WO2023142899A1 - 一种突变型RNase R及其制备方法和应用 - Google Patents

一种突变型RNase R及其制备方法和应用 Download PDF

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WO2023142899A1
WO2023142899A1 PCT/CN2022/144252 CN2022144252W WO2023142899A1 WO 2023142899 A1 WO2023142899 A1 WO 2023142899A1 CN 2022144252 W CN2022144252 W CN 2022144252W WO 2023142899 A1 WO2023142899 A1 WO 2023142899A1
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rnase
seq
mutant
amino acid
acid sequence
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刘明
蔡秋杰
张婉君
张茂雷
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广州吉赛生物科技股份有限公司
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  • the invention belongs to the field of molecular biology, in particular to a mutant RNase R and its preparation method and application.
  • RNase R (Ribonuclease R), with a molecular weight of about 91.4kDa, is a 3'-5' exoribonuclease derived from the RNR superfamily of Escherichia coli, which can gradually cut RNA into two nuclei from the 3'-5' direction nucleotides and trinucleotides.
  • RNase R can digest almost all linear RNA molecules, but it is not easy to digest circular RNA (circRNA), lariat RNA (lariat RNA), and RNA with double-stranded ends.
  • RNase R is often used in the enrichment and identification of circRNA.
  • High-throughput sequencing is the fastest way to discover circRNAs in large quantities, but traditional whole-transcriptome sequencing can only find circRNAs with high abundance, but is powerless for circRNAs with rare abundance.
  • Adding a step of RNase R digestion (RNase R+) to the whole transcriptome sequencing method can make circRNA relatively enriched, and finally make the junction reads of circRNA 5-10 times enriched compared with "RNase R-" samples , greatly increasing the amount of circRNA discovery.
  • circRNA has also been listed by major biopharmaceutical companies as RNA vaccine candidates with great research potential, and RNase R is expected to become one of the important raw materials for large-scale preparation and purification of circRNA.
  • RNase R is often used by researchers in related fields to identify circRNA and lariat RNA.
  • the reaction conditions of RNase R are generally 37°C, 10-30min, and the reaction system requires a lower NaCl concentration. When the concentration of NaCl in the reaction system>100mM, the activity of RNase R will be significantly inhibited. This limitation of conditions puts forward higher requirements on the purity of RNA samples, and at the same time increases the cost and loss of RNA preparation.
  • the present invention provides a mutant RNase R and its preparation method and application.
  • the mutant RNase R provided by the present invention improves the expression yield and salt tolerance of RNase R, and is conducive to meeting the requirements of diverse RNA samples.
  • a mutant RNase R the amino acid sequence of which is shown in SEQ ID NO.5.
  • the nucleotide sequence encoding the mutant RNase R amino acid sequence is as shown in SEQ ID NO.6.
  • the amino acid sequence is obtained by site-directed mutation of the amino acid sequence of the wild-type RNase R derived from E.coli; the amino acid sequence of the wild-type RNase R derived from E.coli is shown in SEQ ID NO.1.
  • nucleotide sequence encoding the amino acid sequence of the wild-type RNase R derived from E.coli is as shown in SEQ ID NO.2.
  • the site-directed mutation is obtained by truncating amino acid 601 to 608 of the amino acid sequence of the wild-type RNase R derived from E.coli.
  • the present invention also provides a kind of preparation method of described mutant RNase R, comprises the steps:
  • step S2 Transform the vector obtained in step S1 into the expression strain BL21 Escherichia coli cells to obtain the expression strain;
  • step S1 the process of constructing the vector described in step S1 is specifically as follows:
  • PCR technology was used to amplify the nucleotide sequence encoding mutant RNase R, using RNase R-F/RNase R_ ⁇ M8-R and RNase R_ ⁇ M8-F/RNase R-R as primers respectively to contain Escherichia coli
  • the plasmid of the RNase R_WT gene was used as a template to obtain a PCR product;
  • step (3) After the PCR product obtained in step (2) is separated by agarose gel electrophoresis, gel cutting and purification are performed to obtain two DNA fragments;
  • step (4) Place the mixed solution obtained in step (4) in a low-temperature ice box for ice bathing for 3 minutes, heat shock in a water bath at 42°C for 30 seconds, immediately ice-bath for 2 minutes, add 200 ⁇ L of LB culture solution, and spread it on a plate containing ampicillin; Cultivate overnight at 37°C, pick 3 single clones the next day, and verify by sequencing.
