CN113249334B - 一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株sftsn5g12 - Google Patents
一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株sftsn5g12 Download PDFInfo
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Abstract
本发明公开了一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12,在中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:C2020133,所述的杂交瘤细胞株SFTSN5G12分泌的抗发热伴血小板减少综合症(SFTS)病毒核衣壳蛋白的单克隆抗体用于制备检测或诊断SFTS病毒感染的试剂盒,试剂盒可以是免疫胶体金试纸条或ELISA试剂盒,本发明提供的杂交瘤细胞株SFTSN5G12可稳定分泌抗SFTS病毒的单克隆抗体(MAb),对单克隆抗体(MAb)的反应性与特异性进行了鉴定,为今后SFTS病毒感染的诊断、感染机制的研究奠定了基础。
Description
技术领域
本发明涉及生物技术领域,具体是涉及一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12。
背景技术
SFTS病毒(SFTSV),是发热伴血小板减少综合症(Fever with throbocytopeniasyndrome,SFTS)的病原体,为布尼亚病毒科白蛉热病毒属的一种病毒,其临床表现以发热伴血小板、白细胞减少和消化道症状为主要特征,中国国家疾控中心、河南省疾控中心、江苏省疾控中心等单位先后成功的分离到该病病毒,并对其进行了病原学、流行病学、临床医学等研究。目前该病毒序列已经阐明,但是对SFTS尚无特效药治疗和疫苗预防。
SFTS为***共患自然疫源性疾病,多发生于山区和丘陵等医疗卫生条件相对较差的地区,SFTS很容易与血液、消化、呼吸等多***疾病相混淆,加上临床医生对其缺乏认知,给该病的诊断和鉴别诊断带来困难。
发明内容
针对上述存在的技术问题,本发明提供了一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12。
本发明的技术方案是:
一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12,在中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:C2020133。
进一步地,所述的杂交瘤细胞株SFTSN5G12的制备方法为:
S1、取SFTS病毒日本分离株Yamakuchi株接种Vero-E6细胞,培养7天后取培养上清,按Trizol试剂说明书提取的总RNA,反转录合成cDNA第一链,以其为模板,设计特异性引物,序列如下:
P1:5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3’(下划线部分为SphI酶切位点)
P2:5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3’-3′(下划线部分为Hind III酶切位点)
S2、以P1、P2为引物,PCR扩增SFTS病毒全长N基因片段,反应条件为:94℃30s;52℃30s,72℃延伸1min,共35个循环;获得908bp的PCR扩增产物,用SphI和Hind III分别双酶切PCR产物和pQE30表达载体,回收目的基因片段和载体片段;
S3、用T4 DNA连接酶连接,连接产物转化感受态大肠杆菌XL1-blue,涂LB氨苄青霉素平板,37℃静止培养过夜,次日分别挑取单菌落,接种于3ml LB培养液中,37℃振荡培养16h,提取质粒,双酶切鉴定,琼脂糖凝胶电泳分析,阳性质粒进行基因测序;
