CN116445453B - 一种高效热稳定的核糖核酸酶SiRe_0917及其编码基因和应用 - Google Patents
一种高效热稳定的核糖核酸酶SiRe_0917及其编码基因和应用 Download PDFInfo
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- CN116445453B CN116445453B CN202310191991.5A CN202310191991A CN116445453B CN 116445453 B CN116445453 B CN 116445453B CN 202310191991 A CN202310191991 A CN 202310191991A CN 116445453 B CN116445453 B CN 116445453B
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Abstract
本发明公开了一种高效热稳定的核糖核酸酶SiRe_0917及其编码基因和应用。本发明从嗜热古菌冰岛硫化叶菌REY15A中克隆得到核糖核酸酶基因SiRe_0918，其核苷酸序列如SEQ ID NO.1所示，其编码的氨基酸序列如SEQ ID NO.2所示。核糖核酸酶SiRe_0917具有非常高效且专一的水解RNA活性，对DNA完全没有非特异性切割。此外，该酶来源于嗜热古菌，经检测其热稳定性好，在22～90℃范围内都具有非常好的活性。因此，核糖核酸酶SiRe_0917是一种新的热稳定性核糖核酸酶，在常温与高温条件下都具有极高的RNA水解活性，符合临床、生产以及分子克隆等领域的需求。
Description
技术领域
本发明属于生物化工和生物技术领域，具体涉及一种高效热稳定的核糖核酸酶SiRe_0917及其编码基因和应用。
背景技术
核糖核酸酶(ribonuclease，RNase)指能水解RNA磷酸二酯键的酶，不同的RNase其专一性不同。目前常用的核糖核酸酶有RNaseA，RNase H以及RNase T1等。核糖核酸酶为具有球蛋白性状的一种蛋白质，溶于水、50％丙酮。此酶最适宜温度为60℃，85℃以上失去作用，最适宜的pH值为7.6。其中核糖核酸酶A(RNase A)：来源于牛胰脏，是一种内切核糖核酸酶，可特异攻击RNA上嘧啶残基的3'端，切割胞嘧啶或尿嘧啶与相邻核苷酸形成的磷酸二酯键，反应终产物是3'嘧啶核苷酸和末端带3'嘧啶核苷酸的寡核苷酸。无辅助因子及二价阳离子存在时，核糖核酸酶A的作用可以被胎盘RNA酶抑制剂(RNasin)或氧钒一核糖核苷复合物(vanadyl-ribonuclosidecomplex，VRC)所抑制。核糖核酸酶T1(RNase T1)：来源于米曲霉(Aspergillus orjzae)，它特异地作用于鸟嘌呤3'端磷酸，切割位点在鸟嘌呤的3'磷酸和相邻核苷酸的5'羟基之问的磷酸二酯键，反应终产物为3'鸟苷酸和末端带3'鸟苷酸的寡核苷酸片段。核糖核酸酶H(RNase H)：最早是从小牛胸腺组织中发现的，其编码基因已被克隆到大肠杆菌中。它能特异地降解DNA:RNA杂交双链中的RNA链，产生具有3'-OH和5'-磷酸末端的寡核苷酸和单核苷酸，它不能降解单链或双链的DNA或RNA。
目前核糖核酸酶在分子克隆，临床以及食品和药品等各个领域都有广泛应用。在分子克隆中，核糖核酸酶可以用在①从DNA：RNA杂交体或DNA制备物中去除RNA分子；②确定RNA或DNA中的单碱基突变的位置；③RNA检测用于RNA酶保护分析法(RNase protectionassay)中。此外，据报道核糖核酸酶能改变宿主细胞新陈代谢，抑制病毒合成，在体外能抑制流感病毒增殖，在鸡胚内能抑制痘苗、疱疹病毒形成。临床用核糖核酸酶每天肌注180毫克，对治疗流行性脑炎有益。但目前市面可用的核糖核酸酶种类有限，仍然对新功能的核糖核酸酶有较大的需求。因此，本发明旨在提供一种新型高效的核糖核酸酶的制备方法，从嗜热古菌冰岛硫化叶菌(Sulfolobus islandicus)REY15A中直接制备高效、耐热且稳定的核糖核酸酶。
发明内容
本发明的目的是提供一种高效热稳定的核糖核酸酶SiRe_0917及其编码基因和应用。
