包含化学修饰。衍生物和修饰的肽/多肽/蛋白质都被认为是“结构相似的”,这意味着它们保留了母体分子的结构(例如二级,三级或四级结构),并预期以与母体分子相同的方式与抗原相互作用。
一类合成衍生的抗体或抗原结合部分可通过保守突变亲本分子上的残基以产生保持与亲本分子相同活性的肽,多肽或蛋白而产生。代表性的保守取代在本领域中是已知的,并且在此也被总结。
通常,只要保留所需的活性,就可以在任何位置进行保守取代。可以进行所谓保守交换,其中被置换的氨基酸具有与原始氨基酸相似的性质,例如Asp对Glu的交换,Asn对Gin的交换,Val对Lie的交换,Leu对Lie的交换和Ser对Thr的交换。
例如,具有类似性质的氨基酸可以是脂肪族氨基酸(例如甘氨酸,丙氨酸,缬氨酸,亮氨酸,异亮氨酸);羟基或含硫/硒的氨基酸(例如丝氨酸,半胱氨酸,硒代半胱氨酸,苏氨酸,蛋氨酸);环状氨基酸(例如脯氨酸);芳族氨基酸(例如苯丙氨酸,酪氨酸,色氨酸);碱性氨基酸(例如组氨酸,赖氨酸,精氨酸);或酸性及其酰胺(如天冬氨酸,谷氨酸,天冬酰胺,谷氨酰胺)。缺失是指氨基酸被直接键取代。缺失的位置包括多肽的末端和各个蛋白质结构域之间的连接。插入是将氨基酸引入多肽链中,直接键形式上被一个或多个氨基酸取代。氨基酸序列可以借助于本领域已知的计算机模拟程序进行调节,所述计算机模拟程序可以产生具有例如改进的活性或改变的调节的单抗。
进一步的,抗体可包含具有至少约75%的免疫球蛋白重链可变区,至少约80%,至少约85%,至少约90%,至少约95%,至少约96%,至少约97%,至少约98%,至少约99%,至少约99.5%,至少约99.9%,或与SEQ ID NO:2 100%序列同一性,和具有至少约75%的免疫球蛋白轻链可变区,与SEQ ID NO:1具有至少约80%,至少约85%,至少约90%,至少约95%,至少约96%,至少约97%,至少约98%,至少约99%,至少约99.5%,至少约99.9%或100%的序列同一性。
本发明还公开了上述的HBV抗体在制备ELISA检测样本中的HBV的试剂盒中的应用。
本发明还公开了上述的HBV抗体在双抗夹心ELISA检测样本中HBV含量中的应用。
优选的,所述应用的程序为:包被HBV抗体,洗板,明胶封闭,洗板,加检测样本孵育,洗板,加生物素标记的HBV抗体孵育,洗板,加HRP标记的亲和素孵育,洗板,加TMB显色,加终止液后酶标仪读数。
本发明还公开了一种HBV检测试剂盒,其特征在于:包含上述的HBV抗体。
进一步的,为了标记单克隆抗体,例如,各种色素类,胶体类,作为将酶等标记物与单克隆抗体结合的色素,例如,FITC(Fluorescein5―isothiocyanate),罗丹明,可以使用荧光素等荧光标识用色素类,通用性强,易于获取,FITC优选的FITC作为能够从Sigma(株)购买的胶体类,例如,作为能够使用金胶体等的酶,例如,通过过氧化物酶,碱性磷酸酶,荧光素酶,β-半乳糖酶等能够使用的标记物与上述单克隆抗体结合,可以利用公知的方法。
进一步的,本发明提供了一种HBV胶体金检测试纸条,所述试纸条内包括胶体金标记抗体,所述胶体金标记单克隆抗体为胶体金与本发明的单克隆抗体偶联制得;
所述胶体金的制备包括如下步骤,将氯金酸加热,加入柠檬酸三钠,继续加热至溶液由淡黄色转为蓝黑色最终转为亮红色,颜色稳定后继续加热、之后冷却即得到胶体金溶液;
所述胶体金标记单克隆抗体的制备包括如下步骤:将胶体金溶液调节至弱碱性,加入本发明的单克隆抗体,之后依次加入PEG以及BSA继续反应,即得到胶体金标记单克隆抗体沉淀,采用PBS重悬即得胶体金标记单克隆抗体;
所述试纸条的组件包括底板、样品垫、硝酸纤维素膜、吸水垫,所述底板、样品垫、硝酸纤维素膜、吸水垫依次连接;所述硝酸纤维素膜上设置有检测线和质控线。
所述试纸条的组装为将样品垫、硝酸纤维素膜、吸水垫依次搭接在底板上,样品垫和吸水垫分别压在硝酸纤维素膜上方1~2mm。
有益效果