S4、测序正确的重组表达质粒转化XL1-blue菌,接种于含100μg/ml Amp的LB培养基中管中,37℃振荡培养过夜;按1%接种量分别接种于含100μg/ml Amp的5ml LB培养基中管和200ml LB培养基三角瓶中扩大培养,37℃振荡培养至菌液A600值约为0.6时,加入终浓度为100μg/ml的IPTG,37℃振荡培养诱导4h;收集菌体经超声破碎,离心,取上清,用Ni2+亲和层析柱进行纯化。纯化表达产物经进行12% SDS-PAGE及Western blot鉴定分析,得到纯化后的重组SFTS核蛋白;
S5、按每只每次小鼠100微克的剂量免疫,首次免疫使用弗氏完全佐剂乳化,二次免疫和三次免疫使用不完全佐剂乳化,免疫间隔时间为两周,三次免疫7d后经小鼠尾部采血测血清抗体效价;融合前3d加强免疫一次,取免疫小鼠脾细胞与SP2/0按照常规方法进行细胞融合,HAT选择培养基筛选培养融合杂交瘤细胞;
S6、生长的杂交瘤细胞经间接ELISA及间接免疫荧光法检测杂交瘤细胞上清筛选分泌抗SFTS病毒核衣壳蛋白单克隆抗体的杂交瘤细胞;
S7、分泌抗SFTS病毒核衣壳蛋白单克隆抗体的杂交瘤细胞接种小鼠腹腔制备腹水,对腹水的ELISA及免疫荧光效价、单克隆抗体的特异性及所分泌单克隆抗体亚类鉴定。
本发明还提供了上述杂交瘤细胞株SFTSN5G12在制备检测或诊断SFTS病毒感染试剂中的应用。
进一步地,所述杂交瘤细胞株SFTSN5G12分泌的抗SFTS病毒核衣壳蛋白单克隆抗体与SFTS病毒的N蛋白发生抗原抗体反应,属IgG型抗体。
本发明还提供了一种检测或诊断SFTS病毒感染的试剂盒,该试剂盒包含所述杂交瘤细胞株SFTSN5G12分泌的抗SFTS病毒核衣壳蛋白单克隆抗体。
可选地,所述试剂盒为免疫胶体金试纸条。
更进一步地,所述试纸条按照连接顺序依次包括样品垫,胶体金垫,硝酸纤维素膜,吸水纸及位于下方的作为组装平台使用的PVC底板,所述硝酸纤维素膜与胶体金垫之间有搭接,所述硝酸纤维素膜与吸水纸之间有搭接,所述胶体金垫是由吸附有胶体金标记的抗SFTS N蛋白单克隆抗体的玻璃纤维素膜组成,所述硝酸纤维素膜上具有山羊抗鼠IgG多克隆抗体包被的质控线,以及抗SFTS N蛋白单克隆抗体包被的检测线。
更进一步地,所述质控线是通过将山羊抗鼠IgG多克隆抗体以0.8mg/ml的浓度包被在硝酸纤维素膜上得到的;所述检测线是通过将抗SFTS N蛋白单克隆抗体以0.5mg/ml的浓度包被在硝酸纤维素膜上得到的。
更进一步地,所述检测线和质控线间隔0.5cm。
更进一步地,上述免疫胶体金试纸条用于SFTS病毒检测的检测方法为:
S1、在点样孔滴加测试样品200μL,反应10-15min观察结果;
S2、结果判定:
质控线和检测线都出现红色条带,则为阳性;
仅在质控线出现现一条红色条带,则为阴性;
若质控线无条带出现,不论检测线是否出现红色条带结果均判定为无效。
还可以选择第,所述试剂盒为ELISA试剂盒。
本发明的有益效果是:本发明克隆了SFTSV N蛋白全长,在大肠杆菌中表达重组N蛋白,并进行纯化,及用纯化的重组核蛋白免疫Balb/c小鼠,以免疫鼠脾淋巴细胞与鼠骨髓瘤SP2/0细胞融合,筛选出1株分泌抗SFTS病毒核衣壳蛋白单克隆抗体的杂交瘤细胞株。此株杂交瘤细胞株可稳定分泌抗SFTS病毒的单克隆抗体(MAb),对单克隆抗体(MAb)的反应性与特异性进行了鉴定,为今后病毒感染的诊断、感染机制及疫苗开发的研究奠定了基础。
附图说明
图1是重组质粒在大肠杆菌表达出的-N蛋白产物经12%SDS-PAGE分析的结果。第1列:标准分子量蛋白;第2列:破碎后大肠杆菌上清;第3列:破碎后大肠杆菌沉渣;第4列:纯化后重组SFTSV N蛋白。
图2是SFTSN5G12株杂交瘤细胞分泌的单克隆抗体与未感染Vero-E6细胞及SFTS病毒感染Vero-E6细胞的反应结果。