本发明的第一个目的是提供一种高效热稳定的核糖核酸酶SiRe_0917，其氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供一种编码核糖核酸酶SiRe_0917的编码基因SiRe_0917，优选，其核苷酸序列如SEQ ID NO.1所示。
本发明的第三个目的是提供一种含有编码基因SiRe_0917的重组载体。
本发明的第四个目的是提供一种核糖核酸酶SiRe_0917的制备方法，其特征在于，将纯化的SiRe_0917经磷酸二酯酶PDE水解去除核酸化修饰，然后再次纯化获得无核酸化修饰的核糖核酸酶SiRe_0917。
优选地，将重组载体pSeSD-SiRe_0917-0918-Nhis电转化进入冰岛硫化叶菌REY15A表达菌株进行蛋白诱导表达，用镍离子亲和层析柱进行蛋白纯化；纯化的SiRe_0917经磷酸二酯酶PDE 4℃过夜水解去除核酸化修饰，然后再次用镍离子亲和层析柱纯化获得无核酸化修饰的核糖核酸酶SiRe_0917。
本发明的第五个目的是提供所述的核糖核酸酶SiRe_0917在水解RNA中的应用。
优选地，所述的核糖核酸酶SiRe_0917浓度为75μg/mL
优选地，所述的水解温度为22-90℃。
优选地，所述的水解温度为60-70℃。
本发明的核糖核酸酶基因SiRe_0917来自嗜热古菌冰岛硫化叶菌REY15A。本发明通过分子克隆，活性验证等方法发现基因SiRe_0917编码高效的热稳定核糖核酸酶。在正常情况下，该酶被下游基因SiRe_0918编码的核酸专一酶所修饰沉默。经实验验证发现，核糖核酸酶基因SiRe_0917与下游SiRe_0918共同表达纯化时，对宿主生长无影响，且两者之间并无稳定的相互作用，因此可以纯化到大量的核酸化修饰的核糖核酸酶SiRe_0917。本专利提供的核糖核酸酶SiRe_0917具有非常高效且专一的水解RNA活性，对DNA完全没非特异性切割。此外，该酶来源于嗜热古菌，经检测其热稳定性好，且在22～90℃范围内都具有非常好的活性。因此，核糖核酸酶SiRe_0917是一种新的热稳定性核糖核酸酶，在常温与高温条件下都具有极高的RNA水解活性，符合临床、生产以及分子克隆等领域的需求。
嗜热古菌冰岛硫化叶菌(Sulfolobus islandicus)REY15A是模式生物，其公开于文献：Genome analyses of Icelandic strains of Sulfolobus islandicus,modelorganisms for genetic and virus-host interaction studies，本申请人也持有，并保证自申请日起20年内向公众公开发放。
附图说明
图1是制备无修饰的活性核糖核酸酶SiRe_0917的流程。
图2是核糖核酸酶SiRe_0917的SDS-PAGE电泳图。其中，Line为蛋白marker，泳道1为IPTG诱导后的含有pSeSD-SiRe_0917-0918-NHis的REY15A粗蛋白，泳道2为纯化后的SiRe_0917蛋白(存在核酸修饰)，泳道3为经过PDE处理后再次纯化的活性SiRe_0917蛋白。
图3是浓度对核糖核酸酶SiRe_0917(75μg/mL)切割RNA探针活性的影响。
图4是核糖核酸酶SiRe_0917(75μg/mL)切割单链DNA探针的能力检测。
图5是反应时间对核糖核酸酶SiRe_0917(75μg/mL)切割双链DNA探针活性的影响。
图6是反应时间对核糖核酸酶SiRe_0917(75μg/mL)切割RNA探针活性的影响。
图7是核糖核酸酶SiRe_0917水解细胞total总RNA中rRNA的能力检测。
图8是温度对核糖核酸酶SiRe_0917水解RNA底物能力的影响。
图9是不同温度下核糖核酸酶SiRe_0917水解RNA底物的效率。
具体实施方式
以下实施例是对本发明的进一步说明，而不是本发明的限制。
实施例1：核糖核酸酶SiRe_0917的克隆及载体构建
