CN103215236A - 尘螨过敏原Derf8和Derf20及其基因和应用 - Google Patents
尘螨过敏原Derf8和Derf20及其基因和应用 Download PDFInfo
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- CN103215236A CN103215236A CN201310033693XA CN201310033693A CN103215236A CN 103215236 A CN103215236 A CN 103215236A CN 201310033693X A CN201310033693X A CN 201310033693XA CN 201310033693 A CN201310033693 A CN 201310033693A CN 103215236 A CN103215236 A CN 103215236A
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Abstract
本发明涉及一种尘螨过敏原Derf8和Derf20及其基因和应用，属于生物医学技术领域。尘螨过敏原Derf8和Derf20、分别由SEQ ID NO:1和SEQ ID NO:3所示的氨基酸序列组成。编码尘螨过敏原Derf8和Derf20的基因，其特征在于分别由SEQ ID NO:2和SEQ ID NO:4所示的核苷酸序列组成。所述的尘螨过敏原Derf8和Derf20在制备治疗尘螨过敏性疾病药物中的应用。本发明的有益效果在于：提供尘螨过敏原Derf8和Derf20及其基因，尘螨过敏原Derf8和Derf20能作为制备治疗尘螨过敏性疾病药物的应用。
Description
技术领域：
本发明涉及一种尘螨过敏原Der
f 8 和Der f 20及其基因和应用，属于生物医学技术领域。
背景技术：
过敏性疾病（如哮喘、过敏性鼻炎等）是临床上的常见病、多发病，世界各国过敏性疾病的总发病率高达10-30%，是当前世界性的重大卫生学问题，目前我国现有哮喘患者2000多万，过敏性鼻炎患者5000多万，并且其发病率和死亡率仍呈上升趋势，近二十年来发病率几乎翻了一倍。在引起过敏性疾病的众多吸入性过敏原中，尘螨是导致呼吸道变态反应性疾病的最重要因素。自从Voorhorst和Spieksma（1964）首次证实尘螨及代谢产物是屋尘中的主要过敏原以来，世界各国（欧美国家、日本、中国等）变态反应学工作者通过大量调查也一致证实尘螨是世界性的主要变应原。尘螨在过敏性疾病患者特异性免疫诊断中阳性率约70-80%。自从Noon和Freeman（1911）首次应用梯牧草花粉变应原浸液（allergen extract）用于治疗花粉症以来，脱敏治疗至今已有90多年的历史。随着对变态反应疾病发病机制的深入研究以及免疫治疗机制的进一步了解，WHO（1997）建议将变应原浸液改称为变应原疫苗（allergen vaccine），并且在《WHO有关免疫治疗的指导文件》（1998）指出：（1）鼓励应用和发展标准化的变应原疫苗；（2）成功的免疫治疗取决于高质量的变应原疫苗。因此，研制出新一代高效变应原疫苗仍是国内外该领域研究热点。
由于尘螨系医学节肢动物，结构和成份复杂，尽管目前人们在尘螨数百种蛋白中已初步鉴定出16种过敏原成份，研究显示尘螨含有的过敏原多大30余种。就某一个尘螨过敏病人而言，他可能仅对尘螨中的一类或数类过敏原蛋白产生过敏反应。而目前在临床上用于尘螨过敏患者诊断和脱敏治疗的仍然为尘螨粗浸液，其中含有大量对患者非特异的过敏原及其它杂质，这严重阻碍了尘螨过敏原试剂标准化的制定及其在临床上的使用。所以进一步探明尘螨过敏原的确切组分，将会为尘螨过敏患者带来个体化的脱敏治疗。
发明内容：
本发明的目的在于提供一种尘螨过敏原Der f 8 和Der f 20及其基因和应用。
本发明的技术方案如下：
Der f 8是我们首次从尘螨匀浆中鉴定得到的一种新的过敏原蛋白，它属于一类谷胱甘肽转移酶家族（Glutathione S-transferase），分子量约32 kDa。Der f 8由197个氨基酸组成，其氨基酸序列如下（SEQ ID NO:1）：
MLAYAGVDYVDKRYNIGPNFDRSEWLNEKFNLGLDFPNLPYYMDGDVKITQSMAILRYLA 60
RKYNMDGTNEQERLRISMAEQQTHDMFMAMVRICYDPNMENLRVDYLKTLPDSLKLMEKF 120
LANHDYIAGSKISYADFYLYEYLCRMKVMVPEVYGQFENLKKFVERIESLPRIAEYIKKQ 180
KPTTFNASLAKWNGSYA 197
尘螨过敏原Der f 8基因克隆步骤包括：尘螨总RNA提取，mRNA纯化，mRNA反转录及cDNA文库构建，设计引物，利用PCR方法扩增编码基因。基因测序结果表明尘螨过敏原Der f 8的编码基因（GenBank accession KC305499）由 595个核苷酸组成，该基因的5’端至3’端序列为(核苷酸序列为SEQ ID NO:3)：
