CN106146640B - 尘螨变应原及其应用 - Google Patents
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Abstract
本发明提供了一种尘螨变应原及其应用。本发明提供的粉尘螨变应原属于翻译延伸因子蛋白家族,可用于过敏性疾病的诊断、治疗、预防,特别是粉尘螨过敏性疾病,尤其特别的,是用于粉尘螨变应原第35组分引起的过敏性疾病的诊断、治疗、预防。本发明通过基因克隆、蛋白质表达制备得到粉尘螨变应原重组蛋白,蛋白纯度高、特异性较好、产量丰富。本发明提供的本发明粉尘螨变应原用于制备诊断、预防或治疗尘螨变应原引起的过敏性疾病的试剂,具有特异性高、成本低的特点;特别地,可高效用于诊断患者是否对粉尘螨变应原第35组分的过敏。
Description
技术领域:
本发明属于生物医学领域,尤其涉及一种尘螨变应原及其应用。
背景技术:
在引起过敏性疾病的众多变应原中,尘螨是最主要的变应原。尘螨在过敏性疾病患者特异性免疫诊断中阳性率约70-80%。由于天然变应原提取液的组分非常复杂,恒定其组分非常困难,且容易受外源性有毒物质、病原微生物的污染,影响其重复性与安全性。而重组变应原疫苗具有纯度高、无杂蛋白、易标准化、无外源性毒性物质和病原微生物污染的优势,临床上已有用于免疫治疗。
由于尘螨系医学节肢动物,结构和成分复杂,尽管人们从数百种蛋白中已初步鉴定出24中过敏原成分,而研究显示尘螨含有过敏原多达30余种;此外,每种过敏原的氨基酸序列和核苷酸序列还具有序列多型性(sequence polymorphisms),比如具有146个氨基酸残基的Der p2变应原具有8种变体,各变体各自具有独特的氨基酸残基取代(Hales BJ,Hazell LA,Smith W,Thomas WR.Genetic variation of Der p 2allergens:effects onT cell responses and immunoglobulin E binding.Clin Exp Allergy 2002;32(10):1461-7.),这些变体对于研究尘螨过敏之免疫发病机制,以及研发用以诊断及治疗尘螨过敏的试剂具有重要意义。
发明内容:
为解决上述问题,本发明提供了一种尘螨变应原及其应用,本发明提供的尘螨变应原属于翻译延伸因子蛋白家族,可用于过敏性疾病的诊断、治疗、预防,特别是粉尘螨过敏性疾病,尤其特别的,是用于粉尘螨变应原第35组分引起的过敏性疾病的诊断、治疗、预防。
第一方面,本发明提供了一种尘螨变应原,包含具有与SEQ ID NO:1所示氨基酸序列至少60%同源性的氨基酸序列,或与如SEQ ID NO:1所示的氨基酸序列具有免疫交叉反应性的尘螨变应原。
第二方面,本发明提供了一种尘螨变应原,包含至少一个T细胞受体特异性识别的抗原表位,所述T细胞受体特异性识别的抗原表位为如SEQ ID NO:1所示的氨基酸序列编码的多肽被T细胞受体特异性识别的抗原表位。
在本发明一个实施例中,如第一方面或第二方面的所述尘螨变应原的氨基酸序列为与SEQ ID NO:1同源性不低于70%、优选为不低于80%,进一步优选为不低于90%,更进一步优选为不低于95%或99%的氨基酸序列。
在本发明一个优选实施例中,编码如第一方面或第二方面的所述尘螨变应原的氨基酸序列如SEQ ID NO:1所示。
具体地,所述SEQ ID NO:1如下所示:
MVNFTVDEIRVLMNKKRNIRNMSVIAHVDHGKSTLTDSLVSKAGIIAAAKAGEMRFTDTRKDEQERCITIKSTAISMYFEMREQDMVFITSADQKESDEKGFLINLIDSPGHVDFSSEVTAALRVTDGALVVVDCVSGVCVQTETVLRQAIAERIKPVLFMNKMDLAMLTLQLEQEDLYQKFTRIVENVNVIISTYADENGPMGDIRVDPSKGSVGFGSGLHGWAFSLKQFAELYSEKFKIDVDKLMNRLWGENFYNPTAKKWSKRFDEGYKRAFCMFVLDPIFKVFDAIMNFKKEETAKLLEKLNIVLKGEDKEKDGKNLLKVVMRTWLPAGDSLLQMIAIHLPSPITAQKYRMELLYEGPHDDEAAVAIKSCNPEGPLMMYISKMVPTSDKGRFYAFGRVFSGIVASGQKVRIMGPNYVHGKKEDLVEKAIQRTVLMMGRYVESIENVPCGNICGLVGVDQFLVKTGTISTFKDAHNMKVMKFSVSPVVRVAVEPKNPADLPKLVEGLKRLAKSDPMVQCIIEESGEHIVAGAGELHLEICLKDLEEDHAQIPIKTSDPVVSYRETVSEESEIMCLSKSPNKHNRLFMKACPLQDGIAEDIDKGDINPRDDFKVRARFLADKYNWDATDARKIWAFGPEGTGPNLLVDVTKGVQYLNEIKDSVVAGFQWATKESVLCEENMRGVRFNIHDVTLHADAIHRGGGQIIPTARRCLYACLLTAQPRLLEPVYLVEIQCPEQAVGGIYGVLNRRRGHVFEESQVVGTPMFTVKAYLPVNESFGFTADLRSNTGGQAFPQCVFDHWQILPGDPLDGKSRPYQIVMDTRKRKGLKDSLPELDNYFDKL
第三方面,本发明提供了一种诊断个体对尘螨过敏的组合物,包含如第一方面或第二方面所述的粉尘螨变应原。
在本发明一个实施例中,所述组合物还包括但不限于行业内在诊断个体对尘螨过敏时采用的其他常规成分,比如稀释剂、免疫佐剂、医药可接受之赋形剂或载体。
第四方面,本发明提供了一种诊断个体对尘螨过敏的方法,所述方法包含检测该个体是否会与第一方面或第二方面所述的粉尘螨变应原产生免疫反应。
第五方面,本发明提供了一种治疗或预防个体过敏性疾病的方法,所述方法包含对所述个体投与第一方面或第二方面所述的粉尘螨变应原。
第六方面,本发明提供了一种如第一方面或第二方面所述的尘螨变应原在制备诊断、预防、治疗尘螨变应原引起的过敏性疾病的试剂中的应用。
第七方面,本发明提供了一种编码如第一方面或第二方面所述的尘螨变应原的核苷酸序列。
第八方面,本发明提供了一种重组载体,所述重组载体含有编码如第一方面或第二方面所述的尘螨变应原的基因表达盒。
第九方面,本发明提供了一种制备粉尘螨Der f35变应原的方法,包括将如SEQ IDNO:1所示的氨基酸序列的DNA编码序列克隆到表达载体中进行蛋白质表达、纯化,获得粉尘螨Derf35变应原。
第十方面,本发明提供了一种检测粉尘螨Der f35变应原的编码基因或粉尘螨Derf35变应原编码基因的变体的方法,包括以下步骤:1)提取粉尘螨总RNA,mRNA纯化以及反转录得cDNA;2)设计引物,以cDNA为模板,利用PCR方法扩增编码基因,获得所述粉尘螨Derf35变应原的编码基因或粉尘螨Der f35变应原编码基因的变体。
基因测序结果表明尘螨过敏原Der f 35的编码基因由2535个氨基酸组成,该基因的5’端至3’端的序列为(核苷酸序列为SEQ ID NO:2):
ATGGTCAATTTTACTGTCGACGAAATCCGTGTTCTTATGAACAAAAAACGGAATATTCGTAACATGTCTGTCATTGCTCATGTCGATCATGGTAAATCGACATTGACCGATTCATTGGTATCGAAAGCCGGTATCATTGCTGCAGCTAAAGCTGGTGAAATGCGTTTTACCGATACCCGTAAGGATGAACAAGAACGTTGTATTACGATCAAATCGACCGCTATTTCGATGTATTTCGAAATGCGTGAACAAGATATGGTTTTCATCACTAGTGCCGATCAAAAAGAATCCGACGAAAAAGGTTTCTTGATCAATTTGATTGATAGTCCCGGCCACGTTGATTTTTCATCCGAAGTTACGGCTGCTCTTCGTGTAACCGATGGTGCTTTGGTTGTCGTTGATTGTGTGTCTGGTGTTTGTGTCCAAACTGAAACTGTATTACGTCAAGCTATTGCCGAACGTATCAAGCCGGTATTGTTCATGAACAAAATGGACTTGGCCATGCTTACTCTTCAATTGGAGCAAGAAGATTTGTATCAAAAATTCACTCGTATCGTCGAAAATGTCAACGTCATCATTTCGACATATGCTGATGAAAATGGGCCCATGGGCGACATTCGTGTCGATCCATCCAAAGGTTCCGTTGGTTTTGGTTCCGGTTTACATGGCTGGGCTTTTTCATTGAAACAATTTGCTGAATTGTATTCGGAAAAATTCAAAATTGATGTCGATAAATTGATGAATCGATTATGGGGTGAAAACTTTTACAATCCTACAGCGAAAAAGTGGTCAAAACGTTTTGATGAAGGATACAAACGTGCTTTCTGTATGTTTGTCTTGGATCCAATCTTTAAAGTTTTCGATGCCATCATGAACTTTAAAAAAGAGGAGACTGCTAAATTGTTGGAGAAATTAAACATCGTATTGAAAGGTGAAGATAAAGAAAAAGATGGCAAGAATTTATTGAAAGTTGTTATGCGAACCTGGTTGCCTGCTGGTGATTCATTGCTTCAGATGATTGCCATTCATTTGCCATCACCAATCACAGCTCAAAAGTATCGTATGGAATTGTTGTATGAAGGACCACATGATGATGAAGCTGCTGTTGCCATTAAATCTTGTAATCCGGAAGGTCCATTGATGATGTACATTTCGAAAATGGTACCGACATCTGATAAAGGACGTTTCTATGCTTTTGGTCGTGTTTTCTCTGGTATTGTCGCTTCTGGACAAAAAGTCCGTATAATGGGACCAAATTATGTGCATGGTAAAAAAGAGGATTTGGTTGAGAAGGCCATTCAACGAACTGTATTGATGATGGGTCGTTATGTGGAATCAATTGAAAATGTACCATGCGGTAATATTTGTGGTTTAGTTGGTGTTGATCAATTTTTGGTCAAAACCGGTACCATTTCAACATTTAAAGATGCACACAACATGAAAGTGATGAAATTCTCCGTATCGCCTGTTGTGCGTGTTGCTGTTGAACCGAAAAATCCTGCCGATTTACCTAAATTGGTAGAAGGTTTAAAACGTTTGGCTAAATCTGATCCTATGGTACAATGTATCATTGAAGAATCGGGTGAACATATTGTAGCAGGTGCTGGTGAACTTCATTTGGAAATTTGTCTGAAAGATTTGGAAGAAGATCATGCCCAAATTCCAATCAAAACATCGGATCCAGTTGTATCGTATCGAGAAACTGTTTCAGAAGAATCTGAAATTATGTGCTTGTCTAAATCACCAAACAAACATAATCGTTTGTTCATGAAAGCATGTCCTCTTCAGGATGGTATTGCCGAAGACATTGATAAAGGTGACATCAATCCACGTGATGATTTCAAAGTGCGTGCTCGATTCTTAGCTGATAAATATAATTGGGATGCAACCGATGCCCGTAAAATCTGGGCTTTTGGACCCGAAGGTACTGGACCAAATCTTTTGGTCGATGTAACCAAAGGTGTGCAATATTTAAACGAAATCAAAGATAGCGTTGTTGCTGGATTTCAATGGGCCACCAAAGAGAGTGTACTTTGTGAAGAAAACATGCGTGGTGTTCGTTTCAACATTCATGATGTAACTTTGCATGCTGATGCTATCCATCGTGGTGGTGGTCAAATCATTCCGACAGCTCGTCGTTGTCTTTATGCCTGCCTTTTGACCGCTCAACCTCGTCTTTTGGAACCGGTCTATTTGGTGGAAATTCAATGTCCTGAACAAGCCGTTGGTGGTATCTATGGTGTGTTGAATAGACGTCGTGGCCATGTATTTGAAGAATCACAAGTTGTCGGTACACCTATGTTCACTGTCAAAGCCTATTTGCCAGTAAATGAATCATTCGGTTTTACTGCCGATCTTCGTTCAAACACTGGTGGCCAAGCTTTCCCACAATGTGTATTTGATCATTGGCAAATTTTGCCTGGCGATCCGTTGGATGGTAAATCCCGTCCATATCAAATTGTCATGGATACACGTAAACGTAAAGGTCTTAAGGATTCATTGCCCGAATTGGACAATTATTTCGATAAACTTTGA
本发明的效益:
(1)本发明提供的粉尘螨变应原属于翻译延伸因子蛋白家族,可用于过敏性疾病的诊断、治疗、预防,特别是粉尘螨过敏性疾病,尤其特别的,是用于粉尘螨变应原第35组分引起的过敏性疾病的诊断、治疗、预防;
(2)本发明通过基因克隆、蛋白质表达制备得到粉尘螨变应原重组蛋白,蛋白纯度高、特异性较好、产量丰富;
(3)本发明提供的本发明粉尘螨变应原用于制备诊断、预防或治疗尘螨变应原引起的过敏性疾病的试剂具有特异性高、成本低的特点;特别地,可高效用于诊断患者是否对粉尘螨变应原第35组分的过敏。
附图说明:
图1是本发明实施例提供的粉尘螨Der f 35质粒酶切结果;
图2是本发明实施例提供的Der f 35电泳图;
图3是Der f35与尘螨过敏病人血清IgE作用的Elisa结果;
图4是Der f35与尘螨过敏病人血清IgE作用的immublotting结果。
具体实施方式:
实施例一、尘螨过敏原翻译延伸因子的发现
通过分析比对深圳大学与香港中文大学合作所测出来的粉尘螨全基因组序列,获得粉尘螨翻译延伸因子的基因序列。
实施例二、尘螨过敏原的分子克隆
一、粉尘螨总RNA的提取
挑取干净的活粉尘螨,用Qicgen公司的RNeasy Mini Kit进行总RNA的提取,操作步骤按说明书进行。
二、Der f 35全长cDNA克隆
以提取的总RNA为模板,逆转录cDNA,进行PCR扩增反应。反应体系如下(50μL):10×Ex Taq Buffer 5μL;TaKaRa ExTaq 0.25μL;dNTP Mixture,4μL;上下游引物各2μL,cDNA为模板1μL;加去离子水至50μL。PCR反应条件:94℃变性1min;50℃退火1min;72℃延伸1min;35个循环,PCR产物经1%琼脂糖电泳验证并拍照。
三、重组质粒构建及酶切鉴定
将上述PCR产物与pMD-18T连接后,热转化至E.coli Top10中,涂布于含氨卞霉素(100mg·L-1)的LB平板上,37℃培养过夜,从LB平板上挑选白色菌落放入含氨卞霉素的LB培养液中扩大培养,提取质粒。用BamH I酶切鉴定,重组质粒,委托华大基因工程(深圳)有限公司进行序列,测序正确后进一步将酶切产物与pET-28a表达载体37℃反应4h进行连接转化至E.coli BL21,37℃培养过夜。挑取单个菌落,提取质粒后双酶切鉴定。
粉尘螨Der f 35基因重组质粒酶切结果如图1所示;M为DNA marker,泳道1为粉尘螨Der f 35。