其中,图A显示SFTSN5G12杂交瘤细胞分泌的单克隆抗体与SFTS病毒感染Vero-E6细胞之间反应,并在细胞浆呈现特异性免疫荧光;图B显示SFTSN5G12杂交瘤细胞分泌的单克隆抗体与未感染Vero-E6细胞无反应。
图3是SFTSN-5G12单抗与纯化表达重组SFTSV-N蛋白呈现阳性反应的Westernblot分析结果。第1列:标准分子量蛋白;第2列:纯化重组SFTSV N蛋白。
图4是胶体金标记SFTSN-5G12单克隆抗体制备的抗原检测诊断试剂的特异性试验结果。试纸条只与SFTS病毒反应,呈现阳性条带,而与四种血清型登革热病毒(D1,D2,D3,D4)、乙型脑炎病毒(JEV)、黄热病病毒(YFV)及裂谷热病毒(RVFV)无反应。
图5是SFTSN-5G12单克隆抗体胶体金抗原检测试剂灵敏度试验结果。试纸条可以检测到105个PFU以上的病毒颗粒。
图6是基于SFTSN-5G12单克隆抗体捕获法IgM抗体检测试剂盒的一次检测结果图。本捕获法IgM抗体检测试剂盒阳性血清颜色反应深,阴性血清背景低,阳性与阴性对比明显,肉眼就可以判断结果。
具体实施方式
实施例1
分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12,在中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:C2020133。
该杂交瘤细胞株SFTSN5G12通过以下方法制备得到:
1.材料与方法
S1、取SFTS病毒日本分离株Yamakuchi株接种Vero-E6细胞,培养7天后取培养上清,按Trizol试剂说明书提取的总RNA,反转录合成cDNA第一链,以其为模板,设计特异性引物,序列如下:
P1:5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3’(下划线部分为SphI酶切位点)
P2:5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3’-3′(下划线部分为Hind III酶切位点)
S2、以P1、P2为引物,PCR扩增SFTS病毒全长N基因片段,反应条件为:94℃30s;52℃30s,72℃延伸1min,共35个循环;获得908bp的PCR扩增产物,用SphI和Hind III分别双酶切PCR产物和pQE30表达载体,回收目的基因片段和载体片段;
S3、用T4 DNA连接酶连接,连接产物转化感受态大肠杆菌XL1-blue,涂LB氨苄青霉素平板,37℃静止培养过夜,次日分别挑取单菌落,接种于3ml LB培养液中,37℃振荡培养16h,提取质粒,双酶切鉴定,琼脂糖凝胶电泳分析,阳性质粒进行基因测序;
S4、测序正确的重组表达质粒转化XL1-blue菌,接种于含100μg/ml Amp的LB培养基中管中,37℃振荡培养过夜;按1%接种量分别接种于含100μg/ml Amp的5ml LB培养基中管和200ml LB培养基三角瓶中扩大培养,37℃振荡培养至菌液A600值约为0.6时,加入终浓度为100μg/ml的IPTG,37℃振荡培养诱导4h;收集菌体经超声破碎,离心,取上清,用Ni2+亲和层析柱进行纯化。纯化表达产物经进行12% SDS-PAGE及Western blot鉴定分析。
2.重组RSV-N蛋白的表达及鉴定
成功将-N基因扩增并克隆岛表达载体pQE-30,重组质粒在大肠杆菌表达出-N蛋白产物经12%SDS-PAGE分析,可见相对分子量约25kD的特异蛋白条带,大小与预期相符。见图1,其中,第1列:标准分子量蛋白;第2列:破碎后大肠杆菌上清;第3列:破碎后大肠杆菌沉渣;第4列:纯化后重组SFTSV N蛋白。
3.杂交瘤细胞株的建立与鉴定
