CN115161296A - 一种刺虾荧光素酶Nluc的突变体及其应用 - Google Patents
一种刺虾荧光素酶Nluc的突变体及其应用 Download PDFInfo
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- CN115161296A CN115161296A CN202110358434.9A CN202110358434A CN115161296A CN 115161296 A CN115161296 A CN 115161296A CN 202110358434 A CN202110358434 A CN 202110358434A CN 115161296 A CN115161296 A CN 115161296A
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Abstract
本发明公开了一种刺虾荧光素酶Nluc的突变体及其应用。所述突变体与如SEQ ID NO:2所示的氨基酸序列相比在第19位、第20位、第27位以及第28位中的一位或多位发生突变。本发明提供的新型刺虾荧光素酶Nluc提升了现有的刺虾荧光素酶发光亮度，且仪器不能检测到其催化高斯荧光素酶Gluc底物腔肠素的发光，其解决了现有的刺虾荧光素酶Nluc底物特异性宽泛的问题，有利于将来在生物监测等领域Nluc与Gluc两种荧光素酶的联合使用。
Description
技术领域
本发明属于基因工程领域，具体涉及一种刺虾荧光素酶Nluc的突变体及其应用。
背景技术
荧光素酶是生物体内催化荧光素或脂肪醛氧化发光的一类酶的总称。通常发现于低等动物体内。目前常用的荧光素酶有萤火虫荧光素酶、海肾荧光素酶、高斯荧光素酶等。萤火虫荧光素酶发光需要ATP和Mg2+的辅助，海肾荧光素酶虽然不依赖于ATP和Mg2+等辅助因子，但发光强度较弱，应用时需要更灵敏的检测。高斯荧光素酶很好的弥补了萤火虫荧光素酶和海肾荧光素酶的缺点，发光时既不需要ATP和Mg2+等辅助因子，也有高于海肾荧光素酶100倍的发光效率。近年，具有增强光输出的合成的深海虾细脚刺虾酶(Nanolucluciferase,Nluc)因其具有分子量小，发光效率较高等特点，亦被广泛应用于科研及产业中。
利用荧光素酶自发光的特点，荧光素酶常应用于活细胞检测、蛋白与蛋白相互作用、蛋白定位、小干扰RNA沉默技术、高通量药物筛选等领域。在生物监测技术领域，荧光素酶可用于检测化学污染物的有无。另外，在免疫检测、生化诊断等领域亦具有广阔的应用前景。另外，作为检测不同启动子下外源基因表达强度和转录调控研究的报告基因，需要多种自发光亮度相近，且催化底物不同的荧光素酶联合使用。因此，更多可以催化不同底物、不依赖于高灵敏度检测技术、且发光更亮的荧光素酶需要尚待被开发。
目前合成的刺虾荧光素酶Nluc和高斯荧光素酶Gluc是两种已知的分子量最小且发光最强的荧光素酶。然而，虽然Nluc具有高活性，高量子产量，但是Nluc具有宽泛的底物特异性，可以催化Gluc的底物腔肠素及其多种类似物发光。导致Nluc及Gluc在需要多种荧光素酶联合使用的生化检测等领域中，不便于同时使用。所以现有Nluc的发光亮度仍有提升空间，且现有Nluc催化底物特异性过于宽泛，与其他荧光素酶联合使用时，有可催化同一底物，背景高等风险。
发明内容
为了解决现有技术中存在的Nluc发光亮度仍需提高、Nluc催化底物特异性过于宽泛等问题，本发明提供一种新型刺虾荧光素酶，该酶具有更高的催化活性，且仪器不能检测到催化Gluc底物腔肠素的发光，同条件下，该酶较现有的刺虾荧光素酶发光亮度更强。且该蛋白具有制备方法简单，易于生产的特点。
本发明通过将Nluc荧光素酶进行蛋白定向进化，得到了发光亮度最强2.89倍的新型刺虾荧光素酶。此荧光素酶纯化工艺简单，有利于规模生产；活性检测方式简单，易于检测；以Furimazine或氟代腔肠素为催化底物，且不催化高斯荧光素酶Gluc的底物腔肠素发光。该荧光素酶将在基础科研、生物监测及生化诊断等多领域具有广阔的应用前景。
本发明第一方面提供一种刺虾荧光素酶Nluc的突变体，所述突变体与如SEQ IDNO:2所示的氨基酸序列相比在第19位、第20位、第27位以及第28位中的一位或多位发生突变。
所述第19位的突变优选为D19N/P/E/G/S；所述第20位的突变优选为Q20R；所述第27位的突变优选为V27A/N/L/T/M/S；所述第28位的突变优选为S28A/N/V/D/P/G/I/M；
较佳地，所述突变体包含D19G、V27L/T/M、和/或S28A/N/M/V/D；
更佳地，所述突变体包含D19G、V27L、V27T、V27M、S28A、S28N、S28M、S28V、S28D，或同时包含D19P、V27L和S28P。
本发明第二方面提供一种分离的核酸，其编码如本发明第一方面所述的突变体；较佳地，所述核酸的碱基序列如SEQ ID NO:3-12任一个所示。
本发明第三方面提供一种重组表达载体，其包含本发明第二方面所述的分离的核酸，所述重组表达载体的骨架优选pET28a或pcDNA3.1质粒。
在使用pcDNA3.1载体时，可在刺虾荧光素酶Nluc的突变体序列前增加编码信号肽的核苷酸序列以便于其在真核细胞中分泌和表达。
本发明第四方面提供一种转化体，其含有如本发明第二方面所述的分离的核酸，或如本发明第三方面所述的重组表达载体；所述转化体构建时使用的宿主细胞优选为大肠杆菌或哺乳动物细胞；所述大肠杆菌优选为E.coli BL21(DE3)；所述哺乳动物细胞优选为CHO细胞或HEK293细胞。
本发明第五方面提供一种制备如本发明第一方面所述的突变体的方法，其包括培养如本发明第四方面所述的转化体得发酵产物，并从发酵产物中获得所述突变体。
本发明第六方面提供一种催化底物发光的方法，其包括以下步骤：利用如本发明第一方面所述的突变体进行底物的催化。
所述突变体的浓度优选为0.1μg/ml～10μg/ml，更优选1μg/ml；