pSeSD质粒(公布于文献“A synthetic arabinose-inducible promoter confershigh levels of recombinant protein expression in hyperthermophilic archaeonSulfolobus islandicus”)为嗜热古菌冰岛硫化叶菌REY15A的高拷贝表达载体，常用于目的蛋白在胞内的表达纯化等。利用Tiangen试剂盒提取野生型冰岛硫化叶菌REY15A基因组DNA作为后续PCR克隆的模板；设计PCR扩增SiRe_0917-0918操纵子(SiRe_0917核苷酸序列如SEQ ID NO.1所示；SiRe_0918核苷酸序列如SEQ ID NO.3所示)的引物，引物序列如下：上游引物SiRe_0917-his-F(pSeSD)：5’CCATCATCACGAGTTCTTAAAAAATAATGC 3’；下游引物SiRe_0918-R(pSeSD)：5’ACGCGTCGACCTAAAATTCCTCGTAAACGTCTAT 3’。以SiRe_0917-his-F(pSeSD)和SiRe_0918-R(pSeSD)为引物，野生型冰岛硫化叶菌REY15A基因组DNA为模板，PCR扩增SiRe_0917-0918操纵子的编码区，PCR扩增条件为：先95℃5min；然后95℃30s，56℃30s，72℃30s，共30个循环；最后72℃10min。反应结束后，将PCR扩增产物进行1.0％琼脂糖凝胶电泳，回收并纯化约700bp的目的基因片段。利用BccI和SalI内切酶对回收片段进行双酶切处理，将酶切产物进行1.0％琼脂糖凝胶电泳，胶回收纯化。再将其与同样经BccI和SalI酶切处理的载体pSeSD用T4 DNA连接酶(TaKaRa公司)进行连接，将连接产物转入大肠杆菌BW25113感受态细胞，筛选阳性转化子，PCR验证并送测序公司测序，获得重组质粒pSeSD-SiRe_0917-0918-Nhis以及含有该重组质粒的目的菌株。
表1PCR反应体系
实施例2：核糖核酸酶SiRe_0917的高效表达
2.1冰岛硫化叶菌REY15A感受态细胞制备
1.在准备感受态细胞前一天，将2mL冰岛硫化叶菌REY15A起始培养物接种到100mLSCVU(U 10μg/mL)培养基中，78℃培养过夜；
SCVU：在1ml SCV(表2)中加入10μl/ml Uracil(2mg/ml stock)。
2.测定OD600在0.2之时停止培养(为便于操作OD600在0.2～0.3之间也可以)，常温下将培养物转移到2×50mL的离心管中。6000rpm室温下离心10min收集细胞。弃上清(吸掉所有的上清)。
3.每管加入15mL室温的蔗糖溶液(20mM)，轻柔地吸吹，重悬细胞。6000rpm室温下离10min，再重复两次。
4.用室温的蔗糖溶液(20mM)重悬细胞(勿涡旋)，使感受态细胞的OD600值为5-10。将感受态细胞在室温保存一周或-80℃保存。
2.2将目的重组质粒电转化进表达菌株REY15A
1.取实施例1中得到的重组质粒pSeSD-SiRe_0917-0918-Nhis 0.8μg(0.5～1μg)与50μL冰岛硫化叶菌REY15A感受态细胞混合，室温孵育30min。
2.将50μl感受态细胞-DNA悬浮液转入到相应的电转杯中。电转化条件是：1.2KV，600Ω，25μF(Bio-Rad Gene pulserⅡ)，时间常数12ms。
3.将转化完的细胞转移到含有800μL孵育培养基(见表1)的且预热到75℃的1.5mL离心管中。75℃孵育1小时(Without shaking)后取出样品，放置在室温下直至铺平板。
4.加热所需的0.4％Gelrite(Sigma，CAS号:71010-52-1)溶液和2×SCV，将两者按1:1的比例混合以得到1×SCV的顶层胶。将6mL 1×SCV顶层胶分别加入50μL电转后的细胞混匀加到预热的铺有底层胶SCV的平板上，室温固定30min。75℃培养7天，检查转化子；
底层胶：按表1-3中所示配制，1.4％的Gelriter溶液与等体积的2×SCV混合。
顶层胶：0.4％Gelrite与2×SCV等体积混合，两者终浓度分别为0.2％和1×SCV。
2.3蛋白诱导表达
将含有pSeSD-SiRe_0917-0918-Nhis的冰岛硫化叶菌REY15A表达菌株在SCV培养基中培养至OD600为0.5左右，加诱导剂D-阿拉伯糖至终浓度10mM，75℃培养48h。500mL菌液4500rpm，常温离心10min收集菌体，用30mLPBS缓冲液(50mM，pH 7.4)重悬菌体，超声400w，超5s，停5s，破碎10min，4℃条件下10000rpm离心30min并收集上清。