用纯化重组核衣壳蛋白免疫BALB/C小鼠,取免疫脾细胞与SP2/0骨髓瘤细胞融合,经HAT选择培养筛选杂交瘤细胞。使用间接免疫荧光及间接ELISA筛选阳性杂交瘤细胞,3次有限稀释法亚克隆后,获得1株稳定分泌抗-N蛋白单克隆抗体的杂交瘤细胞株,分别命名为SFTSN-4G12。5株杂交瘤细胞分泌的单克隆抗体与未感染Vero-E6细胞无反应,与SFTS病毒感染Vero-E6细胞反应,在细胞浆呈现特异性免疫荧光,见图2,其中,图A显示SFTSN5G12杂交瘤细胞分泌的单克隆抗体与SFTS病毒感染Vero-E6细胞之间反应,并在细胞浆呈现特异性免疫荧光;图B显示SFTSN5G12杂交瘤细胞分泌的单克隆抗体与未感染Vero-E6细胞无反应。间接免疫荧光法特异性鉴定显示本株单抗均只与感染细胞反应,与登革热病毒(D1,D2,D3,D4),流行性乙型脑炎病毒,黄热病病毒及裂谷热病毒感染细胞均无反应。本株杂交瘤细胞注入小鼠腹腔,制备腹水的单抗效价,以包被重组核蛋白的间接ELISA法测定,5G12的效价为1:1,000,000;间接免疫荧光法测定,效价为1:100000;均明显高于免疫血清的效价。经单抗亚类的鉴定,本株单抗重链为IgG1,轻链为κ链。
经Western blot分析显示,SFTSN5G12单抗与纯化表达重组SFTSV-N蛋白呈现阳性反应,识别SFTSV-N蛋白,见图3,其中,第1列:标准分子量蛋白;第2列:纯化重组SFTSV N蛋白。
SFTSN-5G12单抗识别SFTSV-N蛋白,识别的氨基酸序列是:MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNL。
实施例2
本实施例提供了一种检测或诊断SFTS病毒感染的试剂盒,该试剂盒为免疫胶体金试纸条,该试剂盒包含所述杂交瘤细胞株SFTSN5G12分泌的抗SFTS核衣壳蛋白的单克隆抗体。
该试纸条按照连接顺序依次包括样品垫,胶体金垫,硝酸纤维素膜,吸水纸及位于下方的作为组装平台使用的PVC底板,所述硝酸纤维素膜与胶体金垫之间有搭接,所述硝酸纤维素膜与吸水纸之间有搭接,所述胶体金垫是由吸附有胶体金标记的抗SFTS N蛋白单克隆抗体的玻璃纤维素膜组成,所述硝酸纤维素膜上具有山羊抗鼠IgG多克隆抗体包被的质控线,以及抗SFTS N蛋白单克隆抗体包被的检测线。
所述质控线是通过将山羊抗鼠IgG多克隆抗体以0.8mg/ml的浓度包被在硝酸纤维素膜上得到的;所述检测线是通过将SFTSN-5G12抗SFTS N蛋白单克隆抗体以0.5mg/ml的浓度包被在硝酸纤维素膜上得到的。胶体金标记的SFTSN-5G12单抗则加在结合垫上,如样本中含有SFTSV抗原,液体移行至结合垫处则与胶体金标记的SFTSN-5G12单抗结合,再移行至检测线处,就会形成双抗体夹心抗原的免疫复合物,从而形成肉眼可见的红色条带。样本中不含SFTSV抗原,则无金标的抗原抗体复合物,无红色条带。
实施例3
本实施例提供的是以胶体金标记SFTSN-5G12单克隆抗体制备抗原检测诊断试剂,用于SFTS病毒的检测,包括以下步骤:
S1、在点样孔滴加测试样品200μL,反应10-15min观察结果;
S2、结果判定:
质控线和检测线都出现红色条带,则为阳性;
仅在质控线出现现一条红色条带,则为阴性;
若质控线无条带出现,不论检测线是否出现红色条带结果均判定为无效。
实施例4
本实施例是针对实施例2的试剂盒进行特异性试验
如图4所示,以胶体金标记SFTSN-5G12单克隆抗体制备抗原检测诊断试剂,胶体金诊断试剂特异性识别SFTS病毒,与4种血清型的登革热病毒(D1,D2,D3,D4),流行性乙型脑炎病毒,黄热病病毒及裂谷热病毒无交叉反应。
实施例5
本实施例是针对实施例2的试剂盒进行灵敏度试验
如图5所示,SFTSN5G12单克隆抗体胶体金抗原检测试剂敏感性分析显示,本抗原检测试剂可以检测到105个PFU以上的病毒颗粒。
实施例6
本实施例提供了一种双抗体夹心法检测SFTS病毒抗原以诊断SFTS病毒感染ELISA试剂盒。
