CN113403327A - 结核分枝杆菌H37Rv新基因Rv2706及其编码蛋白和应用 - Google Patents
结核分枝杆菌H37Rv新基因Rv2706及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明涉及一种结核分枝杆菌H37Rv新编码基因Rv2706(‑|3020323‑3020511|)及其编码蛋白。Rv2706所编码蛋白部分或者全部具有B细胞抗原性,可单独或者联合用于肺结核患者的临床快速筛查和保护人体免受结核分枝杆菌感染。
Description
技术领域
本发明涉及生物技术领域,具体涉及结核分枝杆菌的新基因和应用。
背景技术
结核病是一个全球性的重大公共卫生问题,结核分枝杆菌(Mycobacteriumtuberculosis,MTB)是引起人类结核病的病原菌。据WHO最新发布的2019年全球结核病报告数据显示,2018年新增约1000万新发活动性肺结核患者,结核病仍然是感染性疾病中的头号杀手。除了活跃的结核病患者外,世界上约有1/4人口感染了MTB,面临着进一步发展为活动性结核病的风险。故建立快速、准确、特异、敏感、廉价的结核病检测方法,是有效治疗、控制结核病蔓延的必要前提。
目前结核病的诊断主要依赖1)痰液细菌学检查及胸片影像学检查,但是存在敏感性低或耗时长的缺点;2)免疫学诊断法,但因与BCG疫苗或其它环境分枝杆菌产生交叉免疫反应,从而导致了高假阳性的产生,尤其是BCG疫苗接种区尤为明显。至今国内外已有多个类似于T-SPOT.TB的试剂盒或依托蛋白质芯片免疫诊断辅助技术的试剂盒,但均由于缺乏可鉴别结核分枝杆菌有效特异性新抗原这一核心难题,难于解决“阳性不能确证,阴性难于排除”的难题。因此结核分枝杆菌特异性高效抗原的开发是业界亟待解决的核心难题,任何进步都将为结核病临床诊断技术的发展和试剂盒的研制提供原始创新性成果和技术支撑。
发明内容
本发明的目的是提供一种结核分枝杆菌H37Rv漏注释基因Rv2706(-|3020323-3020511|)。
本发明的第二个目的是提供上述基因编码的蛋白产物。
本发明的第三个目的是提供一种结核分枝杆菌IgG抗体检测抗原。
本发明的第四个目的是提供一种结核分枝杆菌检测试剂。
本发明的第五个目的是提供一种结核分枝杆菌抗原。
本发明的第六个目的是提供上述结核分枝杆菌IgG抗体检测抗原和结核分枝杆菌抗原的应用。
根据本发明的一方面,一种分离的核苷酸,为结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|),所述编码基因的核苷酸序列如SEQ ID NO.1所示。
我们通过精准蛋白质基因组学技术,鉴定到其编码基因表达产物的一条高质量肽段“AKLPSGAELLFCQHHANEHEAK”(图1)。通过人工合成该肽段,液相色谱-质谱联用分析,原始谱图与合成肽段谱图基本一致(图2)。新基因Rv2706(-|3020323-3020511|)的编码蛋白经NCBI-BLASTP后,数据库中没有比对到任何序列,属于功能未知蛋白。Bepipred LinearEpitope Prediction(v2.0)和BioSun分别预测到1和3个高分值B细胞表位,覆盖了整个蛋白质序列的85.48%(表1),意味着其可能具有B细胞抗原性,可用于结核分枝杆菌体液免疫检测,其蛋白序列如SEQ ID NO.2所示。
表1.Rv2706理论预测的高可信表位
根据本发明的又一方面,一种Rv2706编码蛋白,由上述结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)编码,其氨基酸序列如SEQ ID NO.2所示。
根据本发明的再一方面,一种结核分枝杆菌IgG抗体检测抗原,包含所述的Rv2706编码蛋白的全部或部分氨基酸,或者含有Rv2706编码的蛋白的全部或部分氨基酸与结核分枝杆菌已知抗原蛋白的组合。
根据本发明的又一方面,一种结核分枝杆菌检测试剂,至少包含下述之一:
1)PCR引物,所述引物用于扩增所述的结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)的部分或其全部DNA序列或者其转录所得RNA经逆转录所得的部分或者全部模板序列;
2)上述的肽段,其氨基酸序列如SEQ ID NO.3所示;
3)上述的结核分枝杆菌IgG抗体检测抗原;
4)由上述结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)的部分或其全部DNA或其转录所得RNA。
