CN111018998B - 一种莱姆重组融合蛋白OspC-VlsE及其应用 - Google Patents

一种莱姆重组融合蛋白OspC-VlsE及其应用 Download PDF

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CN111018998B
CN111018998B CN201911225389.9A CN201911225389A CN111018998B CN 111018998 B CN111018998 B CN 111018998B CN 201911225389 A CN201911225389 A CN 201911225389A CN 111018998 B CN111018998 B CN 111018998B
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阮美丽
吴晓杰
吴银飞
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HANGZHOU AORUI BIOMEDICINE TECHNOLOGY CO LTD
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Abstract

本发明公开了一种莱姆重组融合蛋白OspC‑VlsE，其特征在于，所述重组融合蛋白OspC‑VlsE的氨基酸序列如SEQ ID NO.1所示。本发明利用基因工程重组技术，在大肠杆菌系统中表达重组融合蛋白OspC‑VlsE，具有生产周期短，产量高，成本低，特异性好的优点。本发明的重组融合蛋白可作为莱姆病重组蛋白检测试剂盒的一部分。

Description

一种莱姆重组融合蛋白OspC-VlsE及其应用
技术领域
本发明涉及基因工程领域，特别是一种在大肠杆菌系统中表达重组莱姆OspC-VlsE的融合蛋白及其生产方法和应用。
背景技术
莱姆病是一种蜱传播的人兽共患病，其病原体为伯氏疏螺旋体。莱姆病临床症状包括:游走性红斑，面神经麻痹，神经根炎，心脏病，关节炎和慢性萎缩性肢皮炎等，严重的导致终身残疾，甚至死亡。因为莱姆病的临床症状复杂多样，所以莱姆病的实验室辅助诊断尤为重要。
莱姆病的实验室诊断主要依靠于血清学诊断方法，包括IFA、ELISA、WB等。用于莱姆病血清学诊断的抗原包括全菌抗原和重组抗原，全菌抗原在检测莱姆病时敏感度较高，但全菌蛋白在诊断过程中易与梅毒血清、钩端螺旋体血清有交叉反应，特异性较低。而莱姆病重组蛋白检测试剂盒可以在一定程度上提高莱姆病检测的特异性，有很好的应用前景。
有不少研究报道指出，OspC(伯氏疏螺旋体外膜蛋白C)在宿主体内可以引起早期免疫反应，因此有研究者致力于利用OspC作为抗原用来诊断早期的莱姆病。早年，Hauser等培养了大量不同莱姆病的菌株，并从中提取了大量的OspC作为抗原，利用ELISA技术检测了222例莱姆病患者的血清和133例对照供血者的血清。其结果显示，有几株菌株间的IgG的检测灵敏度非常相似。
目前看来，OspC作为伯氏疏螺旋体的主要抗原之一，OspC虽然具有比较大的差异，但其具有很强的特异性。因此，在诊断学临床应用中，将OspC作为诊断抗原，具有很大的临床优势。同时，如果将OspC与其他的抗原相结合，进行配伍诊断，那么这种诊断方法不仅能够提高诊断的灵敏度，还能减少与其他病原体的交叉反应。这将在将来莱姆病的诊断中显示出很强的优势与潜力。预计在今后的莱姆病防治中也会发挥一定的作用。
本发明旨在将OspC与VlsE(变量主要蛋白样序列表达蛋白E)相结合，表达一种莱姆重组融合蛋白OspC-VlsE，不仅能够提高诊断的灵敏度，还能减少与其他病原体的交叉反应。
发明内容
本发明目的在于提供一种莱姆重组融合蛋白OspC-VlsE，此重组融合蛋白是利用基因工程重组技术，在大肠杆菌系统中表达，具有生产周期短，产量高，成本低的优点。本发明的莱姆重组融合蛋白OspC-VlsE可做为莱姆病重组蛋白检测试剂盒的一部分。
一种莱姆重组融合蛋白OspC-VlsE，其特征在于，所述莱姆重组融合蛋白OspC-VlsE的氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1:
NTSANSADESVKGPNLTEISKKITDSNAVLLAVKEVEALLSSIDEIAAKAIGKKIHQNNGLDTENNHNGSLLAGAYAISTLIKQKLDGLKNEGLKEKIDAAKKCSETFTNKLKEKHTDLGKEGVTDADAKEAILKTNGTKTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSPVVAESPKKPGSGSGEGAIKEVSELLDKLVKAVKTAEGASSGTAAIGEVVADADAAKVADKASVKGIAKGIKEIVEAAGGSEKLKAVAAAKGENNKGAGKLFGKAGAAAHGDSEAASKAAGAVSAVSGEQILSAIVTAADAAEQDGKKPEEAKNPIAAAIGDKDGGAEFGQDEMKKDDQIAAAIALRGMAKDGKFAVKDGEKEKA。
本发明还提供了编码SEQ ID NO.1的莱姆重组融合蛋白OspC-VlsE基因，其核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2:
AACACCAGCGCGAACAGCGCGGATGAAAGCGTGAAAGGCCCGAACCTGACCGAAATTAGCAAAAAAATTACCGATAGCAACGCGGTGCTGCTGGCGGTGAAAGAAGTGGAAGCGCTGCTGAGCAGCATTGATGAAATTGCGGCGAAAGCGATTGGCAAAAAAATTCATCAGAACAACGGCCTGGATACCGAAAACAACCATAACGGCAGCCTGCTGGCGGGCGCGTATGCGATTAGCACCCTGATTAAACAGAAACTGGATGGCCTGAAAAACGAAGGCCTGAAAGAAAAAATTGATGCGGCGAAAAAATGCAGCGAAACCTTTACCAACAAACTGAAAGAAAAACATACCGATCTGGGCAAAGAAGGCGTGACCGATGCGGATGCGAAAGAAGCGATTCTGAAAACCAACGGCACCAAAACCAAAGGCGCGGAAGAACTGGGCAAACTGTTTGAAAGCGTGGAAGTGCTGAGCAAAGCGGCGAAAGAAATGCTGGCGAACAGCGTGAAAGAACTGACCAGCCCGGTGGTGGCGGAAAGCCCGAAAAAACCGGGCAGCGGCAGCGGCGAAGGCGCGATTAAAGAAGTGAGCGAACTGCTGGATAAACTGGTGAAAGCGGTGAAAACCGCGGAAGGCGCGAGCAGCGGCACCGCGGCGATTGGCGAAGTGGTGGCGGATGCGGATGCGGCGAAAGTGGCGGATAAAGCGAGCGTGAAAGGCATTGCGAAAGGCATTAAAGAAATTGTGGAAGCGGCGGGCGGCAGCGAAAAACTGAAAGCGGTGGCGGCGGCGAAAGGCGAAAACAACAAAGGCGCGGGCAAACTGTTTGGCAAAGCGGGCGCGGCGGCGCATGGCGATAGCGAAGCGGCGAGCAAAGCGGCGGGCGCGGTGAGCGCGGTGAGCGGCGAACAGATTCTGAGCGCGATTGTGACCGCGGCGGATGCGGCGGAACAGGATGGCAAAAAACCGGAAGAAGCGAAAAACCCGATTGCGGCGGCGATTGGCGATAAAGATGGCGGCGCGGAATTTGGCCAGGATGAAATGAAAAAAGATGATCAGATTGCGGCGGCGATTGCGCTGCGCGGCATGGCGAAAGATGGCAAATTTGCGGTGAAAGATGGCGAAAAAGAAAAAGCG。
本发明的第三个目的是提供含有莱姆重组融合蛋白OspC-VlsE基因的重组载体，其特征在于，在质粒pET30a中包含有SEQ ID NO.2核苷酸序列。
本发明第四个目的是提供莱姆重组融合蛋白OspC-VlsE的表达方法，其特征在于，包括步骤：
1)构建莱姆重组融合蛋白OspC-VlsE质粒：在质粒pET30a中插入SEQ ID NO.2核苷酸序列；
2)将步骤1的莱姆重组融合蛋白OspC-VlsE质粒转化至大肠杆菌BL21，诱导表达莱姆重组融合蛋白OspC-VlsE；表达时的诱导温度在37度，诱导转速为200rpm，诱导的IPTG浓度为1mM。
本发明重组蛋白基因由人工合成。所用的载体为pET30a，为卡那抗性，融合表达组氨酸标签，蛋白定位于细胞周质。
本发明的表达系统为大肠杆菌BL21表达系统，它具有周期短，费用低，表达量大等特点。