atgttggcat atgccggtgt tgattatgtt gataaacgtt ataatattgg tccgaatttt 1
gatcgttcag aatggttgaa tgaaaaattc aatttgggtt tagatttccc aaatttgcca 61
tattacatgg atggtgatgt taaaattacc caatcgatgg ccattcttcg ttatttggcc 121
cgtaaatata acatggatgg tactaatgaa caggaacgtt tacgaatttc aatggctgaa 181
caacaaacac atgatatgtt tatggccatg gtccgtattt gttatgatcc aaatatggaa 241
aacttacgag ttgattattt gaaaacattg cccgattcat tgaaattgat ggaaaaattt 301
ttagctaatc atgattatat tgccggttca aaaatttctt atgcagattt ttatttgtat 361
gaatatttgt gccgtatgaa agtcatggta ccagaagtat atggacaatt tgaaaatttg 421
aaaaaatttg ttgaacgcat tgaatcattg ccacgtattg ccgaatacat taagaaacaa 481
aaaccaacaa cattcaatgc atcattggct aaatggaatg gttcttatgc ctaa 595
Der f 20属于一类精氨酸激酶（Arginine kinase），分子量约40 kDa。Der f 20由356个氨基酸组成，其氨基酸序列如下（SEQ ID NO:2）：
MVDQATLSKLEAGFQKLQNAQDCHSLLKKYLTRDVLDQLKTKKTDMGATLLDVIQSGVEN 60
LDSGVGIYAPDAQSYKTFAALFDPIIDDYHKGFKPTDKHPQTDFGNIEHFVNVDPKNEYV 120
ISTRVRCGRSLKGYPFNPMLTEAQYKEMETKVKGQLATFEGELKGTYYPLLGMDKATQQK 180
LIDDHFLFKEGDRFLQAANACRYWPVGRGIFHNDKKTFLMWVNEEDHLRIISMQKGGDLK 240
EVFGRLVKAVKHIEQKIPFSRDDRLGYLTFCPTNLGTTIRASVHIKLPKLAADRKKLEEV 300
AARYNLQVRGTAGEHTESVGGIYDISNKRRMGLTEYQAVKEMQDGIIELIKMEKSL 356
尘螨过敏原Der f 20基因克隆步骤包括：尘螨总RNA提取，mRNA纯化，mRNA反转录及cDNA文库构建，设计引物，利用PCR方法扩增编码基因。基因测序结果表明尘螨过敏原Der f 20的编码基因（GenBank accession EU106619.1）由1071个核苷酸组成，该基因的5’端至3’端序列为(核苷酸序列为SEQ ID NO:4)：
atggtcgatc aagctaccct gagtaaattg gaagccggtt tccaaaaatt acagaatgct 60
caagattgtc attcattgtt gaaaaagtat ttgactcgcg atgtgttaga tcaactcaag 120
acgaaaaaga ccgacatggg cgcaacatta ttggatgtta tccaatctgg cgtggaaaac 180
ctggacagtg gtgttggtat ctatgctcct gatgctcaat catacaaaac atttgctgca 240
ttgttcgatc caatcattga tgattaccat aaaggcttca aaccgaccga taaacatccg 300
caaactgatt tcggcaatat cgaacacttt gtcaatgttg atcctaaaaa cgaatacgtc 360
atttctactc gtgttcgatg tggccgttcg ttgaaaggct atccattcaa ccctatgttg 420
acagaggctc aatacaaaga aatggaaacc aaagtgaaag gacaattggc cacattcgaa 480
ggcgaattga aaggcaccta ttacccattg ttgggaatgg ataaagctac tcaacagaaa 540
ttgatcgacg atcatttctt gttcaaggaa ggtgatcgat tcttgcaagc tgccaatgca 600