在优选的实施方案中,上述检测试剂所用的PCR引物序列可以为但不限于下述序列:
F1:5’-GGCGGTTGCAGACCGAAATTCGGGA-3’(SEQ ID NO.5);
R1:5’-AAACTGCGCGCGCTACCGGCATTGT-3’(SEQ ID NO.6)。
在另一个优选的实施方案中,上述检测试剂所用的PCR引物序列可以为但不限于下述序列:
F2:5’-CCGGAATTCATGAACGCAACTCTGA-3’(SEQ ID NO.7);
R2:5’-CAAGCTTCTATTCGCTCCCGCTGAC-3’(SEQ ID NO.8)。
本领域技术人员可以理解的是,可以根据需要将本发明的检测抗原制备成各种检测试剂,例如通过核酸杂交(例如基因芯片的杂交试验、RNA印迹、斑点印迹等)和/或通过核酸扩增(例如PCR、qPCR、RT-PCR、qRT-PCR、质谱法等),检测试剂也可以是抗体,通过免疫化学试验(例如ELISA、化学发光、免疫荧光试验等)进行检测。
在本发明的具体实施方案中,对有需要的受试者使用上述检测试剂检测结核分枝杆菌,检测方法可以包括:(a)从得自受试者的生物学样品中分离和/或扩增DNA或RNA;(b)使上述分离和/或扩增的DNA或RNA与所述检测试剂接触;(c)检测H37Rv编码基因Rv2706(-|3020323-3020511|)和/或Rv2706编码蛋白的水平;和(d)必要时与参考检测试剂结合。
根据本发明的再一方面,一种结核分枝杆菌抗原,包含选自下述组之一的物质:
a)上述结核分枝杆菌H37Rv基因Rv2706(-|3020323-3020511|)编码蛋白的全部或部分氨基酸;
b)上述Rv2706编码蛋白的全部或部分氨基酸;
c)上述的肽段。
可以将上述的结核分枝杆菌抗原通过免疫动物或基因工程手段制备结核疫苗。
有益效果:本发明提供一种用作结核病诊断的新抗原,该抗原能有效地将结核病患者和健康人区分开来。应用该抗原的诊断方法克服了现有结核病确诊过程中菌株培养时间长、痰样本受限制等缺点。新抗原Rv2706具有快速、准确、特异、敏感、廉价的特点,可用于结核分枝杆菌感染结核病临床患者辅助诊断和大规模结核流行病学调查。
本发明提供了Rv2706抗原的制备过程,通过免疫印迹(Western Blot)、酶联免疫(ELISA)、化学发光等检测,比较肺结核患者及正常人血清中IgG抗体的水平来判断待检者是否为肺结核患者。检测结果表明,本发明提供的新抗原辅助诊断肺结核感染患者的灵敏度为66.25%,特异度62.50%,表明基于Rv2706新蛋白具有抗原性,可用于结核病患者的筛查,在结核病诊断和防治中具有应用价值。
附图说明
图1:质谱检测肽段二级谱图;
图2:合成肽段质谱图与原鉴定肽段镜像比对谱图;
图3:新基因Rv2706(-|3020323-3020511|)特异性引物序列设计区序列;
图4:新基因Rv2706(-|3020323-3020511|)特异性扩增琼脂糖凝胶电泳图;
其中,各泳道样品具体信息见表2。
图5:Rv2706(-|3020323-3020511|)目的基因PCR扩增核酸电泳图;
图6:Rv2706(-|3020323-3020511|)目的基因双酶切核酸电泳图;
图7:H37Rv菌株新基因Rv2706(-|3020323-3020511|)PCR扩增结果和标准序列BLASTN比对结果;
图8:Rv2706(-|3020323-3020511|)蛋白诱导表达10%SDS-PAGE胶图;
图9:Rv2706(-|3020323-3020511|)蛋白纯化10%SDS-PAGE胶图;
图10:Rv2706(-|3020323-3020511|)纯化蛋白质谱检测肽段覆盖度;
图11:血清样本Rv2706抗原ELISA检测的ROC曲线。
具体实施方式
下面结合具体实施方式对本发明做进一步说明,但不限制本发明权利要求范围。本发明所用试剂均为市售。
实施例1:新基因Rv2706(-|3020323-3020511|)的发现及验证
我们利用深度覆盖蛋白质组技术对结核分枝杆菌H37Rv菌株进行了高覆盖蛋白质组研究。用中国科学院计算技术研究所开发的pAnno软件对H37Rv在NCBI发表的全基因组(NC_000962.3)文件按照正链和互补链上连续的3个核苷酸翻译一个氨基酸的规则翻译,使用终止子到终止子的翻译模式,采用MTB特殊的3种起始密码子ATG、GTG和TTG,共翻译得到141,851个开放阅读框(Opening reading frame,ORF)。利用这个数据库对H37Rv高覆盖蛋白质组数据进行搜索。为降低假阳性率,我们在数据过滤的过程中分别从已注释肽段和新肽段两个层面进行严格质控。在H37Rv负链3020293-3020509处鉴定到1条其编码产生的高质量肽段AKLPSGAELLFCQHHANEHEAK,如图1所示,谱图质量很好,b/y离子连续匹配,杂峰信号较低,结果很可信。