所述底物优选为Furimazine或氟代腔肠素；其浓度优选为1μM～1000μM，更优选100μM。
本发明第七方面提供一种如本发明第一方面所述的突变体、第二方面所述的分离的核酸、第三方面所述的重组表达载体或者第四方面所述的转化体在催化底物发光中的应用。
本发明第八方面提供一种用于筛选刺虾荧光素酶Nluc的突变体的试剂盒，所述试剂盒中包括检测如本发明第一方面所述的突变体的试剂，检测如本发明第二方面所述的核酸的试剂，检测如本发明第三方面所述的重组表达载体的试剂，检测如本发明第四方面所述的转化体的试剂。
在本发明的具体实施过程中：编码刺虾荧光素酶Nluc的基因Nluc由北京华大六合进行针对大肠杆菌表达系统的密码子优化并合成。本发明中将优化后的Nluc碱基序列构建入原核表达载体pET28a(或真核表达载体pcDNA3.1)中，两侧酶切位点为BamHI和EcoRI，c端有多聚组氨酸(6×His)标签，得到重组质粒命名为pET28a-Nluc，图谱如图1所示。该质粒作为定向进化的模板，运用定点饱和突变的方法，通过单点或组合突变得到了刺虾荧光素酶Nluc的突变体库。将此突变体库转化入BL21(DE3)感受态细胞、CHO细胞或HEK293细胞中，并挑取单克隆进行刺虾荧光素酶Nluc突变体的表达纯化。该蛋白具备催化底物Furimazine(如图2所示)发光的能力，且发光较现有的刺虾荧光素酶Nluc更强，现有的刺虾荧光素酶Nluc蛋白序列如SEQ ID NO:2所示，碱基序列如SEQ ID NO:1所示，新型刺虾荧光素酶Nluc碱基序列如SEQ ID NO:3-12中任一个所示。
本发明技术方案带来的有益效果：
本发明提供一种新型刺虾荧光素酶Nluc，其具有催化底物Furimazine发光的功能，发光较现有的刺虾荧光素酶强，且仪器不能检测到催化高斯荧光素酶Gluc底物腔肠素的发光。其提升了现有的刺虾荧光素酶发光亮度，解决了现有合成的刺虾荧光素酶Nluc底物特异性宽泛的问题。有利于将来在生物监测等领域Nluc与Gluc两种荧光素酶的联合使用。
附图说明
图1为野生型Nanoluc荧光素酶质粒图谱。
图2为底物Furimazine(左)与底物氟代腔肠素(右)的结构图。
图3为定点突变PCR后1％琼脂糖胶图。
图4为刺虾荧光素酶纯化后SDS-PAGE图。
图5为新型刺虾荧光素酶突变体酶活性测试结果图。
图6为Nanoluc荧光素酶凝胶检测结果图。
具体实施方式
下述实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。以下结合实施例，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的实施例仅是为了解释本发明，并非为了限制本发明。实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
实施例1：Nanoluc荧光素酶突变体库的构建
以pET28a-Nluc为模板，其中Nluc的碱基序列为如SEQ ID NO:1所示，对现有的刺虾荧光素酶Nluc进行D19、V27、Q20、S28四个位点的定点或组合突变，包括但不仅限于以下位点/组合：D19N、D19P、D19E、D19G、D19S、Q20R、V27A、V27N、V27L、V27T、V27M、V27S、S28A、S28N、S28V、S28D、S28P、S28G、S28I、S28M、S28T。
定点突变体的制备使用KOD FX neo酶，并按照其说明书进行PCR反应体系的配制及PCR反应，以D19N突变体的制备为例，具体条件如下：
PCR反应体系：
表1
其中引物的序列如SEQ ID NO:13和14所示。
PCR反应条件：
表2
反应完成后跑1％琼脂糖胶(如图3所示)，向反应体系中加入0.5μL Dpn I酶消化掉模板。转化入DH5α中，涂布于含终浓度为50μg/mL卡那霉素抗性的平板，次日从平板上挑取单克隆，37℃过夜扩大培养后抽提质粒。测序结果证明序列已突变。
实施例2：Nanoluc荧光素酶突变体的表达和纯化
将质粒转入BL21(DE3)感受态细胞中，涂平板，从平板上挑取单菌落，37℃过夜培养，后转接新培养基，1:100稀释，培养至OD值为0.5-0.6，加入IPTG至终浓度1mM，16℃过夜诱导。
收集30ml菌体，加入2.7ml结合缓冲液(50mM Tris，pH 8.0，250mM NaCl)、300μL溶菌酶，冰上裂解30min，超声(2s on 3s off，60％功率)破碎10min，4℃、12000rpm离心30min分离上清(细胞裂解液)和沉淀。在手工柱(购自生工，型号为F506607-0001#亲和层析柱空柱)中加入750μL HisTrap FF填料，用3ml结合缓冲液冲洗平衡填料。然后加入3ml过滤后的细胞裂解液。用漂洗液(50mM Tris，pH 8.0，250mM NaCl,10mM咪唑)冲洗10次(3ml/次)，然后用洗脱液(50mM Tris，pH 8.0，250mM NaCl,300mM咪唑)500μl洗脱蛋白4-5次，收集洗脱后的蛋白。
将Ni柱洗脱后的蛋白用透析缓冲液(25mM Tris，pH 8.0，250mM NaCl)4℃过夜透析。12％SDS-PAGE检测洗脱得到的蛋白(如图4所示)。
实施例3：Nanoluc荧光素酶生物发光检测
用BCA定量试剂盒(Thermo ScientificTM PierceTM BCA Protein Assay Kit)精确测定蛋白的浓度，将荧光素酶用稀释液(50mM Tris-HCl pH 8.0，100mM NaCl，0.1％(v/v)Tween-20)稀释至1μg/ml，取10μL加入黑色96孔板。再加入用相同溶液稀释至100μM的底物Furimazine(购自百奥莱博)90μL，用酶标仪自发光模块读取发光强度。所得突变后相比于野生型(序列如SEQ ID NO:2所示，也称为序列2)的结果如图5和表3所示。
表3
实施例4：Nanoluc荧光素酶凝胶检测。