  • the RNase R-F primer sequence in step (1) is as shown in SEQ ID NO.9, and the primer sequence of RNase R_ ⁇ M8-R is as shown in SEQ ID NO.10;
  • the primer of described RNase R_ ⁇ M8-F The sequence is shown in SEQ ID NO.11;
  • the primer sequence of the RNase R-R is shown in SEQ ID NO.12.
  • the low temperature described in step (5) is -20°C.
  • the present invention also provides an application of the mutant RNase R in the identification of circRNA and lariat RNA.
  • nucleotide sequence encoding the above-mentioned mutant RNase R is not limited to the sequence shown in SEQ NO.
  • the nucleotide sequence shown in .5 is codon-optimized to obtain a nucleotide sequence that can encode the same amino acid sequence.
  • the present invention has the following technical advantages: compared with the wild-type RNase R, the protein expression of the mutant RNase R provided by the present invention is higher, and can tolerate 150mM NaCl.
  • Figure 1 is an amino acid comparison diagram of RNase R (WP_038432731, PDB ID: 5XGU) derived from Escherichia coli and RNase R (MK624989) derived from Psychrobacter sp.ANT206;
  • Fig. 2 is RNase R_WT-pET21a (+) plasmid map
  • Fig. 3 is SDS-PAGE protein electrophoresis figure detection RNase R expression and purification result figure;
  • Fig. 4 is BSA standard curve figure
  • Figure 5 is a diagram of the salt tolerance test results of RNase R_WT and RNase R_ ⁇ M8.
  • the wild-type RNase R (amino acid sequence shown in SEQ.NO.1) of Escherichia coli origin and the RNase R (amino acid sequence shown in SEQ.NO.3) of a salt-tolerant Psychrobacter sp.strain ANT206 origin were carried out amino acid Sequence alignment (the alignment result is shown in Figure 1). Determine the amino acid residues that may have an important impact on salt tolerance, and perform truncation mutations on them.
  • the mutated RNase R is named RNase R_ ⁇ M8, and its amino acid sequence is shown in SEQ.NO.5.
  • RNase R-F TAACTTTAAGAAGGAGATATACATATGCATCATCATCATCATTCACAAG (SEQ.ID NO.9);
  • RNase R_ ⁇ M8-R TGCCGGTTTCAGTGGTGTTGCCCTG (SEQ.NO.10);
  • RNaseR_ ⁇ M8-F GCAACACCACTGAAACCGGCATGCTGCAACTGGGTCAGCAC (SEQ.NO.11);
  • RNase R-R TCAGTGGTGGTGGTGGTGGTGCTCGAGTCACTCTGCCACTTTTTTCTTCG (SEQ.NO.12);
  • the PCR process uses KOD OneTM PCR Master Mix-Blue (TOYOBO, product number KMM-201) kit for reagent preparation, PCR amplification conditions: denaturation at 98°C for 10s, annealing at 58°C for 5s, extension at 68°C for 2s, and the number of cycles is 40 .
  • the gel was cut and purified.
  • the specific process was to cut off the gel containing the target DNA, and then use the agarose gel DNA small recovery kit (Magen, catalog number D2111-03) to carry out DNA purification.
  • the two DNA fragments obtained after purification and the pET21a(+) vector digested with NdeI/XhoI were passed through Hieff Plus One Step Cloning Kit (Yesean, Cat. No. 10911ES20) for homologous recombination reaction.
  • the process of digesting pET21a(+) vector with NdeI/XhoI is to prepare 30 ⁇ L of enzyme digestion system: 3 ⁇ g of pET21a(+) plasmid, 3 ⁇ L of 10X FastDigest Green Buffer, 1.5 ⁇ L of FastDigest NdeI, 1.5 ⁇ L of FastDigest XhoI, make up to 30 ⁇ L with H2O; After swirling and mixing, react at 37°C for 2h.
  • the cloned strain whose sequencing result matched with SEQ NO.6 was named RNase R_ ⁇ M8(Trans1-T1), inoculated into 500 ⁇ L LA culture medium, cultured on a shaker at 37°C for 5 hours, then added 500 ⁇ L 50% sterile glycerol, and mixed well Then store at -80°C.
  • Example 2 The expression strain RNase R_ ⁇ M8 (BL21(DE3)) obtained in Example 1 was inoculated into 5 mL of LA culture medium, and placed in a shaker at 37° C. at 200 rpm for overnight shaking culture.
