CN109406776B - 用于特异性检测结核分枝杆菌感染的蛋白Rv2818c - Google Patents
用于特异性检测结核分枝杆菌感染的蛋白Rv2818c Download PDFInfo
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- CN109406776B CN109406776B CN201710710809.7A CN201710710809A CN109406776B CN 109406776 B CN109406776 B CN 109406776B CN 201710710809 A CN201710710809 A CN 201710710809A CN 109406776 B CN109406776 B CN 109406776B
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Abstract
本发明公开了用于特异性检测结核分枝杆菌感染的蛋白Rv2818c。本发明提供了Rv2818c蛋白在制备检测或辅助检测结核分枝杆菌产品中的应用;所述Rv2818c是如下a)或b)的蛋白:a)序列表中的序列1所示的氨基酸序列组成的蛋白质;b)将序列表中的序列1的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与序列1所示蛋白具有相同功能的由a)衍生的蛋白质。本发明的实验证明,能够更有效的提高结核分支杆菌感染检出率。
Description
技术领域
本发明属于生物医药领域,具体涉及用于特异性检测结核分枝杆菌感染的蛋白Rv2818c。
背景技术
多个世纪以来,结核病持续在全球成为一个不容忽视的公共卫生问题。目前全球已有三分之一的人口携带结核分枝杆菌,仅2010年一年就新增结核病病例880万,死 亡145万,平均不到22秒即有一人死于肺结核病,结核病高居传染病死亡人数之首。
科学证明表明由于活动性肺结核的极易传播性,对活动性肺结核的诊断及尽快消除传染源,控制结核病流行有非常重大的意义。然而肺结核的根治目前还不能完全做 到,据研究表明活动性结核病患者经不同的短程化疗治愈后,有1%-9%的患者将会复 发,而在一些特殊人群中,高达20%(Hong Kong Chest Service/British Medical ResearchCouncil.Am Rev Respir Dis,1991,143:700-706),而且耐药性结核病进一 步传播与复发未及时诊断的结核患者有很大的关系。所以关于结核治愈患者的活动性 肺结核诊断对于防治结核的整体战略具有很大的意义。目前活动性判断应综合临床、X 线表现和痰菌决定,而主要依据是痰菌和X线。痰涂片检查简便易行,准确性较高, 是活动性肺结核确诊的金标准,但某些研究中其检出率甚至低于10%。基于核酸扩增 的诊断方法,如实时定量PCR以及DNA芯片的优点是灵敏度高且耗时短,其问题在于 特异性较难保证,容易导致假阳性结果。而基于抗原抗体反应的血清学诊断,由于其 简便性、快速性,日益成为重要的活动性肺结核临床辅助性诊断手段,并且能满足区 分结核康复人群与活动性肺结核的要求。因此,从长远角度综合来看,应致力于发现 敏感性和特异性均较好的结核标志物。
理想的结核诊断标志物应符合以下条件:(1)敏感性高;(2)特异性高;(3)存 在于体液,特别是血液中,易于检测。但是目前肺结核分支杆菌血清学诊断所针对的 抗原如抗原5、38KD抗原、30/31KD抗原以及抗原60等,均存在阳性检出率低,与其 它分支杆菌的交叉免疫性等问题,虽然在疗效监测、提示复发、判断预后和高危人群 的普查中具有一定的价值,但目前仍不能用于活动性肺结核的确诊。为了实现对活动 性肺结核的高灵敏性,高特异性的诊断,急需在分子水平上寻找到更敏感、更特异的 活动性肺结核生物标志物。
发明内容
本发明的一个目的是提供Rv2818c蛋白的用途。
本发明提供Rv2818c蛋白在制备检测或辅助检测结核分枝杆菌产品中的应用;
所述Rv2818c是如下a)或b)的蛋白:
a)序列表中的序列2所示的氨基酸序列组成的蛋白质;
b)将序列表中的序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与序列2所示蛋白具有相同功能的由a)衍生的蛋白质。