分别取4μg Nluc D19G、0.4μg Nluc D19G、4μg Gluc、0.4μg Gluc跑12％SDS-PAGE蛋白电泳。电泳后将胶于去离子水中浸泡10min，然后用25％异丙醇洗胶3min，共洗胶两次，再用去离子水洗胶3min，共洗两次。再用5ml荧光素酶用稀释液(50mM Tris-HCl pH 8.0，100mM NaCl，0.1％(v/v)Tween-20)浸泡胶3min。浸泡后加入100μL、5mM的Nanoluc荧光素酶底物氟代腔肠素(购自百奥莱博)或5mM的高斯荧光素酶的底物腔肠素(购自百奥莱博)，润洗1min。用化学发光凝胶成像仪下检测发光(见图6中的a和b)。检测后将蛋白胶用考马斯亮蓝染色检测蛋白(见图6中的c)，可见新型Nluc刺虾荧光素酶不能检测到高斯荧光素酶的底物腔肠素。
SEQUENCE LISTING
<110> 深圳华大生命科学研究院
<120> 一种刺虾荧光素酶Nluc的突变体及其应用
<130> P20017817C
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> Nluc碱基序列
<400> 1
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt gagcagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 2
<211> 169
<212> PRT
<213> Artificial Sequence
<220>
<223> Nluc蛋白序列
<400> 2
Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr
1 5 10 15
Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln
20 25 30
Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly
35 40 45
Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly
50 55 60
Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val
65 70 75 80
Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr
85 90 95
Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg
100 105 110
Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr
115 120 125
Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn
130 135 140
Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly
145 150 155 160
Trp Arg Leu Cys Glu Arg Ile Leu Ala
165
<210> 3
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> D19G突变体
<400> 3
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctgggacag 60
gtgctggaac agggcggcgt gagcagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 4
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> V27L突变体
<400> 4
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcct gagcagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 5
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> V27T突变体
<400> 5
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcac aagcagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 6
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> V27M突变体
<400> 6
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcat gagcagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 7
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> S28A突变体
<400> 7
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt ggcaagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 8
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> S28N突变体
<400> 8
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt gaacagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 9