本发明基于不同HBV的S蛋白的氨基酸序列,基于多序列比对,以及抗原表位在线软件筛选,最终选择较好的抗原表位肽,免疫小鼠制备了广谱单克隆抗体,所述抗体具有较好的结合特性,并且基于S蛋白的在病毒中的作用,通过中和实验证实了本发明制备的单克隆抗体也具有较好的中和病毒的效果,具有较好的应用前景。
附图说明
图1广谱性多肽位点的比对结果图
图2单克隆抗体Western-blot检测结果图
图3单抗亚型鉴定结果图
图4单抗特异性鉴定结果图
图5单抗中和实验结果图
具体实施方式
本发明可通过后续对于本发明一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。在进一步叙述本发明之前,应明了本发明不会被局限于所述特定实施方案中,因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案,而非作为限制,因为本发明的范围将会被仅仅界定在所附的权利要求中。
实施例1HBV S蛋白抗原表位肽的筛选
基于不同HBV的S蛋白的氨基酸序列,基于多序列比对,以及抗原表位在线软件筛选,最终选择较好的抗原表位肽为cpgyrwmclrrfiiflfilllclifllvlld(命名为HBV-S广谱肽)。虽然S蛋白的a决定簇cttpaqgnsmf具有存在与任何血清亚型中,但是从图1可以看出,其在不同HBV中具有并不完全保守的序列,因此,本申请筛选的广谱肽具有更广谱的特性。委托上海生工进行多肽合成,用作免疫原。将抗原多肽与KLH偶联备用。
实施例2HBV S蛋白单克隆抗体的制备
选取5只SPF级6周龄雌性Balb/c小鼠,为使其适应环境先饲养两周。用生理盐水稀释实施例1制备的抗原多肽HBV-S广谱肽-KLH偶联复合物,配置成1mg/ml的溶液。首次免疫以免疫原200μg与弗氏完全佐剂按照1:1的比例进行乳化后,皮下多点注射;2周后,再以免疫原200μg与弗氏不完全佐剂按照1:1的比例进行乳化后,皮下多点注射;2周后,免疫原150μg用生理盐水稀释后,进行腹腔注射加强免疫,2周后,取血清检测效价。
免疫小鼠血清抗体效价测定:用包被液将免疫原多肽HBV-S广谱肽溶解,并稀释至20μg/ml,每孔100μl,4℃静置过夜;浸泡洗涤3次,每次3min;每孔加入2%BSA封闭液200μl,37℃孵育2h;浸泡洗涤3次,每次3min;分别加入按1:2500、1:5000、1:10000、1:15000、1:17500、1:20000、1:30000、1:40000梯度稀释的各个小鼠血清,3复孔,37℃孵育1h;浸泡洗涤3次,每次3min;加入有酶标记的兔抗小鼠二抗,1:5000稀释,37℃孵育1h;浸泡洗涤3次,每次3min;加入OPD与过氧化氢混合的显色液,10min后停止反应;测波长490nm处OD值,5只小鼠的血清效价分别是1:15000,1:17500,1:17500,1:20000,1:15000。4号小鼠的血清效价最高,选择4号小鼠进行细胞融合。
处死小鼠,制备脾脏细胞,按照本领域常规的融合方法,将脾细胞与骨髓瘤细胞进行融合,并采用HAT和HT培养基进行培养筛选。采用间接ELISA方法进行阳性克隆的筛选。当两次检测含有克隆的孔的吸光值与阴性对照孔吸光值比值大于2.1时,则可以判定此孔为阳性孔。经统计,共计有23个阳性较好的克隆。将阳性克隆细胞进行亚克隆4次,直到96孔细胞培养板中克隆后的细胞全为阳性时,获得了3株单克隆阳性杂交瘤细胞,分别是HBV-S-2F6、HBV-S-4G4和HBV-S-4H7。其中,阳性反应最强的是HBV-S-2F6和HBV-S-4H7进行后续应用。
采用体内诱生法,所用动物为10周的雌性纯系BALB/c小鼠,提前7天给每只小鼠注射500μL液体石蜡,待2个杂交瘤细胞HBV-S-2F6和HBV-S-4H7细胞数量分别达到3×106个时,注入小鼠腹腔中,小鼠腹部胀大后收集腹水,2500rpm低温离心20min,取清澈的腹水进行腹水效价的测定:将每株杂交瘤细胞收集的腹水分别取出50μL按照1:100-1:51200比例进行倍比稀释,按照间接ELISA的方法测定腹水效价,结果如表1所示。