Rv2818c是已知的蛋白,根据个体差异,该蛋白可能有不同的序列,它们可能存 在一个或几个氨基酸的差异,但是都在本发明的范围内,本发明的一个实施例中使用 的蛋白的序列是序列2。
上述Rv2818c蛋白在制备检测或辅助检测待测患者是否感染结核分枝杆菌产品中的应用也是本发明保护的范围。
本发明的另一个目的是提供一种检测或辅助检测待测样本是否感染结核分枝杆菌的产品。
本发明提供的产品,包括Rv2818c蛋白。
上述产品还包括阳性质控和阴性对照孔;
所述阳性质控为植物血凝素PHA;
所述阴性对照孔为无抗原的细胞培养液。
上述产品中,所述产品还包括记载如下内容的可读载体(可为说明书):
若检测结果满足如下条件:阴性对照孔斑点数为0-5,且Rv2818c检测孔斑点数-阴性对照孔斑点数≥6;或若阴性对照孔斑点数大于等于6,且(Rv2818c检测孔斑点 数-阴性对照孔斑点数)≥2×(阴性对照孔斑点数);则待测样本候选感染结核分枝杆 菌,为阳性样本;若检测结果满足如下所述条件,则待测样本候选未感染结核分枝杆 菌;
所述阴性对照孔为阴性对照所在孔,
所述Rv2818c检测孔为所述Rv2818c蛋白所在孔。
上述产品为试剂盒,上述试剂盒具体为T-SPOT试剂盒,也可以为ELISA试剂盒。
上述T-SPOT试剂盒还包括T-SPOT试剂盒中的微孔培养板、浓缩标记抗体、显色 底物溶液。上述试剂盒为Oxford immunotec(英国)的产品。
本发明第三个目的是提供一种用于鉴别、诊断、辅助诊断、筛查和/或辅助筛查 结核分枝杆菌的标志物。
本发明提供的标志物,为Rv2818c蛋白。
本发明的实验证明,本发明筛选了结核分枝杆菌来源的蛋白片段,提供了一种检测结核病的结核分枝杆菌标识物抗原RV2818c,通过该抗原来体外检测特异性T细胞 免疫反应,可以作为诊断结核病患者的参照,用于诊断患者是否被结核分枝杆菌感染。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
部分试剂如下:
lysis buffer:20mM Tris-HCl、500mM氯化钠、10%甘油、pH 8.0
Wash buffer:20mM Tris-HCl、20mM咪唑、500mM氯化钠、10%甘油、pH 8.0
Elution buffer:20mM Tris-HCl、250mM咪唑、500mM氯化钠、10%甘油、pH 8.0
层析缓冲液:取磷酸二氢钾13.609g(配制1000ml),加0.1mol/l氢氧化钠溶液,调节pH至7.5, 得到pH值为7.5的PBS缓冲液。
固化Ni+层析树脂Novagen,Ni-NTA His,(Cat.NO 70691-5,北京华美;2-8度 保存);蛋白结合能力大于5mg/ml resin层析柱(玻璃或者聚丙烯)。superdex 75: (GE)。
包涵体缓冲液:50mM tris,0.05M EDTA;
包涵体洗涤液:50mM tris,0.05M EDTA,2%DOC,1M尿素;
包涵体溶解液:50mM Tris-HCl,8M尿素;
下述实施例采用结核感染T细胞检测试剂盒是Oxford immunotec(英国)的 T-SPOT试剂盒,为ESAT-6和CFP 10双抗原试剂盒。
实施例1、原核表达目的蛋白Rv2818c
目的蛋白Rv2818c的制备:将表达Rv2818c蛋白编码基因的重组载体 pET28a-Rv2818c导入大肠杆菌BL21中,得到重组菌BL21/pET28a-Rv2818c;再IPTG 诱导重组菌BL21/pET28a-Rv2818c,收集上清液,即为目的蛋白Rv2818c。
具体如下:
1、表达目的蛋白重组菌的构建
Rv2818c蛋白的氨基酸序列为序列2,其编码基因的核苷酸序列为序列1。
将序列1所示的Rv2818c蛋白编码基因插入pET28a载体的NdeI和EcoRI位点, 得到重组载体pET28a-Rv2818c;
将重组载体导入大肠杆菌BL21中,得到重组菌BL21/pET28a-Rv2818c。
2、蛋白表达
1)重组菌BL21/pET28a-Rv2818c在37℃静置过夜(16h左右)。
2)接种于5ml液体LB培养基(加入5ul kan),200rpm,37℃过夜培养,12h左 右。