2.4蛋白纯化与SDS-PAGE电泳
用镍离子亲和层析柱纯化2.3中收集的上清，具体实施方案如下：使用10mM的咪唑洗脱5个柱体积，20mM咪唑洗脱20～30个柱体积，使用50mM的咪唑洗脱1个柱体积，最后使用2.5mL 300mM咪唑洗脱，收集最后的2.5mL洗脱液。用脱盐柱Sephadex G 25进行脱盐，具体操作方法参照GE公司的操作手册进行。将纯化的表达产物进行SDS-PAGE凝胶电泳，得到纯化的带有核酸修饰的核糖核酸酶SiRe_0917(图2)，纯化的蛋白大小约15kD，符合理论预期。
表1Mineral Salt Solution(pH 3.5)
(加入水与浓硫酸1:1稀释的溶液，调节pH至3。)
表2 SCV培养基
表3 Vitamin Mixture 100×
| 成分 | 含量/L |
| Niacin(烟酸) | 10mg |
| Biotin(维生素H) | 4mg |
| Pantothenate(泛酸盐) | 10mg |
| Lipoic acid(硫辛酸) | 10mg |
| Folic acid(叶酸) | 4mg |
| p-Aminobenzoic acid(氨基苯酸) | 10mg |
| Vitamin B1(thiamin,thiamine) | 10mg |
| Vitamin B2(Riboflavin) | 10mg |
| Vitamin B6(Pyridoxine) | 10mg |
| Vitamin B12(cobalamin) | 10mg |
(Tock solution in mg/L distilled H2O；过滤除菌。)
实施例3：活性状态的核糖核酸酶SiRe_0917的制备
按照活性状态的核糖核酸酶SiRe_0917的制备流程对实施例2.4纯化获得的核糖核酸酶SiRe_0917(SiRe_0917-修饰)进行处理(图1)。首先，将2.5mL脱盐后的蛋白SiRe_0917-修饰与100μL的磷酸二酯酶PDE(2UμL-1)混合，4℃的条件下过夜处理(12～16h)。处理完成后，将蛋白混合液重新用镍离子亲和层析柱进行二次纯化，纯化步骤参见实施例2.4。经过纯化，过滤掉核酸小分子以及磷酸二酯酶PDE，最后收集去除核酸修饰的核糖核酸酶SiRe_0917。将纯化的产物进行SDS-PAGE凝胶电泳，纯化的蛋白大小约15kD(图2)，符合预期。
实施例4：核糖核酸酶SiRe_0917的活性检测
为了实验结果的准确性，研究中对两种状态的核糖核酸酶SiRe_0917进行活性对比。首先利用BCA蛋白定量试剂盒(P0010,碧云天)对两种状态的SiRe_0917(带有核酸修饰的和去除核酸修饰)进行蛋白浓度测定，利用Tris-Hcl(20mM,PH 8.0)缓冲液调整两者的母液浓度均为0.3mg/mL，备用。本研究中选取了34种常用的核酸底物进行检测，包括单链RNA探针，总RNA，单链DNA探针以及双链DNA探针，探针序列见表2。
表2探针序列表
核糖核酸酶SiRe_0917水解底物的反应条件如下：①将两种SiRe_0917蛋白进行梯度稀释，分别稀释成1，5，20，75，300μg/mL，稀释缓冲液为50mM Tris-HCl，pH 7.5，10MMgCl2。②在10μL反应体系中加入1μL核糖核酸酶SiRe_0917，核酸底物的浓度均为50nM，混合体系60℃反应15min。③检测底物的水解情况：将10μL反应体系中加入2μL核酸loading，15％尿素胶，110V电压跑胶30min，FAM信号曝光检测(图3、图4)。
图3和图4结果表明存在核酸修饰的SiRe_0917无核糖核酸酶活性，对RNA或DNA探针均没有作用。而去除核酸修饰的SiRe_0917则具有高效的核糖核酸酶活性，能够专一性的切割RNA底物，且切割效率高，在极低的酶浓度下(75μg/mL)，15min就可以完成底物的水解。
实施例5：不同条件对核糖核酸酶SiRe_0917活性的影响
5.1反应时间对核糖核酸酶SiRe_0917活性的影响