  • Protein purification was performed using a protein purification system (Unique AutoPure, Inscinstech):
  • Ni-NTA column purification After flushing the system pipeline and Ni-NTA column (BBI, product number C600792, specification 1mL) with DEPC treated water, the column was equilibrated with buffer for equilibrium washing. Load the sample at a flow rate of 0.8mL/min, wash the impurity protein with the balance washing buffer, and finally use the elution buffer (50mM phosphate, 500mM NaCl, 500mM imidazole, 0.05% Tween 20, 10% Glycerol, pH 8.0) to elute and collect target protein.
  • BBI product number C600792, specification 1mL
  • Ultrafiltration tube concentration use ultrafiltration tubes (Millipore, UFC805024, 50K MWCO) and a refrigerated centrifuge to properly concentrate the target protein collected above to 2 mL.
  • Desalting column desalting After flushing the system pipes and desalting column (GE, product number 29048684, specification 5mL) with DEPC treated water, use glycerol-free 2 ⁇ storage buffer (100mM Tris-HCl (pH 7.5), 200mM NaCl, 0.2mM EDTA, 2mM DTT, 0.2% X-100) balance column. Draw the sample with a disposable syringe, inject it into the quantitative loop in the "manual sample loading" mode, and then pass through the column at a flow rate of 2mL/min, collect the sample when the protein peak appears, and stop collecting the sample when the salt peak appears.
  • glycerol-free 2 ⁇ storage buffer 100mM Tris-HCl (pH 7.5), 200mM NaCl, 0.2mM EDTA, 2mM DTT, 0.2% X-100
  • RNase R represents the cell lysate before RNase R_WT induction; 2) represents the cell lysate after RNase R_WT is induced by IPTG; 3) represents the RNase purified by Ni-NTA column R_WT protein; 4) represents the cell lysate before RNase R_ ⁇ M8 induction; 5) represents the cell lysate after RNase R_ ⁇ M8 was induced by IPTG; 6) represents the RNase R_ ⁇ M8 protein purified by Ni-NTA column.
  • the obtained RNase R was subjected to SDS-PAGE electrophoresis simultaneously with BSA of different qualities. After electrophoresis, stain with Coomassie Brilliant Blue G250, take pictures after decolorization, and use Quantity One software for grayscale analysis. Take the gray value as the Y axis, and take the BSA loading quality as the X axis to draw a standard curve (as shown in Figure 4). Calculate the concentration and yield of RNase R enzyme solution according to the BSA standard curve (as shown in Table 1). The resulting enzyme solution was diluted to 1 ⁇ g/ ⁇ L with Storage buffer and stored at -20°C.
  • Linear_RNA1/2-F 5'TAATACGACTCACTATAGGGAAAAAAGGAGGTTTTAGTCTAGGGAAAGTCATTCA 3'(SEQ NO.13);
  • Linear_RNA1-R 5'TTGAAAAAATCATGAGATTTTCTCTCTA 3' (SEQ NO.14);
  • Linear_RNA2-R 5'GGGAAAAAATCATGAGATTTTCTCCTTA 3' (SEQ NO.15);
  • Linear_RNA1/2-F (SEQ NO.13) and Linear_RNA1-R (SEQ NO.14) were used for PCR amplification, and the PCR products were separated by agarose electrophoresis and recovered by tapping Template DNA1
  • the PCR amplification process here also uses the KOD One TM PCR Master Mix-Blue-(TOYOBO, product number KMM-201) kit to prepare the system, the amplification conditions are denaturation at 98°C for 10s, annealing at 58°C for 5s, and 68°C Extend for 1 s, and the number of cycles is 40.
  • RNA synthesis and purification methods are as follows:
  • step (3) Transfer the product obtained in step (2) to an RNase-free 1.5mL centrifuge tube, add 1mL Trizol for RNA purification, and follow-up operations are the same as Trizol for extracting RNA from cells.
  • Embodiment 6RNase R activity and salt tolerance are measured
  • Wild-type RNase R (RNase R_WT) and mutant RNase R (RNase R_ ⁇ M8) basically do not digest RNA2 with a complete double-stranded structure at the end, but can digest RNA1 with a 3' protruding structure. Compared with the wild-type RNase R (RNase R_WT), the mutant RNase R (RNase R_ ⁇ M8) has stronger digestion ability and can tolerate the final concentration of 150mM NaCl.

