CN114807199A - 重组胱硫醚β-裂解酶的制备方法及其应用 - Google Patents
重组胱硫醚β-裂解酶的制备方法及其应用 Download PDFInfo
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- CN114807199A CN114807199A CN202210163462.XA CN202210163462A CN114807199A CN 114807199 A CN114807199 A CN 114807199A CN 202210163462 A CN202210163462 A CN 202210163462A CN 114807199 A CN114807199 A CN 114807199A
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- lyase
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- cystathionine beta
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Abstract
本发明涉及基因工程技术领域，尤其涉及重组胱硫醚β‑裂解酶的制备方法及其应用。本发明通过分子克隆技术扩增大肠杆菌胱硫醚β‑裂解酶的目的基因，并将其与质粒pET‑28a(+)连接，并转化至表达宿主E.coli BL21(DE3)中，并成功获得表达菌株Eoli BL21(DE3)(PET28a‑CBL)，经过IPTG诱导，可使CBL重组蛋白表达，经过His‑tag进行镍亲和层析获得纯度高活性好的CBL重组蛋白，并将其应用于HCY循环酶法试剂盒的开发。实验表明，本发明提供的制备方法可溶性表达量达到350mg/L，试剂盒的准确率、精密度皆良好。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及重组胱硫醚β-裂解酶的制备方法及其应用。
背景技术
CBL(胱硫醚β-裂解酶)是一种磷酸吡哆醛依赖的酶，是蛋氨酸生物合成途径中的关键酶，能够催化胱硫醚裂解产生同型半胱氨酸(Hcy)、丙酮酸和氨。血浆中Hcy的测定已经成为心脑血管等疾病的早期诊断常见方法。
目前，Hcy主要的检测方法之一是酶循环法，该法操作简便，检测速度快，准确度高。酶循环法的原理是通过胱硫醚β-合成酶(Cystathioninβ-synthetase， CBS)催化L-丝氨酸(L-Ser)和Hcy形成L-胱硫醚(L-Cystathionine，L-Cth)，而L-Cth又能在胱硫醚β-裂解酶(Cystathionineβ-lyase，CBL)的催化作用下，形成Hcy、丙酮酸和氨。因此，CBS和CBL的品质是影响酶循环法测定Hcy效果的关键。
现有技术中已有一些采用基因工程的手段，制备重组胱硫醚β-裂解酶的方案，但一方面的蛋白的表达量较低，且所得蛋白的纯度、活性有限，不能很好的用于HCY循环酶法试剂盒的制备。
发明内容
有鉴于此，本发明要解决的技术问题在于提供高表达量、高纯度、高活性的重组胱硫醚β-裂解酶的制备方法及其应用。
本发明提供的表达胱硫醚β-裂解酶的核酸序列如SEQ ID NO:1所示，其来自大肠杆菌。
本发明提供了胱硫醚β-裂解酶的表达载体，其以pET-28a(+)为骨架载体，其中含有SEQ ID NO:1所示序列的核酸。
在本发明所述的表达载体中SEQ ID NO:1所示序列的核酸的插入位点为BamHI和NotI。
本发明提供的表达载体表达SEQ ID NO:1所示序列后，形成的蛋白为融合蛋白，其在胱硫醚β-裂解酶的N端添加6×His标签，以及T7检测标记。
本发明还提供了一种重组大肠杆菌，其为转化本发明所述的质粒载体的大肠杆菌E.coliBL21(DE3)。
本发明以E.coliBL21作为表达的宿主细胞，在培养后获得的蛋白表达量较高、纯度较高，从而获得了良好的回收率。并且，在表达过程中，折叠正确，形成了正确的二级结构以及三级结构，从而维持了胱硫醚β-裂解酶的生物活性，使其能够用于制备HCY循环酶法检测试剂盒。
本发明还提供了胱硫醚β-裂解酶的制备方法，其包括：诱导培养本发明所述的重组大肠杆菌，诱导蛋白表达后，收集菌体经纯化获得胱硫醚β-裂解酶。