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> S28M突变体
<400> 9
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt gatgagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 10
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> S28V突变体
<400> 10
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt ggtgagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 11
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> S28D突变体
<400> 11
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctggatcag 60
gtgctggaac agggcggcgt ggacagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 12
<211> 507
<212> DNA
<213> Artificial Sequence
<220>
<223> D19P/V27L/S28P突变体
<400> 12
tttaccctgg aagattttgt gggcgattgg cgccagaccg cgggttataa tctgccacag 60
gtgctggaac agggcggcct gccaagctta tttcaaaatc tgggcgtgag cgtgaccccg 120
attcagcgca ttgttctgag cggcgaaaat ggcctgaaaa ttgatattca tgtgattatc 180
ccgtacgagg gcctgagcgg cgatcagatg ggtcaaattg aaaaaatttt taaggtggtg 240
tatccggtgg acgatcatca ttttaaagtg atcctgcatt acggcaccct ggtgattgat 300
ggcgtgaccc cgaatatgat tgattatttt ggccgcccgt atgagggcat tgcggtgttt 360
gatggcaaaa aaattaccgt gaccggcacc ctgtggaatg gcaataaaat tattgatgag 420
cgcctgatca acccggatgg cagcttatta tttcgcgtga ccattaatgg cgtgaccggc 480
tggcgtctgt gcgaacgtat tctggcg 507
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物序列-1
<400> 13
gttataatct gaaccaggtg ctggaacagg gc 32
<210> 14
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 引物序列-2
<400> 14
ccagcacctg gttcagatta taacccgcgg tc 32
Claims (10)
1.一种刺虾荧光素酶Nluc的突变体，其特征在于，所述突变体与如SEQ ID NO:2所示的氨基酸序列相比在第19位、第20位、第27位以及第28位中的一位或多位发生突变。
2.如权利要求1所述的突变体，其特征在于，所述第19位的突变为D19N/P/E/G/S；所述第20位的突变为Q20R；所述第27位的突变为V27A/N/L/T/M/S；所述第28位的突变为S28A/N/V/D/P/G/I/M；
较佳地，所述突变体包含D19G、V27L/T/M、和/或S28A/N/M/V/D；
更佳地，所述突变体包含D19G、V27L、V27T、V27M、S28A、S28N、S28M、S28V、S28D，或同时包含D19P、V27L和S28P。
3.一种分离的核酸，其编码如权利要求1或2所述的突变体；
较佳地，所述核酸的碱基序列如SEQ ID NO:3-12中任一个所示。
4.一种重组表达载体，其包含如权利要求3所述的分离的核酸，所述重组表达载体的骨架优选pET28a或pcDNA3.1质粒。
5.一种转化体，其含有如权利要求3所述的分离的核酸，或如权利要求4所述的重组表达载体；
所述转化体构建时使用的宿主细胞优选为大肠杆菌或哺乳动物细胞；所述大肠杆菌优选为E.coliBL21(DE3)；所述哺乳动物细胞优选为CHO细胞或HEK293细胞。
6.一种制备如权利要求1或2所述的突变体的方法，其包括培养如权利要求5所述的转化体得发酵产物，并从发酵产物中获得所述突变体。
7.一种催化底物发光的方法，其包括以下步骤：利用如权利要求1或2所述的突变体进行底物的催化。
8.如权利要求7所述的方法，其特征在于，所述突变体的浓度为0.1μg/ml～10μg/ml，优选1μg/ml；
和/或，所述底物为Furimazine或氟代腔肠素；其浓度为1μM～1000μM，优选100μM。
9.如权利要求1或2所述的突变体、权利要求3所述的分离的核酸、权利要求4所述的重组表达载体或者权利要求5所述的转化体在催化底物发光中的应用。
10.一种用于筛选刺虾荧光素酶Nluc的突变体的试剂盒，其特征在于，所述试剂盒中包括检测如权利要求1或2所述的突变体的试剂，和/或检测如权利要求3所述的核酸的试剂，和/或检测如权利要求4所述的重组表达载体的试剂，和/或检测如权利要求5所述的转化体的试剂。
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