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Abstract

属于分子生物学技术领域，具体提供一种突变型RNase R及其制备方法和应用。提供的突变型 RNase R被命名为RNase R △M8，其氨基酸序列如SEQ ID NO.5所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO.6所示。提供的突变RNase R_△M8 的制备过程包括载体构建、载体转化、蛋白诱导表达、细菌收集、蛋白纯化及活性测定等过程。本发明提供的突变型RNase R，提高了RNase R表达产量及耐盐性，有利于满足多样化的RNA样本要求。

Description

一种突变型RNase R及其制备方法和应用 技术领域
本发明属于分子生物学领域，具体涉及一种突变型RNase R及其制备方法和应用。
背景技术
RNase R(Ribonuclease R)的分子量约为91.4kDa，是一种来源于大肠杆菌RNR超家族的3’-5’核糖核酸外切酶，可从3’-5’方向将RNA逐步切割成二核苷酸和三核苷酸。RNase R能够消化几乎所有的线性RNA分子，但不易消化环状RNA(circRNA)、套索结构RNA(lariat RNA)、末端为双链的RNA。
目前RNase R常被用于circRNA的富集和鉴定研究。高通量测序是大批量发现circRNA最快捷的方法，但传统的全转录组测序只能发现丰度较高的circRNA，对丰度稀少的circRNA则无能为力。而在全转录组测序的方法上再增加一步RNase R消化(RNase R+)，则可以使circRNA相对富集，最终使circRNA的junction reads相对于“RNase R-”样本有5-10倍的富集，大大提升了circRNA的发现量。另外，随着RNA疫苗的兴起，circRNA也被各大生物医药公司列入为极具研究潜能的RNA疫苗候选分子，RNase R将有望成为circRNA规模化制备及纯化的重要原料之一。此外，RNase R也常被相关领域的研究者用于circRNA和lariat RNA的鉴定。
RNase R的反应条件一般为37℃，10-30min，且反应体系要求较低的NaCl浓度。当反应体系中NaCl的浓度>100mM时，RNase R的活性会明显受抑制。这种条件的限制对RNA样本纯度提出了更高的要求，同时增加了RNA的制备成本及损失量。
因此急需开发一种耐盐且蛋白表达产量高的RNase R突变体。
发明内容
针对现有技术普遍存在的缺陷，本发明提供了一种突变型RNase R及其制备方法和应用。本发明提供的突变型RNase R，提高了RNase R表达产量及耐盐性， 有利于满足多样化的RNA样本要求。
为了达到上述目的，本发明采用的技术方案为：
一种突变型RNase R，氨基酸序列如SEQ ID NO.5所示。
优选地，编码所述突变型RNase R氨基酸序列的核苷酸序列如SEQ ID NO.6所示。
优选地，所述氨基酸序列为E.coli来源的野生型RNase R的氨基酸序列定点突变所得；所述E.coli来源的野生型RNase R的氨基酸序列如SEQ ID NO.1所示。
优选地，编码所述E.coli来源的野生型RNase R的氨基酸序列的核苷酸序列如SEQ ID NO.2所示。
优选地，所述定点突变为将E.coli来源的野生型RNase R的氨基酸序列的601至608号氨基酸截断突变所得。
本发明还提供了一种所述的突变型RNase R的制备方法，包括如下步骤：
S1、构建含有编码所述突变型RNase R的核苷酸序列的载体；
S2、将步骤S1获得的载体转化至表达菌株BL21大肠杆菌细胞中，获得表达菌株；
S3、将步骤S2获得的表达菌株扩大培养并进行蛋白诱导表达；
S4、收集扩大培养后的表达菌株，并进行洗涤及裂解；
S5、进行蛋白纯化；
S6、进行蛋白活性测定。
优选地，步骤S1所述构建载体的过程具体如下：
(1)将大肠杆菌来源的野生型RNase R与一株耐盐的Psychrobacter sp.strain ANT206来源的RNase R进行氨基酸序列比对，确定对耐盐有重要影响的氨基酸残基，并对其进行截断突变，获得如SEQ ID NO.5所述的氨基酸序列；
(2)利用PCR技术对编码突变型RNase R的核苷酸序列进行扩增，分别以RNase R-F/RNase R_△M8-R和RNase R_△M8-F/RNase R-R为引物，以含有大肠杆菌来源的RNase R_WT基因的质粒为模板，获得PCR产物；
(3)将步骤(2)获得的PCR产物经琼脂糖凝胶电泳分离后，切胶纯化，得到两段DNA片段；