四、Der f 35的诱导表达及纯化
将上述鉴定的pET28a-Der f 35转化至感受态大肠杆菌BL21(DE3),待细菌生长处于对数生长期(A600nm=0.6~0.9)时,加入20μl 1mol/L的IPTG,诱导蛋白表达。诱导4h后取1mL菌液,离心弃上清液,加100μl去离子水后重悬菌体,加20~25μl 10×SDS-PAGE上样缓冲液混合,沸水与浴10min,按照5μl,10μl,20μl上样,进行SDS-PAGE电泳分析,以检测重组蛋白表达情况。诱导表达的重组蛋白,经裂解、溶菌、超声,将上清液以2ml/min的速度上样于已平衡好的Ni-NTA柱。然后用平衡液充分洗柱,再分别用含40mmol/L、300mmol/L咪唑平衡缓冲液进行洗脱,收集各次洗脱液进行SDS-PAGE分析。
粉尘螨Der f 35SDS-PAGE电泳图结果如图2所示,M为蛋白质marker,1泳道为纯化后的Der f 35尘螨蛋白。
实施例三:尘螨过敏原的过敏原性检测
一、Elisa实验
用包被液把过敏原稀释成10ug/ml,用50ul 4℃包被过夜,病人血清按1:5稀释,二抗是按1:2000稀释,最终测OD450nm的光吸收值。
Der f 35与尘螨过敏病人血清IgE作用的Elisa结果如图3所示,图3中,P1-P3为不同哮喘病人的结果,C1-C2为不同健康人的结果。
二、Western blot实验
以粉尘螨过敏患者血清为一抗进行Western blotting,以带有链酶亲和素-辣根过氧化物酶(HRP)标记的羊抗人IgE为二抗孵育后,加入免疫印迹化学发光试剂(ECL),放射自显影片曝光和洗片处理。
Der f 35与尘螨过敏病人血清Western blot结果如图4所示,M为蛋白质marker,1泳道为Der f 35与尘螨过敏病人血清Western blot结果。
三、皮肤针刺实验
用生理盐水溶解的过敏原滴在患者前臂掌侧皮肤,用特制的点刺针刺破皮肤,使少量的过敏原进入皮肤内,擦干遗留的过敏原,15min后读结果,分别用生理盐水和组胺做阴性和阳性对照。结果如表1.
表1.Der f 35对39名尘螨过敏病人的皮肤针刺实验结果
上述临床皮肤针刺实验显示,对于38位尘螨过敏患者,Der f 35与其中7位的皮肤针刺实验呈阳性反应,阳性反应率为17.9%。此外,上述过敏原检测表明,Der f 35具有较强的过敏原性,是来自尘螨的重要过敏原,能应用于诊断患者是否因为粉尘螨变应原第35组分引起的过敏性疾病及制备因为粉尘螨变应原第35组分引起的过敏性疾病的试剂。
因此,本发明提供的粉尘螨变应原可用于过敏性疾病的诊断、治疗、预防,特别是粉尘螨过敏性疾病的诊断、治疗、预防。
Claims (7)
1.一种尘螨变应原,其特征在于,具有如SEQ ID NO:1所示的氨基酸序列。
2.一种诊断个体对尘螨过敏的组合物,其特征在于,包含如权利要求1所述的尘螨变应原。
3.一种编码如权利要求1所述的尘螨变应原的核酸。
4.一种重组载体,其特征在于,所述重组载体含有编码如权利要求1所述的尘螨变应原的基因表达盒。
5.一种制备粉尘螨变应原的方法,其特征在于,包括将如SEQ ID NO:1所示的氨基酸序列的DNA编码序列克隆到表达载体中进行蛋白质表达、纯化,获得粉尘螨Der f35变应原。
6.一种检测制备粉尘螨Der f35变应原的编码基因或粉尘螨Der f35变应原编码基因的变体的方法,包括以下步骤:1)提取粉尘螨总RNA,mRNA纯化以及反转录得cDNA;2)设计引物,利用PCR方法扩增编码基因,获得所述粉尘螨Der f35变应原的编码基因或粉尘螨Derf35变应原编码基因的变体。
7.权利要求1所述的尘螨变应原在制备诊断、预防、治疗尘螨变应原引起的过敏性疾病的试剂中的应用。
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