为进一步辨别这个肽段的真伪,我们人工合成了这一氨基酸序列的肽段“AKLPSGAELLFCQHHANEHEAK”。液相色谱-质谱联用分析生产了合成肽段的质谱谱图,手工比对了合成肽段的二级质谱谱图(MS2)(图2)和大规模鉴定新肽段的质谱谱图,两者基本一致,谱图相似性(Cosin值)为0.89,表明我们利用蛋白质基因组学从结核分枝杆菌鉴定到了一段DNA序列对应的漏注释基因编码的蛋白质消化产生的肽段。
在确认上述漏注释肽段的序列后,根据上述肽段所在的基因位置,以起始密码子ATG和终止密码子TGA包括的区域为界,得到包含上述新漏注释肽段的开放阅读框(ORF)DNA序列,如SEQ ID NO.1所示。基因序列从ATG开始,共189bp,编码62个氨基酸,其理论分子量6.68kDa,根据该基因在染色体上的位置及结核菌基因命名法则将其命名为Rv2706基因。
ATGAACGCAACTCTGACCAGTCCTGAGCTGACTAGAGCAGACCGCTGCGACCGCTGTGGCGCTGCAGCTCGGGTGCGCGCCAAGCTGCCCTCCGGAGCCGAGCTTCTTTTCTGCCAGCATCACGCCAACGAGCACGAGGCGAAACTGACCGAGATGTCCGCCGTGCTGGAGGTCAGCGGGAGCGAATAG(SEQ ID NO.1)
该基因理论编码产物氨基酸序列如SEQ ID NO.2所示:
MNATLTSPELTRADRCDRCGAAARVRAKLPSGAELLFCQHHANEHEAKLTEMSAVLEVSGSE(SEQ IDNO.2)
蛋白质基因组学技术鉴定到的肽段序列如SEQ ID NO.3所示:
AKLPSGAELLFCQHHANEHEAK(SEQ ID NO.3)
实施例2:建立鉴定目的基因特异性验证的方法
(1)设计引物:
从NCBI数据库下载了已经完成全基因组测序的分枝杆菌属序列(近200个物种,8,046株菌),构建了分枝杆菌属数据库。目的基因进行本地同源性序列比对后,对虽有同源性但序列有异的区域进行引物设计(图3)。
采用Primer Premier 5对目的基因前后各延伸600bp序列(SEQ ID NO.4)比对差异区设计了PCR特异性引物,引物序列如下:
F1:5’-GGCGGTTGCAGACCGAAATTCGGGA-3’(SEQ ID NO.5);
R1:5’-AAACTGCGCGCGCTACCGGCATTGT-3’(SEQ ID NO.6)。
上述引物在与Rv2706(-|3020323-3020511|)基因附近的位置关系如下所示,其中引物对应位置下标波浪线,黑色背景部分为目的基因区,下划线标注序列为PROMPREDICT在线平台理论预测的启动子。
(2)提取包括M.tuberculosis H37Rv、近源分枝杆菌以及其它感染性菌株的总DNA,6株分枝杆菌属标准菌株由中国医学细菌菌种保藏管理中心(CMCC)保藏,其余6株非结核分枝杆菌是中国人民解放军309医院临床分离株,已经完成菌种16S RNA基因测序、比对及NCBI序列提交工作,特异性扩增待测菌株如表2所示:
表2.选用的相关菌株
(3)扩增DNA片段,进行聚合酶链式(PCR)反应,所用上述设计的F1/R1引物进行扩增。
PCR体系(25μL)为dd H2O(9.5μL)、2XTaq PCR MasterMix(TIANGEN,12.5μL)引物F(10μM,1μL)、引物R(10μM,1μL)、DNA模板(1μL);
扩增程序:94℃预变性3min、94℃变性1min、58℃退火1min、72℃延伸2min、35个循环,72℃延伸5min。
(4)扩增产物电泳检测,在琼脂糖凝胶、1×TBE电泳液中电泳检测。结果如图4所示,仅在H37Rv菌株的728bp处出现了特异性扩增条带,且扩增结果和预期相符,意味着此特异性引物可以将结核分枝杆菌与其它分枝杆菌、致病菌进行有效地区分。
实施例3:Rv2706(-|3020323-3020511|)基因的原核克隆、表达、纯化及质谱鉴定
3.1Rv2706(-|3020323-3020511|)基因的克隆
(1)设计引物:
采用Primer Premier 5设计了PCR引物,上、下游引物分别添加EcoR I和Hind III限制性内切酶位点,便于后续克隆构建。引物序列如下:
F2:5’-CCGGAATTCATGAACGCAACTCTGA-3’(SEQ ID NO.7);
R2:5’-CAAGCTTCTATTCGCTCCCGCTGAC-3’(SEQ ID NO.8)。
(2)M.tuberculosis H37Rv菌株的总DNA由中国疾病预防控制中心传染病所结核病室提供。
(3)扩增DNA片段,进行聚合酶链式(PCR)反应,所用上述F2/R2引物进行扩增。
PCR体系(25μL)为dd H2O(9.5μL)、2XTaq PCR MasterMix(TIANGEN,12.5μL),引物F(10μM,1μL)、引物R(10μM,1μL)、DNA模板(1μL);
扩增程序:94℃预变性3min、94℃变性1min、60℃退火1min、72℃延伸2min、35个循环,72℃延伸5min。
(4)扩增产物5μL电泳检测,在琼脂糖凝胶、1×TBE电泳液中电泳检测。结果如图5所示,在205bp处扩增出了与目的条带预期相符的DNA条带。
(5)将剩余20μL×4样品用50mL 1.8%琼脂糖凝胶电泳分离,出现目的条带,进行切胶回收,回收方法采用Takara DNA凝胶回收试剂盒说明操作(最后加入50μL dd H2O),回收产物于-80℃冰箱冻存备用。
(6)pMD18-T载体与扩增的目的基因连接。
连接体系(10μL)为Solution I(5μL)、PMD-18-T载体(Takara,0.5μL)、第(5)步DNA切胶回收产物;16-18℃连接12h。
(7)大肠杆菌DH5α转化
取大肠杆菌DH5α感受态细胞(北京康为世纪生物科技有限公司)置于冰浴中,待感受态细胞融化后,向感受态细胞悬液中加入(6)的连接产物,吹打混匀,冰浴20-30min;
42℃热浴90s,迅速转移到冰浴中,静置1min;
42℃热浴1min,冰浴2min;
向含有DH5α感受态细胞的1.5mL EP离心管中加入800μL LB液体培养基(胰蛋白胨,10g,酵母提取物5g,氯化钠10g,dd H2O补至1升),混匀后置于37℃摇床,200rpm振荡培养1h;
取出EP管,4000rpm离心5min,保留100μL上清,重悬菌体并涂布于LA固体培养基(配置LB固体培养基,高温灭菌冷凝至50-60℃时加入终浓度为100μg/mL的氨苄抗生素(Amp)),先正置培养1h,再倒置,37℃恒温培养12-16h。
(8)PMD18-T目的基因质粒提取。
向无菌试管中加入5mL LA液体培养基(配置LB液体培养基,冷凝至室温后加入终浓度为100μg/mL的氨苄抗生素(Amp)),接种单克隆,于37℃摇床200rpm培养12h后,收集菌体,采用质粒DNA小量纯化试剂盒(Takara)提取质粒,最后加入50μL dd H2O,质粒放-80℃冰箱冻存备用。
(9)双酶切获取目的基因片段
双酶切体系(50μL)为(8)中的质粒(44μL)、NEBufferTM 2.1(5μL)、EcoR I(0.5μL)、Hind III(0.5μL);
37℃酶切5h,1%琼脂糖凝胶电泳检测酶切目的片段。如图6,在目的大小处看到目的条带,采用Takara DNA凝胶回收试剂盒进行胶回收,最后加入50μLdd H2O,回收产物放-80℃冰箱冷冻备用。
(10)pMAL-c2X载体连接目的片段
连接体系(10μL)为(9)中的DNA片段(4μL)、Solution I(5μL)、EcoR I和Hind III双酶切好的pMAL-c2X载体(1μL);16-18℃连接12h。
(11)大肠杆菌DH5α转化
取大肠杆菌DH5α感受态细胞置于冰浴中,待感受态细胞融化后,向感受态细胞悬液中加入(10)的连接产物,混匀后冰浴20-30min;
42℃热激90s,冰浴1min;
42℃热浴1min,冰浴2min;
向含有大肠杆菌DH5α的感受态细胞的EP离心管中加入800μL LB液体培养基,混匀后置于37℃摇床,200rpm振荡培养1h;
取出EP管,4000rpm离心5min,保留100μL上清,重悬菌体并涂布于LA固体培养基,先正置培养1h,再倒置,37℃恒温培养12-16h。
(12)提取pMAL-c2X-目的基因质粒。
向无菌试管中加入5mL LA液体培养基,接种单克隆,于37℃摇床200rpm培养12h后,收集菌体,采用质粒DNA小量纯化试剂盒(Takara)提取质粒,最后加入50μL dd H2O,质粒放-80℃冰箱冷冻备用。
(13)将(12)的质粒送去测序(北京擎科生物科技有限公司),测序结果BLAST比对如图7