(4)将pET21a载体经NdeI/XhoI酶切后，与步骤(3)所得的DNA片段 进行同源重组反应，反应液与克隆菌株Trans1-T1Phage Resistant Chemically Competent Cell轻轻混匀，获得混合液；
(5)将步骤(4)获得的混合液置于低温冰盒中冰浴3min，42℃水浴热激30s，立即冰浴2min，加入200μL LB培养液，涂布在含有氨苄青霉素的平板上；37℃过夜培养，次日分别挑取3个单克隆，测序验证，即得。
优选地，步骤(1)中的RNase R-F引物序列如SEQ ID NO.9所示，RNase R_△M8-R的引物序列如SEQ ID NO.10所示；所述RNase R_△M8-F的引物序列如SEQ ID NO.11所示；所述RNase R-R的引物序列如SEQ ID NO.12所示。
优选地，步骤(5)中所述的低温为-20℃。
本发明还提供了一种所述突变型RNase R在circRNA和lariat RNA鉴定中的应用。
需要说明的是，由于同一氨基酸可能有多种不同的密码子来决定，所以编码上述突变型RNase R的核苷酸序列并不局限于SEQ NO.5所示序列，也可以是由与SEQ NO.5所示核苷酸序列进行密码子优化得到可编码相同氨基酸序列的核苷酸序列。
与现有技术相比，本发明具有如下技术优势：与野生型RNase R相比，本发明提供的突变型RNase R的蛋白表达量更高，可以耐受150mM的NaCl。
附图说明
图1为Escherichia coli来源的RNase R(WP_038432731,PDB ID:5XGU)与Psychrobacter sp.ANT206来源的RNase R(MK624989)氨基酸比对图；
图2为RNase R_WT-pET21a(+)质粒图谱；
图3为SDS-PAGE蛋白电泳图检测RNase R表达及纯化结果图；
图4为BSA标准曲线图；
图5为RNase R_WT和RNase R_△M8耐盐性检测结果图。
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术 方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者，按照本领域常规技术方法和仪器说明书内容进行操作；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1突变型RNase R表达载体的构建
将大肠杆菌来源的野生型RNase R(氨基酸序列如SEQ.NO.1所示)与一株耐盐的Psychrobacter sp.strain ANT206来源的RNase R(氨基酸序列如SEQ.NO.3所示)进行氨基酸序列比对(比对结果如图1所示)。确定可能对耐盐有重要影响的氨基酸残基，并对其进行截断突变，突变后的RNase R命名为RNase R_△M8，其氨基酸序列如SEQ.NO.5。
根据突变点进行引物设计，引物序列如下：
RNase R-F：TAACTTTAAGAAGGAGATATACATATGCATCATCATCATCATCATTCACAAG(SEQ.ID NO.9)；
RNase R_△M8-R：TGCCGGTTTCAGTGGTGTTGCCCTG(SEQ.NO.10)；
RNaseR_△M8-F：GCAACACCACTGAAACCGGCATGCTGCAACTGGGTCAGCAC(SEQ.NO.11)；
RNase R-R：TCAGTGGTGGTGGTGGTGGTGCTCGAGTCACTCTGCCACTTTTTTCTTCG(SEQ.NO.12)；
利用PCR对改造的RNase R_△M8基因(其基因序列如SEQ ID NO.6所示)进行扩增，分别以引物RNase R-F/RNase R_△M8-R和RNase R_△M8-F/RNase R-R以含有大肠杆菌来源的RNase R_WT基因的质粒(如图2所示))为模板，分别扩增1843bp片段和665bp片段。其中，PCR过程采用KOD OneTM PCR Master Mix-Blue(TOYOBO，货号KMM-201)试剂盒进行试剂配制，PCR扩增条件：98℃变性10s，58℃退火5s，68℃延伸2s，循环数为40。
上述PCR产物经琼脂糖凝胶电泳分离后，切胶纯化，具体过程为将含有目标DNA的凝胶切下后，使用琼脂糖凝胶DNA小量回收试剂盒(Magen，货号 D2111-03)进行DNA纯化。纯化后得到的两段DNA片段与NdeI/XhoI酶切后的pET21a(+)载体通过Hieff
Figure PCTCN2022144252-appb-000001