tgtcgttact ggcctgttgg tcgtggtatc ttccacaatg acaaaaaaac attcttgatg 660
tgggtaaacg aagaagatca tttgcgtatc atttccatgc aaaaaggtgg cgatctaaaa 720
gaggtctttg gacgtttggt caaggctgtc aagcacattg aacaaaagat tccattctct 780
cgtgatgacc gtctcggtta tttgacattc tgtccaacca atcttggcac aaccatccgt 840
gcttcggttc atatcaaact tccgaaattg gccgctgacc gtaagaagtt ggaagaagtg 900
gctgctcgtt acaatcttca agtgcgtggc actgcaggtg aacataccga aagtgtgggt 960
ggtatctatg atattagtaa caaacgacga atgggtctca ccgaatacca agctgttaa1020
gaaatgcaag atggcatcat tgaattgatt aaaatggaaa aatcattgta a 1071
通过常规的双向电泳及双向Western blot方法检测，发现Der f 8 和Der f 20具有较强的过敏原性，在制备治疗尘螨过敏性疾病药物中的应用。
本发明的有益效果在于：提供尘螨过敏原Der f 8 和Der f 20及其基因，尘螨过敏原Der f 8 和Der f 20能作为制备治疗尘螨过敏性疾病药物的应用。
附图说明：
图1.是尘螨匀浆过分子筛Sephadex-G75的峰型图。
图2为尘螨匀浆Sephadex-G75 II 峰的Westernblot，其中：点1 Der f 20，点2和3为Der f 8。
图3-A至图3-E是 ESI-Q-TOF质谱图。其中：图3-A至图3-B为Der f 8的质谱图； 图3-C至图3-E为Der f 20的质谱图
具体实施方式：
实施例一：尘螨过敏原的鉴定及其氨基酸序列测定
一、尘螨的饲养和收集及尘螨匀浆的制备
将纯品系的粉尘螨至于25 ℃相对湿度75%的条件下采用磨成粉末状的鼠粮进行批量饲养。由于尘螨具有避光的生活特性，可用白炽灯光照法将尘螨从饲料中分离出来。对于分离的尘螨可加入适量的20mM pH 7.8 Tris-HCL进行充分研磨，然后在转速12000 ×g离心30 min获取上清。
二、尘螨匀浆的分离纯化和双向电泳及其Western blot检测
以上述收集的上清液为原料，上样于预先用20mM pH7.8 Tris-HCL缓冲液平衡好的分子筛凝胶层析Sephadex-G75，用相同的缓冲液进行洗脱。流速为0.3ml/min，用自动收集器每10min收集一管，检测每管在280nm和215nm处的吸收值，制作吸收值变化的峰型图如图1所示。然后将每峰收集冻干浓缩用于下一步的分离纯化或双向电泳及其Western blot检测。双向电泳及其Western blot具体步骤如下：
A.样品处理：采用GE公司的2D clean-up对来自分子筛各峰的样品进行脱盐浓缩等处理，主要过程如下：
1：将含有约60 μg 100 μl的样品至于1.5 ml的微型离心管中，加入300 μl沉淀剂振荡搅匀，冰浴15 min。
2： 以最大转速（12000×g）离心5 min，尽量取净上清。加入40μl共沉淀剂，冰浴5 min。再次相同转速离心5 min，用移液枪将上清移去，加入25 μl加入1 ml 洗涤缓冲液（在-20 ℃至少预冷1小时）和5 μl洗涤添加剂，振荡直至沉淀完全散开。将管在-20 ℃下孵育至少30 min，每10 min 振荡20至30 s。
3：以转速12000×g离心5 min。
4：小心将上清移去，此时可见白色沉淀，将沉淀简单风干。
5：加入150 μl水化液再溶解沉淀，以备第一向等电聚焦电泳（IEF）使用。
B. 等电聚焦（IEF）: 将含有约60 μg 样品100 μl水化液上样于7 cm长Immobiline DryStrip 非线性胶条，在Ettan IPGphor Ⅲ等电聚焦系统上聚焦，等电聚焦条件为20 ℃，每条胶的电流为50 μA，总聚焦伏小时约为6 kVh。将等电聚焦好的胶条分别用含有二硫苏糖醇（DTT）和碘乙酰胺的平衡液各洗15 min，将平衡好的胶条横放在以配好的浓度为12%的SDS-PAGE胶面上，并用琼脂糖封好，准备第二向电泳。
C. 第二向电泳：对于胶宽度只有7 cm的胶块可用SE260进行跑胶。在20 mA/胶的条件下一个半小时即可。
实施例二：尘螨过敏原的基因克隆
一、尘螨总RNA提取
A.称取约80 mg的活尘螨，加入已预冷的1 mL Trizol提取液（美国Invitrogen公司），并加入液氮充分匀浆。
B.加入Trizol 1/5体积的氯仿，剧烈混匀约15秒，室温放置5分钟，4℃，12000 rpm离心10分钟，取上清。