3.2Rv2706(-|3760359-3761516|)基因编码蛋白的诱导表达
(1)表达宿主E.coli BL21(DE3)表达重组质粒
取宿主细胞置于冰浴中,待宿主细胞融化后,向宿主细胞悬液中加入重组质粒0.5μL,混匀后冰浴20-30min;
42℃热激90s,冰浴1min;
42℃热激1min,冰浴2min;
加入800μL LB液体培养基,混匀后置于37℃摇床,200rpm振荡培养1h;
取出EP管,4000rpm离心5min,保留100μL上清,重悬菌体并涂布于LA固体培养基,先正置培养1h,再倒置,37℃恒温培养12-16h。
(2)Rv2706(-|3760359-3761516|)基因编码蛋白IPTG诱导表达
取(1)中的单克隆转接至装有50mL LA培养基的250mL三角瓶中,37℃过夜培养;
按照起始OD=0.1的量转接至装有500mL LA培养基的三角瓶中,37℃、200rpm摇床培养至OD=0.6时,取出14OD菌于-80℃保存,作为诱导前菌体;
剩余菌液加入IPTG诱导(终浓度1mmol/L),28℃诱导培养6h,20倍稀释测OD值,取出14OD的菌于-80℃保存,作为诱导后菌体;
其它诱导后菌体一并收集后-80℃保存;
诱导前和诱导后菌体超声破碎5min(30%功率,超声2s,冰浴4s),13000rpm离心5min,上清转到新的1.5mLEP管;
诱导前和诱导后沉淀处理:加400μL 1XSDS Loading buffer重悬菌体,添加终浓度为5mmol/L DTT,95℃煮样10min,13000rpm离心5min收集上清,-80℃保存;
诱导前后蛋白样本用10%SDS-PAGE进行蛋白检测,电泳胶图如图8。
3.3Rv2706蛋白的纯化向菌体中添加适量裂解液,超声破碎5min,13000rpm离心10min,保留上清;
吸取100μL Amylose beads加入到纯化柱中,向纯化柱中加入500μL清洗液(Washing buffer),平衡纯化柱;
将超声后的细胞裂解液200μL加入平衡好的纯化柱中,迅速套上黄色截留管,4℃冰箱孵育1h,以便充分结合;
4℃孵育后,移走下面的黄色截留管,让其液体缓缓流出,向纯化柱中加入1mLWashing buffer溶液,让其液体缓缓流出,重复三次;
向纯化柱中加入50μL洗脱液(Elution buffer)溶液,让其液体缓缓流入1.5mL离心管中,重复两次,收集所得的目的蛋白;
以上步骤均在冰浴中进行。取5μL收集的洗脱液,加入5μL的2XSDS Loadingbuffer,加入新鲜配置的DTT至终浓度为5mmol/L,95℃变性10min,10%SDS-PAGE电泳检测,考马斯亮蓝染色,结果如图9。
上述纯化过程中所用到的缓冲液的组成见表3-5。
表3.蛋白裂解液成分
表4.清洗液成分
表5.洗脱液成分
3.4 Rv2706纯化蛋白的质谱检测
将纯化的目的条带切成1mm3胶粒,脱水、蒸干、经乙酰化胰蛋白酶(10ng/μL)37℃过夜消化、抽提肽段、蒸干。
蒸干后的肽段使用上样缓冲液(1%乙腈+1%甲酸+98%ddH2O)进行充分溶解。通过超高压液相色谱进行分离,其中分离柱采用内径75μm,长15cm的C18反相色谱分析柱,内部C18填料内径3μm。洗脱组分经纳升级电喷雾离子源接口喷出进入LTQ Orbitrap Velos分析。
毛细管离子传输温度为250℃,电喷雾电压为1.8kV。质谱采用一级质谱数据依赖的二级质谱扫描模式(Data dependent MS/MS scan)碰撞诱导裂解(Colision-induceddissociation,CID)模式碎裂一级离子。一级质谱扫描质核比范围为300-1600(m/z),分辨率设置为30 000;自动增益控制(Automatic gain control,AGC)为106。依次选取一级信号强度最高的20个离子进行二级碎裂分析,碰撞归一化能量为35%;AGC为104;最大离子注入时间为30ms;动态排除为40s。
MaxQuant对质谱产生的数据文件(.raw)进行蛋白质数据库搜索,数据库由从UniProt(Version:2016-01)下载目标E.coli完整蛋白质组序列、结核分枝杆菌H37Rv遗漏注释蛋白Rv2706序列、pMAL-c2X载体标签MBP蛋白序列和常见污染构成,肽段覆盖度如图10。
实施例4:应用Rv2706抗原辅助肺结核感染诊断筛查
4.1待测血清样本的收集与筛
按照WHO指南要求(表6)进行结核及健康志愿者临床外周血样本收集,具体信息见表7-9。