表1单抗的腹水效价
单抗类型 | 效价 |
HBV-S-2F6 | 1:25600 |
HBV-S-4H7 | 1:51200 |
将腹水低温离心,用微孔滤膜过滤腹水,以除去较大的凝块及脂肪滴;用10000g15分钟高速离心(4℃)除去细胞残渣及小颗粒物质。在搅拌下,滴加饱和硫酸铵溶液5.0ml;继续缓慢搅拌30分钟;10000r/min离心15分钟;弃去上清液,沉淀物用1/3饱和度硫酸铵悬浮,搅拌作用30分钟,同法离心;重复前一步2次;沉淀物溶于1.5ml PBS(0.01mol/LPH7.2),得到纯化后的2个抗体。将抗体的浓度调为2mg/mL,4℃保存。
实施例3HBV-S-2F6单克隆抗体Western-blot鉴定
蛋白样品的制备:取HBV-S广谱肽放入EP管中,多肽:SDS:DTT=5:4:1放于离心管中,沸水煮样10min,冰浴5min。蛋白样直接用于Western blot。
Western blot方法步骤如下:
(1)制备SDS-PAGE,下层的15%分离胶和上层的5%浓缩胶;
(2)加样:将多肽进行上样;
(3)电泳:浓缩胶90V电压先跑30分钟左右,分离胶120V电压跑至底端;
(4)转膜:转印时叠放的由上到下的顺序是:滤纸—蛋白胶—NC膜—滤纸,转印条件:56mA,40min左右,转印结束后将膜放入20mL的5%脱脂乳封闭液中,4℃封闭过夜;
(5)抗体孵育:
1)倒掉封闭液,将膜用PBST洗,洗5次,每次5min,以制备的单抗,PBST进行1:1000稀释倍数,4℃封闭孵育8h;
2)一抗回收,用PBST洗,洗5次,每次5min;
3)加山羊抗小鼠IgG-HRP二抗,PBST进行1:5000稀释倍数,37℃摇床孵育2h;
4)二抗回收,用PBST洗,洗5次,每次5min;
5)避光保存现用现配的ECL曝光液,将膜铺于曝光盒中,与曝光液作用30s进行曝光成像。
结果见图2,可以发现HBV-S-2F6与S蛋白多肽反应,在转印膜处出现了明显的特异性条带,说明该HBV-S-2F6单抗与S蛋白发生特异性反应,条带单一。
实施例4HBV-S-2F6单抗亚型鉴定
将准备好的HBV-S-2F6单抗加入板条样品孔中,每孔50μL;无需孵育,将1×羊抗鼠IgA+IgM+IgG-HRP加入样品孔中,每孔50μL。混匀器上轻轻混匀,也可用手轻轻敲击板架两侧1min。盖上封板膜,室温孵育1h。1×PBST洗涤3次,拍干。将现配的显色液加入孔中,每孔100μL。显色液配制方法:A液:B液=1:100。室温避光显色15min。每孔加入终止液,每孔100μL。结果判读。结果通过酶标仪读取OD450。颜色最深或者OD值最高的孔对应的即为相应的亚型。结果如图3所示。
从图3的结果可以看出,HBV-S-2F6单抗是IgG2b亚型,Kappa链。
实施例5HBV-S-2F6单抗亲和力鉴定
利用AMC传感器,将纯化出来的HBV-S-2F6抗体用PBST稀释到10μg/mL,抗原多肽S-广谱肽用PBST进行梯度稀释:250μg/mL、125μg/mL、62.5μg/mL、31.3μg/mL、15.6μg/mL、7.80μg/mL、3.9μg/mL。
运行流程:缓冲液中平衡65s,抗体溶液中固化抗体340s,缓冲液中孵育250s,抗原溶液中结合500s,缓冲液中解离1600s,用10mM pH 1.69GLY溶液及缓冲液进行传感器再生,输出数据。KD表示平衡解离常数即亲和力。结果见下表1。
表1 HBV-S-2F6抗体的亲和力检测结果
抗体名称 | 解离常数 |
HBV-S-2F6抗体 | 9.64E-10 |
从表1数据可以看出,HBV-S-2F6抗体对抗原具有较好的的亲和力。
实施例6HBV-S-2F6单抗可变区测序鉴定