按照实施例4的反应条件，使用50mM Tris-HCl、pH 7.5、10M MgCl2作为缓冲液，RNA作为底物，恒定蛋白浓度为75μg/mL，测定在1-15min内测定SiRe_0917完成底物水解所需的最短时间。尿素胶检测结果发现，在60℃条件下，RNA只需要4min就可以完成单链RNA探针的彻底水解(图5)；与之对比，DNA探针即使处理时间到15min依然没有任何降解条带出现(图6)。对于总RNA来说，75μg/mL浓度下加入底物就可以完成RNA主带的水解，超过4min后SiRe_0917对RNA底物几乎完全水解(图7)。
5.2温度对核糖核酸酶SiRe_0917活性的影响
使用50mM Tris-HCl、pH 7.5、10M MgCl2作为缓冲液，RNA作为底物，按照实施例4中的反应体系，恒定蛋白浓度为75μg/mL，反应时间为5min，在4-90℃范围内测定SiRe_0917的最适反应温度(图8)。
利用“Quantity One”软件对尿素胶中的底物条带以及降解条带进行定量分析发现，在4℃条件下SiRe_0917对RNA底物几乎无切割活性，但在22℃条件下可以达到40％左右。在60-70℃间是SiRe_0917的最适反应温度，对底物的切割效率达到60％，超过70℃后酶活存在一定的降低。但令人意外的是，即使在90℃的高温条件下，SiRe_0917对底物的水解效率也有40％左右。上述结果表明，核糖核酸酶SiRe_0917是一种新的热稳定性核糖核酸酶，在常温与高温条件下都具有极高的RNA水解活性(图9)。
SEQ ID NO.1(核糖核酸酶基因SiRe_0917核苷酸序列)
ATGGAGTTCTTAAAAAATAATGCACTAGATTTCTTAAATTATGCTAGGCTTCTACTACGTGATGGTAGATATAATTTGGCATTATTCTCATTAGAGCAAGCATTGCAGCTAGGTTTAAAATACTACATTTCTAAATTAACGGGTTCTTTCCCGAAAACGCATGACATAGTAGACTTATTAAAGAGAATTATAGAGCTTACGGGAAATAAAAAGCTTAAAGAAATTTTAAACGCAGAAATCTCTACCTTAGATTTGTTAAAACAAGCTTATATTGCCTCAAGATATTTACCTACTAATTATGATAAAGAGGCTGTTGAAAAAGCGTTAAACGTCGTTGAGGCGATATTAAATGAACTGGGAATATCTTAASEQID NO.2(核糖核酸酶SiRe_0917氨基酸序列)
MEFLKNNALDFLNYARLLLRDGRYNLALFSLEQALQLGLKYYISKLTGSFPKTHDIVD LLKRIIELTGNKKLKEILNAEISTLDLLKQAYIASRYLPTNYDKEAVEKALNVVEAILNELGIS SEQ ID NO.3(核糖核酸酶基因SiRe_0918核苷酸序列)
ATGAACTGGGAATATCTTAAAAAGAAGTGGGATGAGAGGAAAGAATTCCTTAATAACGCTAGGCGTTATGTGAAGTTAATTAAAGAGATCTGCGTTAAAAAGGTAGATTCCGAGTGTAGGGTAATACTTTTCGGCTCTGTTGCAAGGGGTAATTATAGAGATGACAGCGATGTAGATGTGCTAATAATTACTGATAAGGCTGAAAGCATATGGGATAAAGTAAACATTGAGGTCATTATCGAGAGGGAATTAAACATAGGAGACCCATTTGAATTCCATATAGTAACAAATAGTGAATATGAGAACTGGTATAAGAAATTCATAGACGTTTACGAGGAATTTTAG
Claims (4)
1.核糖核酸酶SiRe_0917在非疾病诊断和治疗中水解RNA中的应用，其特征在于，所述核糖核酸酶SiRe_0917的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的应用，其特征在于，所述的核糖核酸酶SiRe_0917浓度为75μg/mL。
3.根据权利要求1所述的应用，其特征在于，所述的水解温度为22-90℃。
4.根据权利要求3所述的应用，其特征在于，所述的水解温度为60-70℃。
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