Plus One Step Cloning Kit(Yesean，货号10911ES20)进行同源重组反应。其中，NdeI/XhoI酶切pET21a(+)载体的过程为配制酶切体系30μL：pET21a(+)质粒3μg、10X FastDigest Green Buffer 3μL、FastDigest NdeI 1.5μL、FastDigest XhoI 1.5μL、H2O补足至30μL；涡旋混匀后，37℃反应2h。
10μL的反应液与50μL的克隆菌株Trans1-T1Phage Resistant Chemically Competent Cell(全式金，货号CD501-02)轻轻混匀，低温冰盒中冰浴3min，42℃水浴热激30s，立即冰浴2min，加入200μL LB培养液，涂布在含有氨苄青霉素的平板上。37℃过夜培养，次日分别挑取3个单克隆，测序验证。
将测序结果与SEQ NO.6吻合的克隆菌株命名为RNase R_△M8(Trans1-T1)，接种至500μL LA培养液中，37℃摇床培养5h后加500μL 50％无菌的甘油，混匀后置于-80℃保存。
接种10μL上述甘油菌至5mL LA培养液中，37℃过夜振荡，次日使用HiPure Plasmid EF Mini Kit(Magen，货号P1111-03)抽提质粒，获得的质粒即为RNase R_△M8-pET21a(+)载体。将100ng的RNase R_△M8-pET21a(+)质粒热激法转化大肠杆菌BL21(DE3)，即获得蛋白表达菌株RNase R_△M8(BL21(DE3))，表达菌株以同样的方法加甘油保存。
实施例2蛋白表达
将实施例1获得的表达菌株RNase R_△M8(BL21(DE3))接种至5mL LA培养液中，置于37℃，200rpm摇床中过夜震荡培养。
次日，将5mL过夜培养物接种于新的500mL LA培养液中，37℃，200rpm摇床培养3h(OD值约0.5)，加入500μL IPTG(1M)于培养液中(终浓度1mM)，37℃，200rpm摇床继续培养3h。10000g离心5min收集菌体，用5mL的无菌PBS洗涤一次。
实施例3蛋白纯化
向实施例2中收集到的菌体中加入40mL平衡清洗buffer(50mM磷酸盐， 500mM NaCl，20mM imidazole，0.05％Tween 20，10％Glycerol，pH 8.0)，涡旋至菌体充分重悬，将离心管固定在冰水浴中，超声探头插入液面1-2cm液面下，超声至菌液澄清透明(75％功率，超声4s停歇6s，总时间10min)，18000g，4℃离心60min，将上清液(即RNase R_△M8蛋白裂解液)转移至新的离心管。
利用蛋白纯化系统(Unique AutoPure，Inscinstech)进行蛋白提纯：
Ni-NTA柱纯化：以DEPC treated water冲洗系统管道和Ni-NTA柱(BBI，货号C600792，规格1mL)后，以平衡清洗buffer平衡柱子。以0.8mL/min的流速上样，以平衡清洗buffer清洗杂蛋白，最后用洗脱buffer(50mM磷酸盐，500mM NaCl，500mM imidazole，0.05％Tween 20，10％Glycerol，pH 8.0)洗脱并收集目标蛋白。
超滤管浓缩：使用超滤管(Millipore,UFC805024，50K MWCO)和冷冻离心机将上述收集到的目标蛋白进行适当浓缩，至2mL。
脱盐柱脱盐：以DEPC treated water冲洗系统管道和脱盐柱(GE，货号29048684，规格5mL)后，以无甘油的2×storage buffer(100mM Tris-HCl(pH 7.5)，200mM NaCl，0.2mM EDTA，2mM DTT，0.2％
Figure PCTCN2022144252-appb-000002
X-100)平衡柱子。用一次性注射器吸取样品，在“手动上样”模式下注入定量环中，之后以2mL/min的流速过柱，当蛋白峰出现时收样，盐峰出现时停止收样。
将脱盐后的酶液加入等体积的甘油，轻轻颠倒混匀后，短暂离心后置于-20℃冰箱中保存。具体的RNase R表达及纯化情况见图3，其中，1)代表RNase R_WT诱导前细胞裂解液；2)代表RNase R_WT经过IPTG诱导后细胞裂解液；3)代表经过Ni-NTA柱纯化后的RNase R_WT蛋白；4)代表RNase R_△M8诱导前细胞裂解液；5)RNase R_△M8经过IPTG诱导后细胞裂解液；6)代表经过Ni-NTA柱纯化后的RNase R_△M8蛋白。
实施例4蛋白定量
将获得的RNase R与不同质量的BSA同时进行SDS-PAGE电泳。电泳结束后采用考马斯亮蓝G250染色，脱色后拍照，采用Quantity One软件进行灰度分析。以灰度值为Y轴，以BSA上样质量为X轴，作出标准曲线(如图4所示)。根据BSA标准曲线计算RNase R酶液的浓度及产量(如表1所示)。将所得的 酶液用Storage buffer稀释成1μg/μL，置于-20℃保存。