C.上清加入等体积的异丙醇，室温放置10分钟，4 ℃，12000 rpm离心10分钟，沉淀用75%乙醇洗一次，晾干，管底沉淀物即为地鳖虫消化道中肠总RNA。
二、尘螨mRNA的纯化
尘螨mRNA分离纯化采用美国PROMEGA公司的 PolyATtract® mRNA Isolation Systems 试剂盒。
A. 取尘螨总RNA 500 μg 溶于 500 μL DEPC水中， 65 ℃水浴10 min，加人 3 μL的Oligo(dT) 探针和13 μL 20×SSC 溶液，混匀，放置室温冷却，称为A液。
B. 磁珠(SA-PMP)的洗涤：将磁珠轻弹混匀，至磁力架吸附30秒，弃上清，加 0. 5× SSC 300 μL，至磁力架吸附30秒，最后加100 μL 0. 5×SSC 悬浮，称为B液。
C. 将A液加入B液中，室温放置10 min，至磁力架吸附30秒，弃上清，用0. 1×SSC洗涤4次，最后弃上清，加100 μL DEPC水悬浮，至磁力架上吸附30 s，将上清移至新的试管，再加入150 μL DEPC水重悬，至磁力架吸附30 s，移上清至上述试管，即为纯化的地鳖虫消化道中肠mRNA。
D. 加入1/10体积的pH5. 2，3 M的乙酸钠溶液，等体积异丙醇，于-70℃放置30分钟，4℃，12000 rpm离心10 min，弃上清，沉淀溶解于10 μL DEPC水中。
三、尘螨cDNA文库构建
采用CLONTECH公司 CreatorTM SMART TM cDNA Library Construction Kit 构建尘螨cDNA文库。
A. cDNA第一链合成
于无菌PCR管加入2μl 尘螨mRNA、1 μl SMART IV引物、1 μl CDS III/3’ PCR引物、1 μl RNA-free水，使总体积达到 5 μl ，混匀并短暂离心。
1.72℃ 保温 2 min，冰上孵育2 min。
2.在上述PCR管中加入2 μl 5×第一链buffer、1 μl 20 mmol/L DTT、1 μl 10 mmol/L dNTP 混合物、1 μl PowerScript逆转录酶，混匀并短暂离心。
3.置于PCR仪中42℃保温1 hr后，冰浴终止第一链的合成。
B. 采用长末端聚合酶链式反应（LD- PCR）方法扩增cDNA第二链
1．将1 μl cDNA第一链、40 μl 去离子水、5 μl 10×Advantage 2 PCR 缓冲液、1 μl 50×dNTP 混合物、1 μl 5’PCR引物、1 μl CDS III/3’PCR引物以及1 μl聚合酶于PCR管中混匀。
2．在PCR仪中按以下程序扩增： 95℃，20 sec；22个循环：95℃，5 sec；68℃，6 min。
3．扩增结束后，将合成的双链cDNA置于-70℃保存。
C. 酶切、连接以及连接产物的转化：
1. 在微量离心管中加入1 μL pMD19-T 载体（日本Takara公司）、4 μL尘螨cDNA双链溶液，全量为5 μL。
2. 加入5 μL（等量）的连接酶缓冲混合物。
3. 16℃反应2小时。
4. 全量（10 μL）加入至100 μL DH5α感受态细胞（北京天根生化科技有限公司）中，冰浴30分钟。
5. 42℃加热90秒钟后，再在冰中放置1分钟。
6. 加入37℃温浴过的LB培养基900 μL，37℃缓慢振荡培养60分钟。
7. 取200 μL涂布于含有X-Gal、IPTG、Amp的LB培养基上37℃培养16小时，形成单菌落。
8. 每个LB平皿用5 mL LB液体培养基洗涤菌落，加30%甘油冻存。构建的cDNA大约含2×106个单独克隆。
四、尘螨过敏原基因序列扩增和测定：
根据实施例一中分离纯化所得尘螨过敏原Der f 8的氨基酸序列，我们设计了一条简并引物Der f 8-F和Der f 20-F，与构建地鳖虫消化道中肠cDNA 文库时所使用的接头引物CDS III/3’ PCR配对，正反向引物序列为:
CDS III/3’ PCR：5’-ATT CTA GAG GCC GAG GCG GCC ATG-3’
Der f 8-F: 5’-ATGCT(A/T/C/G)GC(A/T/C/G)TA(T/C)GC(A/T/C/G)GG(A/T/C/G)
GT(A/T/C/G)GA(T/C)-3’
Der f 20-F: ’-GG(A/T/C/G)GG(A/T/C/G)GA(T/C)CT(A/T/C/G)AA(A/G)GA(A/G)
GT(A/T/C/G)TT(T/C)GG(A/T/C/G)-3’
其中，括号内的碱基表示简并引物。
简并引物Der f 8-F和接头引物CDS III/3’ PCR配对使用，以尘螨cDNA为模板，进行PCR。其反应条件为：95℃预变性4 min，接着在如下条件进行35轮循环，94℃ 30 sec，55℃ 30 sec，72℃ 40 sec，然后72℃ 10 min。然后将PCR产物连接到T载体上，导入大肠杆菌DH5α，筛选单克隆进行测序。结果表明编码过敏原Der f 8的cDNA 序列由595个核苷酸组成，编码过敏原Der f 20的cDNA 序列由891个核苷酸组成。