3)将过夜培养的菌液按接种量150-200ul接种于已加入相应抗性的200ml LB液体培养基中,在37℃,200rpm条件下培养至菌液OD600为0.6-0.8;
4)加入终浓度0.4mM IPTG开始诱导表达,在200rpm,37℃条件下至OD600在1.5 左右,4000rpm离心10min开始收菌。
3、蛋白纯化
1)菌体重悬:每300ml培养基所收集的菌体用20ml的包涵体缓冲液重悬;
2)菌体破碎:超声3S,间隔6S,功率为38%,超声15min;
3)收集包涵体:4℃10000g高速离心20min;
4)洗包涵体:沉淀用15ml包涵体缓冲液重悬,4℃10000g高速离心10min;共 洗3次;
5)包涵体溶解:包涵体用15ml包涵体溶解液重悬,吹打混匀并在斡旋仪上振动 3-5min;
6)将5ml上清置于透析袋中密封,放入到一个装有10倍量上清体积的外围透析 液(200mM Tris-HCl,8M尿素)(即与透析袋内液体体积比为100:1),置于4℃,充 分平衡。然后用蠕动泵向外围透析液中逐渐缓慢滴加清水,直至外围透析液被稀释10 倍;
7)放入二次透析液中透析至蛋白液中尿素浓度低于0.04M。若无明显沉淀产生,则复性成功。
电泳检测复性后产物,为目的蛋白Rv2818c(大小为34.4KDa)。
复性后产物的浓度为0.2mg/ml,其中溶剂为20mM Tris-HCl,pH8.0,10%glycerol,溶 质为目的蛋白Rv2818c。
实施例2、结核分枝杆菌标识物抗原Rv2818c在检测结核分枝杆菌中的应用
1、T-SPOT试剂盒检测
具体方法如下,里面的一些溶液均来自Oxford immunotec(英国)的T-SPOT试 剂盒:
1)使用肝素真空采血管对不同待测患者(表1所示61个均为结核病人例临床检测结核病的患者和表2所示的10例临床检测非结核病人,且知情)直接采外周静脉血, 得到抗凝血样本;
2)将抗凝血样本按体积1:1与RPMI1640培养液混匀;按3:1比例小心将血样加在Ficoll淋巴细胞分离液上层(GE Healthcare 17-1440-02)(利用Ficoll-PaqueTMplus按照操作说明分离PBMCs);室温25℃,1000g离心22分钟;水平转子,缓升缓降;
3)将单核细胞层(位于离心管中间层,为一层较薄的白膜状)从Ficoll分离管 中转移到已加10ml AIM-V培养液(Invitrogen 12055091)的无菌15ml离心管,轻轻 混匀,室温600g离心7min;
4)小心去除上清,加入1ml AIM-V培养液,轻缓重悬细胞,加入AIM-V培养液至10ml,350g离心7min;
5)小心弃上清,加入1ml AIM-V培养液重悬细胞,得到细胞悬液(PBMCs);
6)取10μl细胞悬液加入40μl的1%trypan blue,用AIM-V培养液稀释,得到 细胞稀释工作液;
7)将24孔PVDF膜板从铝封袋中取出,按顺序加入:50μl植物血凝素PHA(sigma) 至阳性对照孔、50μl制备获得的Rv2818c蛋白溶液(初始浓度为60ug/ml,溶剂为AIM V培养液)加入到检测孔、50μl AIM V培养液至阴性对照孔;在上述3孔中分别加入 已制备细胞稀释工作液100μl(每孔细胞数量2.5×105),且样品孔中Rv2818c蛋白溶 液的终浓度为20ug/ml;
8)将培养板放入37℃,5%CO2培养箱孵育16小时;
9)用无菌PBS按1:200制备新鲜酶标抗体工作液;从培养箱取出培养板,弃去 孔内液体。以200μl/well无菌PBS洗涤4遍;
10)加入50μl/well酶标抗体工作液,将培养板在2-8℃孵育1小时。用PBS洗 涤4遍去除未结合酶标抗体;
11)每孔加入室温平衡过的底物工作液50μl,室温反应7分钟。以蒸馏水冲洗终 止反应,在37℃2-3hrs或室温过夜干燥培养板,得到含有反应产物的培养板。
2、斑点记数
1)从培养箱中取出含有反应产物的培养板弃去细胞培养液;
2)每个反应孔加入200ul PBS缓冲液(pH 7.5);
3)弃去PBS缓冲液;用新鲜的PBS缓冲液重复洗涤至少3遍;
4)用PBS缓冲液200倍稀释试剂盒中的浓缩标记抗体试剂(碱性磷酸酶标记的小鼠的抗人γ-干扰素单抗);
5)每个反应孔加入50ul标记抗体工作液,2-8℃孵育1小时;
6)弃去标记抗体工作液,按上述的步骤2和3洗涤;