表1野生型RNase R与突变型RNase R的产量对比
Enzyme Yield(μg)
RNase R_WT 2020
RNase R_△M8 2735
实施例5反应底物的制备
1)利用基因合成的方法，合成PCR所需的引物，其序列如下：
Linear_RNA1/2-F：5'TAATACGACTCACTATAGGGAAAAAAGGAGGTTTTAGTCTAGGGAAAGTCATTCA 3'(SEQ NO.13)；
Linear_RNA1-R：5'TTGAAAAAATCATGAGATTTTCTCTCTTA 3'(SEQ NO.14)；
Linear_RNA2-R：5'GGGAAAAAATCATGAGATTTTCTCTCTTA 3'(SEQ NO.15)；
2)利用PCR合成DNA模板。
以含有circ-ACE2RNA序列的质粒为模板，采用Linear_RNA1/2-F(SEQ NO.13)和Linear_RNA1-R(SEQ NO.14)进行PCR扩增，PCR产物经过琼脂糖电泳分离后，割胶回收获得模板DNA1，此处的PCR扩增过程同样采用KOD One TM PCR Master Mix-Blue-(TOYOBO，货号KMM-201)试剂盒配制体系，扩增条件为98℃变性10s，58℃退火5s，68℃延伸1s，循环数为40。
以含有circ-ACE2RNA序列的质粒为模板，利用采用Linear_RNA1/2-F(SEQ NO.13)和Linear_RNA2-R(SEQ NO.15)进行PCR扩增(具体过程同DNA1)，PCR产物经过琼脂糖电泳分离后，割胶回收获得模板DNA2。
1)利用T7 RNA聚合酶体外合成线性RNA
以DNA1为模板，利用TranscriptAid T7High Yield Transcription Kit(Thermo Scientific，货号K0441)体外转录合成两末端互补配对但3’端有2个碱基突出的Linear_RNA1(SEQ NO.7)，作为RNase R特异性消化反应中的目标RNA。
以DNA2为模板，利用TranscriptAid T7High Yield Transcription Kit(Thermo  Scientific，货号K0441)体外转录合成两末端完全互补配对的Linear_RNA2(SEQ NO.8)，作为RNase R特异性消化反应中的对照RNA。
上述RNA合成及纯化的方法具体如下：
(1)体外转录合成RNA。以DNA1和DNA2为模板，分别合成Linear_RNA1和Linear_RNA2。按照表2所示，按顺序配制反应体系，轻轻混匀后37℃反应2h。
表2体外转录的反应体系
组分 用量
DEPC treated water 补足至20μL
5X TranscriptAid Reaction Buffer 4μL
ATP,Tris buffered 100mM* 2μL
UTP,Tris buffered 100mM* 2μL
GTP,Tris buffered 100mM* 2μL
CTP,Tris buffered 100mM* 2μL
DNA1或DNA2 1μg
TranscriptAid Enzyme Mix 2μL
(2)上述反应结束后，往20μL体系中添加2μL的DNase I(RNase-free,1U/μLDNA)，混匀后37℃反应15min，消化DNA模板。
(3)将步骤(2)所得的产物转移至一个RNase free的1.5mL离心管，加入1mL的Trizol进行RNA纯化，后续操作同Trizol抽提细胞中的RNA。
(4)获得的RNA加适量DEPC treated water溶解，测定浓度后置于-80℃冰箱中保存。
实施例6RNase R活性及耐盐性测定
1)按照表3所示，配制不同浓度NaCl的10×reaction buffer
表3不同浓度NaCl的10×reaction buffer
Figure PCTCN2022144252-appb-000003
Figure PCTCN2022144252-appb-000004
2)按照表4所示，在20μL体系中，以3μg的Linear_RNA1或Linear_RNA2为底物，分别加入不同NaCl浓度的反应buffer，最后加入突变型RNase R(RNase R_△M8)，混匀后于37℃反应15min，70℃热失活10min，插入冰盒中。上述试验以野生型RNase R(RNase R_WT)为对照。分别取3μL反应液加入3μL 2×RNA Loading Dye(NEB，货号B0363A)进行1.5％的琼脂糖凝胶电泳。结果如图5所示，野生型RNase R(RNase R_WT)和突变型RNase R(RNase R_△M8)对末端完全双链结构的RNA2基本不消化，可以消化3’有突出结构的RNA1。对比野生型RNase R(RNase R_WT)，突变型RNase R(RNase R_△M8)的消化能力更强且能耐受终浓度为150mM的NaCl。