为了使得重组蛋白更好的保持活性位点，本发明选择比较温和的培养和诱导条件，其表达时的诱导温度在37度，诱导转速为200rpm，诱导的IPTG浓度为1mM。这样使得重组蛋白有了更加缓慢的表达，有充分的时间进行空间构象的形成。
本发明可以采用超声的方式破碎细菌。在破碎菌体的时候，为了避免太过剧烈，将破碎条件定为400w，超声3s，间隔6s超声一次，共180次。
本发明重组蛋白的纯化方式可以有多种，例如离子交换层析，凝胶过滤层析和亲和层析等方法。本发明优选亲和层析，因为重组蛋白中本发明加入了His标签，一步纯化能够达到较高的纯度。
本发明的最后一个目的是提供上述莱姆重组融合蛋白OspC-VlsE在制备莱姆病重组蛋白检测试剂盒中的应用，其检测方法可以是间接金标法。采用莱姆重组融合蛋白OspC-VlsE作为抗原，用于捕获样本中的莱姆抗体，检测灵敏度高，特异性强，与其他病原体的交叉反应少。
具体实施方式
实施例1含有莱姆重组融合蛋白OspC-VlsE基因表达载体的构建
莱姆重组融合蛋白OspC-VlsE基因是根据OspC(NCBI Sequence ID:CAG44438.1)和VlsE(NCBI Sequence ID:ACC99642.1)的蛋白序列设计的。分析其上的抗原表位，选取了其中最优势的抗原表位进行融合。
根据氨基酸的序列，用大肠杆菌的密码子，反翻译成核苷酸序列，得到的核苷酸序列如下:由上海捷瑞生物工程有限公司合成重组基因序列SEQ ID NO.2，载体为pET30a。
SEQ ID NO.2:
AACACCAGCGCGAACAGCGCGGATGAAAGCGTGAAAGGCCCGAACCTGACCGAAATTAGCAAAAAAATTACCGATAGCAACGCGGTGCTGCTGGCGGTGAAAGAAGTGGAAGCGCTGCTGAGCAGCATTGATGAAATTGCGGCGAAAGCGATTGGCAAAAAAATTCATCAGAACAACGGCCTGGATACCGAAAACAACCATAACGGCAGCCTGCTGGCGGGCGCGTATGCGATTAGCACCCTGATTAAACAGAAACTGGATGGCCTGAAAAACGAAGGCCTGAAAGAAAAAATTGATGCGGCGAAAAAATGCAGCGAAACCTTTACCAACAAACTGAAAGAAAAACATACCGATCTGGGCAAAGAAGGCGTGACCGATGCGGATGCGAAAGAAGCGATTCTGAAAACCAACGGCACCAAAACCAAAGGCGCGGAAGAACTGGGCAAACTGTTTGAAAGCGTGGAAGTGCTGAGCAAAGCGGCGAAAGAAATGCTGGCGAACAGCGTGAAAGAACTGACCAGCCCGGTGGTGGCGGAAAGCCCGAAAAAACCGGGCAGCGGCAGCGGCGAAGGCGCGATTAAAGAAGTGAGCGAACTGCTGGATAAACTGGTGAAAGCGGTGAAAACCGCGGAAGGCGCGAGCAGCGGCACCGCGGCGATTGGCGAAGTGGTGGCGGATGCGGATGCGGCGAAAGTGGCGGATAAAGCGAGCGTGAAAGGCATTGCGAAAGGCATTAAAGAAATTGTGGAAGCGGCGGGCGGCAGCGAAAAACTGAAAGCGGTGGCGGCGGCGAAAGGCGAAAACAACAAAGGCGCGGGCAAACTGTTTGGCAAAGCGGGCGCGGCGGCGCATGGCGATAGCGAAGCGGCGAGCAAAGCGGCGGGCGCGGTGAGCGCGGTGAGCGGCGAACAGATTCTGAGCGCGATTGTGACCGCGGCGGATGCGGCGGAACAGGATGGCAAAAAACCGGAAGAAGCGAAAAACCCGATTGCGGCGGCGATTGGCGATAAAGATGGCGGCGCGGAATTTGGCCAGGATGAAATGAAAAAAGATGATCAGATTGCGGCGGCGATTGCGCTGCGCGGCATGGCGAAAGATGGCAAATTTGCGGTGAAAGATGGCGAAAAAGAAAAAGCG。
实施例2含有莱姆重组融合蛋白OspC-VlsE的表达
将合成好的莱姆重组融合蛋白OspC-VlsE质粒转化至大肠杆菌BL21中，涂布于含50ug/ml卡那霉素(上海生工，货号:K0408)的LB平板上，37度过夜培养，挑取单克隆菌落，用含有相同浓度的卡那霉素的300ml LB培养基37度培养至OD600达0.6左右，用终浓度为1mM的IPTG(上海生工，货号:IB0168)进行诱导表达，诱导条件为:37度，转速200rpm，4h。诱导之后，将培养液4度，转速7000rpm，离心10min，收集菌体。