7)每个反应孔加入50ul底物显色溶液室温孵育7分钟;
8)用蒸馏水或去离子水彻底洗涤培养板终止反应;
9)在通风处或37℃温箱干燥培养板;
10)记数每个反应孔内深蓝色清晰的斑点。
结果判断:
若阴性对照孔斑点数为0-5,且Rv2818c检测孔斑点数-阴性对照孔斑点数≥6;
或若阴性对照孔斑点数大于等于6,且(Rv2818c检测孔斑点数-阴性对照孔斑点数)≥2×(阴性对照孔斑点数);
则待测样本候选感染结核分枝杆菌,为阳性样本;
若不满足上述,则待测样本与Rv2818c不产生免疫反应,待测样本候选未感染结核分枝杆菌。
用上述方法检测表1中61个结核病人待测样本,结果如表1所示。
用上述方法检测表2中10个健康人的待测样本,结果如表2所示。
对比例:
采用Oxford immunotec(英国)的T-SPOT试剂盒检测表1所示的61个结核病人 待测样本和表2中10个健康人的待测样本,方法与实施例1相同,唯一不同的是设置 两个检测孔,且A检测孔为抗原A孔,加入是抗原ESAT-6,B检测孔为抗原B孔,加 入是抗原CFP 10。
根据抗原A和抗原B孔的反应判断结果:
若阴性对照孔斑点数为0-5,且(抗原A斑点数)–(阴性对照孔斑点数)≥6或(抗 原B斑点数)–(阴性对照孔斑点数)≥6;
或当阴性对照孔斑点数≥6,且(抗原A斑点数)≥2×(阴性对照孔斑点数)或(抗原B斑点数)≥2×(阴性对照孔斑点数)。
则待测样本候选感染结核分枝杆菌,为阳性样本;
若不满足上述,则待测样本与抗原A或抗原B不产生免疫反应,待测样本候选未 感染结核分枝杆菌。
表1为样本及其检测结果
表中,-为阴性对照,+为阳性对照孔(加了PHA的培养基),X为Rv2818c蛋白检 测孔,A为抗原A检测孔,B为抗原B检测孔。
表2为健康人样本及其检测结果
样本编号 | - | A | B | + | Rv2818c |
1 | 0 | 0 | 1 | 405 | 5 |
2 | 0 | 0 | 0 | 672 | 2 |
3 | 0 | 0 | 0 | 772 | 4 |
4 | 0 | 1 | 0 | 32 | 4 |
5 | 0 | 45 | 20 | 252 | 4 |
6 | 0 | 3 | 58 | 334 | 0 |
7 | 0 | 29 | 12 | 736 | 4 |
8 | 1 | 0 | 0 | 952 | 5 |
9 | 0 | 2 | 0 | 958 | 2 |
10 | 0 | 2 | 2 | 937 | 0 |
表中,-为阴性对照(不加入任何抗原的培养基),+为阳性对照孔(加了PHA的培 养基),X为Rv2818c蛋白检测孔。
可以看出,
Rv2818c作为抗原检测,61个临床已经鉴定感染结核分枝杆菌的待测样本中50 例为Rv2818c蛋白检测感染结核分枝杆菌的样本,这与临床鉴定结果一致。但是其余 11个样本没有检测出来,因此,本发明用Rv2818c作为抗原检测是否感染结核分枝杆 菌的检出率达82%。
10个健康人样本中均未检出结核分枝杆菌,这与临床鉴定结果一致。
对比例检测61个临床已经鉴定感染结核分枝杆菌的待测样本,其中48例为对比例检测感染结核分枝杆菌的样本,这与临床鉴定结果一致;但是其余13个样本没有检 测出来。因此,对比例检测是否感染结核分枝杆菌的检出率仅为78%,远远低于Rv2818c 作为抗原检测。
对比例中10个健康人样本中3例检出结核分枝杆菌,这与临床鉴定结果不同。
从上述实验可以看出,Rv2818c蛋白可以检测或辅助检测待测患者是否感染结核分枝杆菌,具体如下:以Rv2818c蛋白作为抗原,通过结核感染T细胞检测试剂盒检 测,以细胞培养基作为阴性对照孔,若阴性对照孔斑点数为0-5,且Rv2818c检测孔 斑点数-阴性对照孔斑点数≥6;或若阴性对照孔斑点数大于等于6,且(Rv2818c检 测孔斑点数-阴性对照孔斑点数)≥2×(阴性对照孔斑点数);则待测样本候选感染结 核分枝杆菌,为阳性样本;若不满足上述,则待测样本与Rv2818c不产生免疫反应, 待测样本候选未感染结核分枝杆菌。
序列表
<110>中国科学院生物物理研究所,广东体必康生物科技有限公司
<120>用于特异性检测结核分枝杆菌感染的蛋白Rv2818c
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1149