以1:10000磷酸盐缓冲液(PBS pH7.2)稀释的SFTSN5G12单克隆抗体,每孔100μL加入96孔ELISA反应板,4℃包被过夜,再每孔加入100μL 3%BSA室温封闭1小时,PBS-Tween20洗涤ELISA板3次,加入100μL患者血清,37℃反应1小时,PBS-Tween20洗涤ELISA板3次,加入100μL稀释的辣根过氧化物酶(HRP)标记的SFTSN5G12单克隆抗体,37℃反应1小时,PBS-Tween20洗涤ELISA板3次,加入HRP底物(ABTS或TMB或OPD),37℃显色反应30分钟后终止反应,用酶标仪测定各样本孔吸光度OD值,OD值高于阴性对照2倍以上判断为阳性。
实施例7
本实施例提供了一种IgM捕获法检测病人血清SFTS病毒特异性IgM抗体以诊断SFTS病毒感染的ELISA试剂盒。
以1:500磷酸盐缓冲液(PBS pH7.2)稀释的羊抗人IgM抗体,每孔100μL加入96孔ELISA反应板,4℃包被过夜,再每孔加入100μL 3% BSA室温封闭1小时,PBS-Tween20洗涤ELISA板3次,加入100μL1:100稀释患者血清,37℃反应1小时,PBS-Tween20洗涤ELISA板3次,加入100μL(25ng)重组SFTSV-N蛋白,37℃反应1小时,PBS-Tween20洗涤ELISA板3次,稀释的辣根过氧化物酶(HRP)标记的SFTSN5G12单克隆抗体,37℃反应1小时,PBS-Tween20洗涤ELISA板3次,加入HRP底物(ABTS或TMB或OPD),37℃显色反应30分钟后终止反应,用酶标仪测定各样本孔吸光度OD值,OD值高于阴性对照2倍以上判断为阳性。SFTS病毒特异性IgM抗体阳性可以诊断SFTS
病毒近期感染。用河南省SFTS病毒疫区疑似患者血清115例,用于检验IgM捕获法ELISA的检测效果,检测出IgM抗体阳性85例,阴性30例,与市售检测患者血清总抗体的试剂盒结果完全一致。图6是基于SFTSN-5G12单克隆抗体捕获法IgM抗体检测试剂盒的一次检测结果图。由图6可见,本捕获法IgM抗体检测试剂盒阳性血清颜色反应深,阴性血清背景低,阳性与阴性对比明显,肉眼就可以判断结果。
序列表
<110> 贵州省人民医院
<120> 一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12
<130> 无
<160> 2
<170> PatentIn version 3.5
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<211> 27
<212> DNA
<213> 人工序列
<221> misc_feature
<222> (1)..(27)
<223> 根据实验要求而设计,用于HRSV N基因片段扩增的特异性引物
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<212> RNA
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<223> 根据实验要求而设计,用于HRSV N基因片段扩增的特异性引物
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Claims (4)
1.一种分泌抗发热伴血小板减少综合征病毒单克隆抗体的杂交瘤细胞株SFTSN5G12,其特征在于,在中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:C2020133。
2.一种如权利要求1所述的杂交瘤细胞株SFTSN5G12在制备检测或诊断SFTS病毒感染试剂中的应用。
3.一种如权利要求1所述的杂交瘤细胞株SFTSN5G12分泌的抗SFTS病毒核衣壳蛋白单克隆抗体,其特征在于,所述的单克隆抗体与SFTS病毒的N蛋白发生抗原抗体反应,属IgG型抗体。
4.一种检测或诊断SFTS病毒感染的试剂盒,其特征在于,包含如权利要求3所述的单克隆抗体。
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