实施例3含有莱姆重组融合蛋白OspC-VlsE的纯化及复性
用50ml上样缓冲液Binding Buffer(50mM Tris，0.2M Nacl，pH8.0)50ml破碎菌体，然后超声破碎，条件为400w，超声3s，间隔6s，共180次，最后12000rpm，30min，4度离心收集上清，目的蛋白在上清中。接着过Ni+柱一步纯化，用洗脱缓冲液Elution Buffer(50mMTris，0.2M Nacl，0.5M Imidazole，pH8.0)洗脱目的蛋白。将纯化后的重组蛋白用透析缓冲液(50mM Tris，0.2M Nacl，pH8.0)透析，每隔12h换一次透析液，共3次。取出透析后的蛋白液，经0.22um滤器过滤后，用BCA法测定浓度后，于-20度保存备用。
实施例4间接金标法检测莱姆抗体
4.1间接金标法检测条的制备
在三角烧瓶中加入1000ml超纯水，在磁力加热搅拌器上加热至沸腾，然后加入10％氯金酸(sigma)1ml，再加入10％柠檬酸三钠溶液1ml，继续加热沸腾15min，然后冷却至室温。
取冷却后胶体金溶液100ml放入烧杯中，搅拌加入0.2M K2CO3 1.5ml调节金水pH至7.0，搅拌后加入一定量的OspC-VlsE莱姆重组抗原，室温搅拌30min，加入10ml 10％BSA溶液，室温搅拌30min后12000rpm离心30min，将上清小心吸出弃去，沉淀用金标稀释液(20mM Tris，0.2M NaCl，1％BSA，0.1％TritonX-100，0.01％Proclin300，pH8.0)定容至1ml，此为标记好的莱姆抗原胶体金复合物。
将上述金标复合物用金标稀释液稀释100倍后浸泡玻璃纤维，冻干后即制成金标垫。
用点膜稀释液(10mM PBS，1％蔗糖，pH7.4)稀释proteinA至1.0mg/ml，用相同稀释液稀释羊抗OspC-VlsE多抗至0.5mg/ml，将上述两种稀释之后的溶液划线至硝酸纤维素膜上，37度烘干过夜。
将以上金标垫、包被好的硝酸纤维素膜及滤纸、聚酯板、样品垫等组装成莱姆抗体间接法检测试剂条。
4.2间接金标法检测条的检测
加入50ul待检测样本(血清、血浆)至样品垫处，室温放置10min后判定结果，结果判定标准如下：
①仅质控线出现一条带，在测试区内无条带出现，为阴性；
②有两条带出现，其中一条位于质控区，另一条位于测试区，为阳性；
③质控线未出现条带，表明此测试条已损坏，应当更换新试纸条重新测试。
4.3间接金标法检测莱姆抗体结果
间接金标法一共检测100份阳性莱姆病人血清，及400份正常人血清，其中100份阳性血清中检测出阳性97例，漏检3例，在400份阴性血清中出现假阳10例，灵敏度和特异性分别为97％和97.5％，从结果数据可以说明，本发明的莱姆重组抗原，有着姣好的灵敏度和特异性，可以作为制作莱姆抗体检测试纸条的原料。
4.3间接金标法试纸条检测莱姆和对照试剂结果对比
购买商业化莱姆抗体检测试剂盒(ELISA法)，用本发明胶体金试纸条进行对比，对比结果如下：
间接金标法 ELISA
莱姆病I期(30例确诊病例) 阳性26例 阳性24例
莱姆病II期(30例确诊病例) 阳性30例 阳性30例
莱姆病III期(40例确诊病例) 阳性40例 阳性37例
健康人群(400例) 阴性390例 阴性387例
由上表可以看出，胶体金法试纸条的准确度要高于ELISA法。
序列表
<110> 杭州傲锐生物医药科技有限公司
<120> 一种莱姆重组融合蛋白OspC-VlsE及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 379
<212> PRT
<213> 未知(Unknown)
<400> 1
Asn Thr Ser Ala Asn Ser Ala Asp Glu Ser Val Lys Gly Pro Asn Leu
1 5 10 15
Thr Glu Ile Ser Lys Lys Ile Thr Asp Ser Asn Ala Val Leu Leu Ala
20 25 30
Val Lys Glu Val Glu Ala Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala
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Lys Ala Ile Gly Lys Lys Ile His Gln Asn Asn Gly Leu Asp Thr Glu