采用简并引物扩增法测序获得HBV-S-2F6单抗的重链可变区和轻链可变区序列,具体来说,收集HBV-S-2F6的杂交瘤单克隆细胞,采用试剂盒提取细胞总RNA,1%琼脂糖凝胶电泳检测RNA完整性,核酸定量分析仪测定RNA浓度。使用RNA反转录试剂盒,将lμg的RNA逆转录成cDNA,以5μl cDNA为模板,按照本领域常规的轻重链引物扩增引物和反应体系来配置PCR反应体系和设置PCR程序,其中退火温度采用梯度降温的方法,然后直接以1μ1第一轮PCR产物为模板进行第二轮PCR,PCR反应体系和程序同上,第二轮PCR产物全部上样进行1%琼脂糖凝胶电泳,切下合适大小的特异目的条带,进行胶回收,连接克隆载体,转化感受态细胞,挑取单克隆菌落,菌落活化后委托上海生工进行测序,测序后获得HBV-S-2F6抗体的VL和VH氨基酸序列,分别如SEQ ID NO:1和2所示。
实施例7HBV-S-2F6单抗特异性鉴定
包被免疫原S-广谱肽、S表位肽2(cttpaqgtsmypsccctkpseg)、重组乙型肝炎疫苗(酿酒酵母)(深圳康泰生物制品股份有限公司)、G9P[8]型灭活轮状病毒(购买自北京协和医学院)、BSA、屎肠球菌裂解液、大肠杆菌裂解液的酶标板,加入1:3000稀释的抗体。同时设阴性对照孔。酶标仪测定A450nm。P/N样本孔吸光度值(S)/阴性对照孔吸光度值(N)≥2.1判断为阳性。结果如图4所示。
从图4的ELISA体系的特异性鉴定结果可以看出,HBV-S-2F6单抗针对S-广谱肽以及重组乙型肝炎疫苗的P/N值显著大于2.1,分别达到了(15.33±0.29)和(14.29±0.26),针对其他物质的检测结果为阴性,说明该体系能特异检测HBV的S蛋白,也说明HBV-S-2F6单抗具有较好的用于区分乙肝病毒与其他物质。
实施例8HBV-S-2F6单抗的中和病毒能力检测
由于S蛋白是病毒的主要致病蛋白,因此,抑制S蛋白的单克隆抗体理论上都具有中和病毒的能力。委托中科科学院上海巴斯德研究所进行中和病毒能力检测。具体的,将HepG2-NTCP细胞接种到24孔板中,接种2×105细胞/孔,细胞贴壁;细胞分为实验组和对照组。实验组为ayw血清型病毒1.0×107(MOI=500)和梯度稀释后的HBV-S-2F6抗体(1、0.5、0.1、0.01mg/ml)共孵育,对照组(MOCK组)为病毒和PBS共孵育。各组在DMEM(10%FBS、2.5%DMSO、4%PEG 8000)中混匀后感染细胞过夜;用PBS洗5次,之后每2d换一次培养基,并检测第5天培养基中HBeAg。具体检测采用乙型肝炎病毒e抗原(HBeAg)检测试剂盒(化学发光法)(上海科华生物工程股份有限公司,国械注准20143402150)进行检测。
如图5所示,与对照组相比,实验组中的HBV-S-2F6抗体共孵育的细胞HBeAg均有明显下降,特别是1mg/mL的浓度下,OD450只有(0.19±0.03),而对照组为(1.79±0.06)(差异显著,P<0.01),这说明HBV-S-2F6抗体对HBV病毒有中和作用。随着孵育抗体浓度的下降,HBeAg的量不断上升,表明抗体中和病毒的能力不断减弱,说明HBV-S-2F6抗体对HBV病毒的中和效果具有剂量依赖效应。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (6)
1.一种广谱性的检测HBV的单克隆抗体HBV-S-2F6,其特征在于所述单克隆抗体包含重链可变区和轻链可变区,其中,
所述轻链可变区的氨基酸序列为SEQ ID NO:1所示,
所述重链可变区的氨基酸序列为SEQ ID NO:2所示。
2.如权利要求1所述的单克隆抗体在制备用于检测HBV S蛋白的试剂中的应用。
3.如权利要求1所述的单克隆抗体在制备用于检测HBV的试剂盒中的应用。
4.如权利要求1所述的单克隆抗体在制备用于检测HBV的试纸条中的应用。
5.如权利要求2或3所述的应用,其特征在于所述单克隆抗体被可检测的酶标记。
6.如权利要求4所述的应用,其特征在于所述单克隆抗体被胶体金标记。
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