表6.临床血清样本信息
*结核菌素:结核杆菌的菌体成分,包括纯蛋白衍生物(PPD)和旧结核菌素(OT);判断标准:PPD>10mm;
#干扰素实验:利用结核特异抗原ESAT-6及CFP-10,通过酶联免疫斑点技术ELISPOT检测受试者体内是否存在结核效应T淋巴细胞,从而判断目前该受试者是否感染结核杆菌。判断标准:单抗原斑点数>6。
按照上述判断标准共收集结核病患者血清80例,其中菌阳40例、菌阴40例、健康者的血液样本各40例。
判断标准:
结核病患者:1)胸片(CXR)异常,有临床症状,抗结核治疗有效;2)T-SPOT试验阳性;3)结核菌快速培养结果阳性/阴性;
健康志愿者:1)胸片(CXR)无异常;2)T-SPOT试验阴性;3)结核菌素阴性。
以上样本皆排除人免疫缺陷病毒(HIV)阳性和服用免疫抑制药物的患病志愿者。
表7.结核分枝杆菌培养阳性患者信息
表8.结核分枝杆菌培养阴性患者信息
表9.健康志愿者信息
4.2血清IgG抗体检测
国内应用较好的商用结核分枝杆菌IgG检测试剂盒包含七抗原组合阳性抗原对照(如上海荣盛结核分枝杆菌IgG检测试剂盒)和国际公认的38kD抗原作为阳性对照,对我们融合表达的Rv2706编码蛋白进行包被,统一用上海荣盛结核分枝杆菌IgG检测试剂盒的试剂和流程进行操作筛选。
检测结果发现上海荣盛试剂盒对我们上述收集的120例不同人群临床血清样本的灵敏度为75%(60/80)、特异度为62.5%(25/40),与该试剂盒已报道的灵敏度、特异度比例相差不大,意味着该试剂盒及其检测体系可作为我们后续筛选目的抗原的阳性参考及检测方案。
为了进一步检测目的蛋白的包被条件及检测体系,我们利用和Rv2706目的蛋白一样的融合表达体系表达了国际已知的结核分枝杆菌较好的阳性抗原38kD蛋白,纯化后进行包被、抗原性检测,检测试剂用上海荣盛结核分枝杆菌IgG检测试剂盒中的试剂,具体操作方法如下:
包被:用包被缓冲液(称取2.93g NaHCO3、1.59g Na2CO3,加dd H2O定容至1000mL(pH约为9.6),高压灭菌)稀释Rv2706蛋白,终浓度30μg/mL,每孔100μL包被酶标板(康宁),4℃包被过夜;
洗板:1×PBST(试剂盒)每孔300μL,洗板5次,拍干;
封闭:每孔加入封闭液(3%BSA溶于PBS),37℃封闭2h;
洗板:每孔300μL洗涤液,洗板5次,拍干;
一抗(血清):针对每一个蛋白分别加入阴性、阳性对照血清100μL,重复一次;再设一孔空白对照,加样品稀释液100μL,其余每孔加入100μL样品稀释液及10μL待测血清,37℃孵育30min;洗板:每孔300μL洗涤液,洗板5次,拍干;
二抗(酶结合物):每孔加入100μL(空白对照孔除外),37℃孵育20min;
洗板:每孔300μL洗涤液,洗板5次,拍干;
显色:每孔加显色液A和B各50μL,轻轻震荡,37℃避光显色10min;
终止反应:每孔加50μL终止液终止反应;
读数:酶标仪读数,在450nm波长处读取吸光度值(OD)。
检测结果发现阳性抗原38kD的灵敏度和特异度分别是65.83%和55%,意味着融合表达蛋白经包被后,利用上海荣盛结核分枝杆菌中的试剂及检测体系可行,可用于Rv2706目的蛋白抗原性的检测。鉴于前期预试验结果,我们对Rv2706新包被蛋白进行血清IgG抗体检测,具体检测值见表10。
表10.Rv2706新抗原检测120份血清样本OD检测数值
阴影部分是患者的数据
检测分类:根据ROC曲线(图11)中给出的各切点计算Yu-den指数,Yu-den指数最高值所对应的即为Cut-off值,该蛋白的Cut-off值为0.128,≥0.128的判断为阳性(患者),<0.128为阴性(健康者)。
结果80份患者血清中正确判断为阳性的有53例,灵敏度为66.25%,40份健康人的血清中正确判断为阴性的有25例,特异度62.50%。这表明基于新抗原Rv2706进行结核病患者筛查的方法真实可靠。
SEQUENCE LISTING
<110> 北京蛋白质组研究中心
<120> 结核分枝杆菌H37Rv新基因Rv2706及其编码蛋白和应用
<130> BJ1936-20P150031
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 189
<212> DNA
<213> Mycobacterium tuberculosis H37Rv
<400> 1
atgaacgcaa ctctgaccag tcctgagctg actagagcag accgctgcga ccgctgtggc 60