50 55 60
Asn Asn His Asn Gly Ser Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr
65 70 75 80
Leu Ile Lys Gln Lys Leu Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu
85 90 95
Lys Ile Asp Ala Ala Lys Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu
100 105 110
Lys Glu Lys His Thr Asp Leu Gly Lys Glu Gly Val Thr Asp Ala Asp
115 120 125
Ala Lys Glu Ala Ile Leu Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala
130 135 140
Glu Glu Leu Gly Lys Leu Phe Glu Ser Val Glu Val Leu Ser Lys Ala
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Ala Lys Glu Met Leu Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val
165 170 175
Val Ala Glu Ser Pro Lys Lys Pro Gly Ser Gly Ser Gly Glu Gly Ala
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Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys Ala Val Lys
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Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly Glu Val Val
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Ala Asp Ala Asp Ala Ala Lys Val Ala Asp Lys Ala Ser Val Lys Gly
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Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Gly Ser Glu
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Lys Leu Lys Ala Val Ala Ala Ala Lys Gly Glu Asn Asn Lys Gly Ala
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Gly Lys Leu Phe Gly Lys Ala Gly Ala Ala Ala His Gly Asp Ser Glu
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Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln
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Ile Leu Ser Ala Ile Val Thr Ala Ala Asp Ala Ala Glu Gln Asp Gly
305 310 315 320
Lys Lys Pro Glu Glu Ala Lys Asn Pro Ile Ala Ala Ala Ile Gly Asp
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Lys Asp Gly Gly Ala Glu Phe Gly Gln Asp Glu Met Lys Lys Asp Asp
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Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly Lys