表4 RNase R反应体系
组分 用量
DEPC treated water 补足至20μL
RNA 3μg
10×buf1/2/3/4/5/6 2μL
RNase R(1μg/μL) 0.5μL
需要说明的是，尽管已经对上述各实施例进行了描述，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例做出另外的变更和修改，所以以上所述仅为本发明的实施例，并非因此限制本发明的专利保护范围，凡是利用 本发明说明书所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围之内。

Claims (10)

  1. 一种突变型RNase R，其特征在于，氨基酸序列如SEQ ID NO.5所示。
  2. 如权利要求1所述的突变型RNase R，其特征在于，编码所述突变型RNase R氨基酸序列的核苷酸序列如SEQ ID NO.6所示。
  3. 如权利要求1所述的突变型RNase R，其特征在于，所述氨基酸序列为E.coli来源的野生型RNase R的氨基酸序列定点突变所得；所述E.coli来源的野生型RNase R的氨基酸序列如SEQ ID NO.1所示。
  4. 如权利要求3所述的突变型RNase R，其特征在于，编码所述E.coli来源的野生型RNase R的氨基酸序列的核苷酸序列如SEQ ID NO.2所示。
  5. 如权利要求3所述的突变型RNase R，其特征在于，所述定点突变为将E.coli来源的野生型RNase R的氨基酸序列的601至608号氨基酸截断突变所得。
  6. 一种如权利要求1-5任一项所述的突变型RNase R的制备方法，其特征在于，包括如下步骤：
    S1、构建含有编码所述突变型RNase R的核苷酸序列的载体；
    S2、将步骤S1获得的载体转化至表达菌株BL21大肠杆菌细胞中，获得表达菌株；
    S3、将步骤S2获得的表达菌株扩大培养并进行蛋白诱导表达；
    S4、收集扩大培养后的表达菌株，并进行洗涤及裂解；
    S5、进行蛋白纯化；
    S6、进行蛋白活性测定。
  7. 如权利要求6所述的制备方法，其特征在于，步骤S1所述构建载体的过程具体如下：
    (1)将大肠杆菌来源的野生型RNase R与一株耐盐的Psychrobacter sp.strain ANT206来源的RNase R进行氨基酸序列比对，确定对耐盐有重要影响的氨基酸残基，并对其进行截断突变，获得如SEQ ID NO.5所述的氨基酸序列；
    (2)利用PCR技术对编码突变型RNase R的核苷酸序列进行扩增，分别以RNase R-F/RNase R_△M8-R和RNase R_△M8-F/RNase R-R为引物，以含有大肠杆菌来源的RNase R_WT基因的质粒为模板，获得PCR产物；
    (3)将步骤(2)获得的PCR产物经琼脂糖凝胶电泳分离后，切胶纯化，得到两段DNA片段；
    (4)将pET21a载体经NdeI/XhoI酶切后，与步骤(3)所得的DNA片段进行同源重组反应，反应液与克隆菌株Trans1-T1 Phage Resistant Chemically Competent Cell轻轻混匀，获得混合液；
    (5)将步骤(4)获得的混合液置于低温冰盒中冰浴3min，42℃水浴热激30s，立即冰浴2min，加入200μL LB培养液，涂布在含有氨苄青霉素的平板上；37℃过夜培养，次日分别挑取3个单克隆，测序验证，即得。
  8. 如权利要求7所述的制备方法，其特征在于，步骤(2)中的RNase R-F引物序列如SEQ ID NO.9所示，RNase R_△M8-R的引物序列如SEQ ID NO.10所示；所述RNase R_△M8-F的引物序列如SEQ ID NO.11所示；所述RNase R-R的引物序列如SEQ ID NO.12所示。
  9. 如权利要求7所述的制备方法，其特征在于，步骤(5)中所述的低温为-20℃。
  10. 一种如权利要求1所述的突变型RNase R在circRNA和lariat RNA鉴定中的应用。
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