gctgcagctc gggtgcgcgc caagctgccc tccggagccg agcttctttt ctgccagcat 120
cacgccaacg agcacgaggc gaaactgacc gagatgtccg ccgtgctgga ggtcagcggg 180
agcgaatag 189
<210> 2
<211> 62
<212> PRT
<213> Mycobacterium tuberculosis H37Rv
<400> 2
Met Asn Ala Thr Leu Thr Ser Pro Glu Leu Thr Arg Ala Asp Arg Cys
1 5 10 15
Asp Arg Cys Gly Ala Ala Ala Arg Val Arg Ala Lys Leu Pro Ser Gly
20 25 30
Ala Glu Leu Leu Phe Cys Gln His His Ala Asn Glu His Glu Ala Lys
35 40 45
Leu Thr Glu Met Ser Ala Val Leu Glu Val Ser Gly Ser Glu
50 55 60
<210> 3
<211> 22
<212> PRT
<213> Mycobacterium tuberculosis H37Rv
<400> 3
Ala Lys Leu Pro Ser Gly Ala Glu Leu Leu Phe Cys Gln His His Ala
1 5 10 15
Asn Glu His Glu Ala Lys
20
<210> 4
<211> 1389
<212> DNA
<213> Mycobacterium tuberculosis
<400> 4
aggctttcgg caagatccgt ccggtgacga gcatggtcga ggttaccgcg ctgattgcgc 60
ccggcctgct ggtagagatc gaggccgacg cctacgtagg gtcggcggtt gcagaccgaa 120
attcgggagc cggcccgaag gacccgtcac cagccggtgg gtaggcggcg gccccaatca 180
cagcgcgcac cggcagtggg ccgtagagat gcgggaaaag catcgaccgc ggatcagtag 240
gcacgcccgg ctcccaacgc acgggtgagt cgagcgccgc cgggtcgatg tacagcagca 300
ccaggtcagc acggccacgg taaaggcggt tggcgggcag gtgaacctgc tcgagtgtcg 360
acaggtggat ataccccgtc ttgtcggact cgggatagat cccaccgcgt tctcgggcat 420
gcgaccactc ctgcaccccg cataggtgca ccagcatggc aggatcgggc gtcattctca 480
ccaccctgcc cgattggcgg gggcgaaagt cgtgagaaat gacacacccg acagcggccg 540
gggaacacgg cgagaacccc gaacgtctga gaaggtgaag atacccgaga acggagagcc 600
atgaacgcaa ctctgaccag tcctgagctg actagagcag accgctgcga ccgctgtggc 660
gctgcagctc gggtgcgcgc caagctgccc tccggagccg agcttctttt ctgccagcat 720
cacgccaacg agcacgaggc gaaactgacc gagatgtccg ccgtgctgga ggtcagcggg 780
agcgaataga ccgaactcac ccgtccacaa tgccggtagc gcgcgcagtt ttcggtaatg 840
ctggactggt atgagcgacc aggtccccaa gccacaccgc caccacatct ggcgaatcac 900
ccgtaggact ttgtccaaaa gctgggacga ctcgatcttc tcggagtcag cgcaagcggc 960
tttttggtcg gccttgtctt tgccgccgct actgctggga atgctgggca gtctggccta 1020