实施例三：尘螨过敏原Der f 8 和Der f 20的过敏原性检测
对于实例一中的同一个样品需要跑两块双向电泳，其中一块用于直接染色，一块接着用于Western blot检测其致敏性，具体过程如下：
1：转膜：将2-D-SDS-PAGE胶上的蛋白用转膜槽在200 mA 2 h的条件下转至PVDF膜上。
2：封闭：将转有蛋白的PVDF膜用5%的脱脂奶粉在常温封闭2h。
3：一抗孵育：将封闭好的PVDF膜用PBS洗涤，把一抗（尘螨过敏病人血清）用5%的脱脂奶粉按1:20稀释，在4 ℃孵育过夜。
4：二抗孵育：将一抗孵育的PVDF膜再用PBS洗涤，把二抗（羊抗人IgE）用5%的脱脂奶粉按1:2000稀释，在常温孵育1小时。
5：显影：将二抗孵育好的PVDF膜用PBS洗涤3次，每次5 min，在膜表面加上适量的二抗特异发光底物，用胶片进行显影。
E. 将与胶片上显影点匹配较好的2-D-PAGE蛋白点进行ESI-Q-TOF质谱测序。
结果如图2-B所示，其中点1 Der f 20，点2和3为Der f 8。
SEQUENCE LISTING
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中国科学院昆明动物研究所
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尘螨过敏原Der f 8 和Der f 20及其基因和应用
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PatentIn version 3.3
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atgttggcat atgccggtgt tgattatgtt gataaacgtt
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gatcgttcag aatggttgaa tgaaaaattc aatttgggtt
tagatttccc aaatttgcca 120
tattacatgg atggtgatgt taaaattacc caatcgatgg
ccattcttcg ttatttggcc 180
cgtaaatata acatggatgg tactaatgaa caggaacgtt
tacgaatttc aatggctgaa 240
caacaaacac atgatatgtt tatggccatg gtccgtattt
gttatgatcc aaatatggaa 300
aacttacgag ttgattattt gaaaacattg cccgattcat
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ttagctaatc atgattatat tgccggttca aaaatttctt
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gaatatttgt gccgtatgaa agtcatggta ccagaagtat
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aaaaaatttg ttgaacgcat tgaatcattg ccacgtattg
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aaaccaacaa cattcaatgc atcattggct aaatggaatg
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atggtcgatc aagctaccct gagtaaattg gaagccggtt
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ggcgaattga aaggcaccta ttacccattg ttgggaatgg
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Claims (3)
1.尘螨过敏原Derf 8和Der f 20、其特征在于分别由SEQ ID NO:1和SEQ ID NO:3所示的氨基酸序列组成。
2.编码尘螨过敏原Der f 8和Der f 20的基因，其特征在于分别由SEQ ID NO:2和SEQ ID NO:4所示的核苷酸序列组成。
3.权利要求1所述的尘螨过敏原Der f 8和Der f 20在制备治疗尘螨过敏性疾病药物中的应用。
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