<212> DNA
<213>人工序列
<220>
<223>
<400> 1
atgctattcc tcagcgccga gatagctgcc tttgagaacg cggaccggcg gtactccgcg 60
gcaatcacgc ggctcgcgcc tgagaccgac gttcgcatag tcacctatac caacccgtcg 120
gtgcacaggt tcgacctttt cgtgccggtt ttccgcaacc acctggttga actgtcggct 180
gagttccctg atcgaaccat tctgctgaat accagttccg gcacccctgc gatgcaggcg 240
gcgctggtgg ccataaatgt gtttggcatt cccaggacca ccgctgtgca agtaagcacg 300
cctgcccggg cattgagcaa gcctggcgat cgtgaatccc cagacgctta cgacctcgaa 360
ctaatgtggg acgcaaacga cgacaatcag cctggagccc ccaaccgttg ctttgaggcg 420
acttccgctg cgctcggcgc gctgcttgag cgggccaacc tgaagcagct gatcgtgtcg 480
tacgactact cggcagcagt gacgatcgcg gcagactcgc gcctgcccga tcaagtgagc 540
aatctgatcc gcggcgcgat gcaccgctcg aggctggaac acctcgtagc gccaaagttc 600
tttaaggaca ccgcgttcac gtatgacccc gcgaacaagg tcgctgagta cataagtgct 660
cttgcgctgc tggcaaagcg cgagcaatgg gctgaattcg cacgatcagc taccccggca 720
atcactatcg tgctcagggc ggctgtggca aaacaccttc cggaggaccg ctatctcgac 780
gacatgggcc gcgtcgaccg ccgaaagctg gaaagagagc cggagatacg gtgcgcgctc 840
aagcaccctc cgaaatcgcc aaacgcggag tggtacctct acaccaagga ctggctcgca 900
ctgctccgcc aattcgcacc cgatcgagtt ggtgctcttg aagtactcgg aaggttcgag 960
agccgggtcc gcaacaccgc agcacacgag atcgtctcaa tcagtgagga tcgcatcacg 1020
aaggatggcg gccttctccc agaacaactg ctgaagatcc tcgcccgcga aacaggcgct 1080
gatttgacgc tctatgaccg gttgaacgac gagatcatcc ggcagattga tatggcaccg 1140
ctgggctaa 1149
<210> 2
<211> 382
<212> PRT
<213>人工序列
<220>
<223>
<400> 2
Met Leu Phe Leu Ser Ala Glu Ile Ala Ala Phe Glu Asn Ala Asp Arg
1 5 10 15
Arg Tyr Ser Ala Ala Ile Thr Arg Leu Ala Pro Glu Thr Asp Val Arg
20 25 30
Ile Val Thr Tyr Thr Asn Pro Ser Val His Arg Phe Asp Leu Phe Val
35 40 45
Pro Val Phe Arg Asn His Leu Val Glu Leu Ser Ala Glu Phe Pro Asp
50 55 60
Arg Thr Ile Leu Leu Asn Thr Ser Ser Gly Thr Pro Ala Met Gln Ala
65 70 75 80
Ala Leu Val Ala Ile Asn Val Phe Gly Ile Pro Arg Thr Thr Ala Val