cgttgctccg ctattcggcc cggacacctt gcccgcgatt gaaaagagcg cgctttcgac 1080
ggcccacagc tttttctccc ccagtgtggt caacgagatc atcgagccca ccatcggcga 1140
tatcaccaac aacgcccgcg gtgaggtggc gtcgctgggc ttcttgatct cgctgtgggc 1200
aggatcgtcg gcaatctcgg cgttcgtcga tgcagtggtg gaagcgcacg accagacacc 1260
gctacgccac ccggtccggc aacgcttctt tgcgctcttc ctctacgtgg tgatgttggt 1320
gttcctagta gcgaccgcac cggtaatggt ggtgggtcca cgcaaggtaa gcgagcacat 1380
cccggagag 1389
<210> 5
<211> 25
<212> DNA
<213> Synthetic
<400> 5
ggcggttgca gaccgaaatt cggga 25
<210> 6
<211> 25
<212> DNA
<213> Synthetic
<400> 6
aaactgcgcg cgctaccggc attgt 25
<210> 7
<211> 25
<212> DNA
<213> Synthetic
<400> 7
ccggaattca tgaacgcaac tctga 25
<210> 8
<211> 25
<212> DNA
<213> Synthetic
<400> 8
caagcttcta ttcgctcccg ctgac 25
Claims (10)
1.一种分离的核苷酸,为结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|),所述编码基因的核苷酸序列如SEQ ID NO.1所示。
2.一种Rv2706编码蛋白,由权利要求1所述的结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)编码,其氨基酸序列如SEQ ID NO.2所示。
3.一种多肽,包含在权利要求2所述的Rv2706编码蛋白中,其氨基酸序列如SEQ IDNO.3所示。
4.一种结核分枝杆菌IgG抗体检测抗原,包含权利要求2所述的Rv2706编码蛋白的全部或部分氨基酸,或者含有Rv2706编码蛋白的全部或部分氨基酸与结核分枝杆菌已知抗原蛋白的组合。
5.一种结核分枝杆菌检测试剂,至少包含下述之一:
1)PCR引物,所述引物用于扩增权利要求1所述的结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)的部分或其全部DNA序列或者其转录所得RNA经逆转录所得的部分或者全部模板序列;
2)权利要求4所述的检测抗原;
3)权利要求1所述的结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)的部分或其全部DNA或其转录所得RNA。
6.如权利要求5所述的检测试剂,其中所述PCR引物序列为:
F1:5’-GGCGGTTGCAGACCGAAATTCGGGA-3’(SEQ ID NO.5);
R1:5’-AAACTGCGCGCGCTACCGGCATTGT-3’(SEQ ID NO.6)。
7.如权利要求5所述的检测试剂,其中所述PCR引物序列为:
F2:5’-CCGGAATTCATGAACGCAACTCTGA-3’(SEQ ID NO.7);
R2:5’-CAAGCTTCTATTCGCTCCCGCTGAC-3’(SEQ ID NO.8)。
8.一种结核分枝杆菌抗原,包含选自下述组之一的物质:
a)权利要求1所述的结核分枝杆菌H37Rv基因Rv2706(-|3020323-3020511|)编码蛋白的全部或部分氨基酸;
b)权利要求2所述的Rv2706编码蛋白的全部或部分氨基酸。
9.权利要求1所述的结核分枝杆菌H37Rv编码基因Rv2706(-|3020323-3020511|)或权利要求2所述的Rv2706编码蛋白在结核流行病学调查和/或临床结核病患者快速鉴别诊断中的应用。
10.权利要求8所述的结核分枝杆菌抗原在制备结核疫苗中的应用。
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