355 360 365
Phe Ala Val Lys Asp Gly Glu Lys Glu Lys Ala
370 375
<210> 2
<211> 1137
<212> DNA
<213> 未知(Unknown)
<400> 2
aacaccagcg cgaacagcgc ggatgaaagc gtgaaaggcc cgaacctgac cgaaattagc 60
aaaaaaatta ccgatagcaa cgcggtgctg ctggcggtga aagaagtgga agcgctgctg 120
agcagcattg atgaaattgc ggcgaaagcg attggcaaaa aaattcatca gaacaacggc 180
ctggataccg aaaacaacca taacggcagc ctgctggcgg gcgcgtatgc gattagcacc 240
ctgattaaac agaaactgga tggcctgaaa aacgaaggcc tgaaagaaaa aattgatgcg 300
gcgaaaaaat gcagcgaaac ctttaccaac aaactgaaag aaaaacatac cgatctgggc 360
aaagaaggcg tgaccgatgc ggatgcgaaa gaagcgattc tgaaaaccaa cggcaccaaa 420
accaaaggcg cggaagaact gggcaaactg tttgaaagcg tggaagtgct gagcaaagcg 480
gcgaaagaaa tgctggcgaa cagcgtgaaa gaactgacca gcccggtggt ggcggaaagc 540
ccgaaaaaac cgggcagcgg cagcggcgaa ggcgcgatta aagaagtgag cgaactgctg 600
gataaactgg tgaaagcggt gaaaaccgcg gaaggcgcga gcagcggcac cgcggcgatt 660
ggcgaagtgg tggcggatgc ggatgcggcg aaagtggcgg ataaagcgag cgtgaaaggc 720
attgcgaaag gcattaaaga aattgtggaa gcggcgggcg gcagcgaaaa actgaaagcg 780
gtggcggcgg cgaaaggcga aaacaacaaa ggcgcgggca aactgtttgg caaagcgggc 840
gcggcggcgc atggcgatag cgaagcggcg agcaaagcgg cgggcgcggt gagcgcggtg 900
agcggcgaac agattctgag cgcgattgtg accgcggcgg atgcggcgga acaggatggc 960
aaaaaaccgg aagaagcgaa aaacccgatt gcggcggcga ttggcgataa agatggcggc 1020
gcggaatttg gccaggatga aatgaaaaaa gatgatcaga ttgcggcggc gattgcgctg 1080
cgcggcatgg cgaaagatgg caaatttgcg gtgaaagatg gcgaaaaaga aaaagcg 1137

Claims (4)

1.编码SEQ ID NO.1的莱姆重组融合蛋白OspC-VlsE基因，其核苷酸序列如SEQ IDNO.2所示。
2.一种含有莱姆重组融合蛋白OspC-VlsE基因的重组载体，其特征在于，在质粒pET30a中包含有SEQ ID NO.2核苷酸序列。
3.莱姆重组融合蛋白OspC-VlsE的表达方法，其特征在于，包括步骤：
1）构建莱姆重组融合蛋白OspC-VlsE质粒：在质粒pET30a中插入SEQ ID NO.2核苷酸序列；
2）将步骤1的莱姆重组融合蛋白OspC-VlsE质粒转化至大肠杆菌BL21，诱导表达莱姆重组融合蛋白OspC-VlsE；表达时的诱导温度在37度，诱导转速为200rpm，诱导的IPTG浓度为1mM。
4. 一种莱姆抗体检测试剂盒，其特征在于，所述检测试剂盒包含氨基酸序列如SEQ IDNO.1所示莱姆重组融合蛋白OspC-VlsE。
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