85 90 95
Gln Val Ser Thr Pro Ala Arg Ala Leu Ser Lys Pro Gly Asp Arg Glu
100 105 110
Ser Pro Asp Ala Tyr Asp Leu Glu Leu Met Trp Asp Ala Asn Asp Asp
115 120 125
Asn Gln Pro Gly Ala Pro Asn Arg Cys Phe Glu Ala Thr Ser Ala Ala
130 135 140
Leu Gly Ala Leu Leu Glu Arg Ala Asn Leu Lys Gln Leu Ile Val Ser
145 150 155 160
Tyr Asp Tyr Ser Ala Ala Val Thr Ile Ala Ala Asp Ser Arg Leu Pro
165 170 175
Asp Gln Val Ser Asn Leu Ile Arg Gly Ala Met His Arg Ser Arg Leu
180 185 190
Glu His Leu Val Ala Pro Lys Phe Phe Lys Asp Thr Ala Phe Thr Tyr
195 200 205
Asp Pro Ala Asn Lys Val Ala Glu Tyr Ile Ser Ala Leu Ala Leu Leu
210 215 220
Ala Lys Arg Glu Gln Trp Ala Glu Phe Ala Arg Ser Ala Thr Pro Ala
225 230 235 240
Ile Thr Ile Val Leu Arg Ala Ala Val Ala Lys His Leu Pro Glu Asp
245 250 255
Arg Tyr Leu Asp Asp Met Gly Arg Val Asp Arg Arg Lys Leu Glu Arg
260 265 270
Glu Pro Glu Ile Arg Cys Ala Leu Lys His Pro Pro Lys Ser Pro Asn
275 280 285
Ala Glu Trp Tyr Leu Tyr Thr Lys Asp Trp Leu Ala Leu Leu Arg Gln
290 295 300
Phe Ala Pro Asp Arg Val Gly Ala Leu Glu Val Leu Gly Arg Phe Glu
305 310 315 320
Ser Arg Val Arg Asn Thr Ala Ala His Glu Ile Val Ser Ile Ser Glu
325 330 335
Asp Arg Ile Thr Lys Asp Gly Gly Leu Leu Pro Glu Gln Leu Leu Lys
340 345 350
Ile Leu Ala Arg Glu Thr Gly Ala Asp Leu Thr Leu Tyr Asp Arg Leu
355 360 365
Asn Asp Glu Ile Ile Arg Gln Ile Asp Met Ala Pro Leu Gly
370 375 380
Claims (2)
1.Rv2818c蛋白、阳性质控、阴性对照孔和记载如下内容的可读载体在制备检测或辅助检测待测样本是否感染结核分枝杆菌的产品中的应用;
所述Rv2818c是序列表中的序列2所示的蛋白质;
所述阳性质控为植物血凝素PHA;
所述阴性对照孔为阴性对照所在孔,
所述阴性对照为无抗原的细胞培养液;
所述内容为:
若检测结果满足如下条件:阴性对照孔斑点数为0-5, 且Rv2818c检测孔斑点数-阴性对照孔斑点数≥6; 或若阴性对照孔斑点数大于等于6, 且(Rv2818c检测孔斑点数-阴性对照孔斑点数)≥ 2×(阴性对照孔斑点数);则待测样本候选感染结核分枝杆菌,为阳性样本;若检测结果未满足上述条件,则待测样本候选未感染结核分枝杆菌;
所述Rv2818c检测孔为所述Rv2818c蛋白所在孔。
2.根据权利要求1所述的应用,其特征在于:所述产品为试剂盒。
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