本发明中，所述诱导培养包括，扩大培养和诱导表达的步骤。
本发明中，所述诱导培养的培养基为TB培养基；所述诱导的诱导剂为 IPTG。其中所述扩大培养是指将构建获得重组大肠杆菌接种至TB培养基，于37℃、220rpm条件下培养至OD600值∽1。所述诱导表达的培养基为TB培养基，诱导剂的剂量为1mmol/L，37℃，220rpm诱导8h。
本发明中，所述TB培养基包括水和：11.8g/L胰蛋白胨、23.6g/L酵母提取物、9.4g/LK2HPO4、2.2g/LKH2PO4、4ml/L甘油。
在本发明中，所述纯化包括：
超声破碎菌体后，离心取上清液；所述上清液过镍琼脂糖凝胶柱，以清洗缓冲液清洗然后用洗脱缓冲液洗脱；
所述清洗缓冲液包括水和20mmol/L Tris-HCl，250mmol/L NaCl，50 mmol/LImidazole，100μmol/LPLP；
所述洗脱缓冲液包括水和20mmol/L Tris-HCl，250mmol/L NaCl，250 mmol/LImidazole，100μmol/LPLP。
本发明中，所述超声破碎前，以平衡缓冲液重悬菌体；所述重悬至重悬液中菌体浓度为0.05g/mL；所述平衡缓冲液包括水和20mmol/L Tris-HCl， 250mmol/LNaCl，25mmol/LImidazole，100μmol/LPLP。
在本发明中，所述超声破碎的条件包括：-4～4℃，25％功率，工作3s停 3s。具体的，所述超声破碎采用冰浴破碎。
本发明所述制备方法制得的胱硫醚β-裂解酶。
本发明还提供了HCY循环酶法检测试剂盒，其包括试剂A和试剂B；
所述试剂A包括水和500mmol/L L-Ser、70mmol/L NADH、2％BSA和 50mmol/LTris-HCl；
所述试剂B包括水和5U/mg CBS、60U/mg CBL、5U/μL LDH、2％BSA、 50mmol/LTris-HCl和100μmol/LPLP；
其中，CBL为本发明所述制备方法制得的胱硫醚β-裂解酶。
本发明还提供了HCY的循环酶检测方法，其以本发明提供的试剂盒进行检测。
本发明通过分子克隆技术扩增大肠杆菌胱硫醚β-裂解酶的目的基因，并将其与质粒pET-28a(+)连接，并转化至表达宿主E.coli BL21(DE3)中，并成功获得表达菌株EoliBL21(DE3)(PET28a-CBL)，经过IPTG诱导，可使 CBL重组蛋白表达，经过His-tag进行镍亲和层析获得纯度高活性好的CBL 重组蛋白，并将其应用于HCY循环酶法试剂盒的开发。实验表明，本发明提供的制备方法可溶性表达量达到350mg/L，试剂盒的准确率、精密度皆良好。
附图说明
图1示CBL纯化前后全过程；
图2示以本发明实施例1制备的重组CBL检测HCY，具有良好的线性关系。
具体实施方式
本发明提供了重组胱硫醚β-裂解酶的制备方法及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1重组CBL的表达和纯化
根据NCBI中提供的CBL基因序列(SEQ ID NO:1)，设计引物，以大肠杆菌基因为模板扩增出CBL基因片段，并将该基因片段插入到质粒pET-28a (+)的多克隆位点中间(BamHI和Not I之间)，将重组的质粒转化至表达宿主E.coli BL21(DE3)中，获得重组表达菌株E.coli BL21(DE3) (PET28a-CBL)。37℃，220rpm条件下在TB培养基中扩大培养该重组菌至生长期(OD600∽1)，在37℃，220rpm条件下1mM的IPTG培养8h诱导重组蛋白CBL(SEQ ID NO:2)表达。
收集诱导好的重组菌体，PBS缓冲液清洗3次后，使用平衡缓冲液(20mmol /LTris-HCl，250mmol/LNaCl，25mmol/LImidazole，100μmol/LPLP，pH7.9) 重悬菌体，菌液浓度大约为0.05g/ml，冰浴超声(25％功率，工作3s停3s) 至菌液变透明，13000rpm，4℃离心30min，取上清；将上清液加入到镍琼脂糖凝胶柱中进行亲和层析，然后用清洗缓冲液(20mmol/LTris-HCl，250mmol /LNaCl，50mmol/LImidazole，100μmol/LPLP，pH7.9)清洗非特异性蛋白，最后用洗脱缓冲液(20mmol/L Tris-HCl，250mmol/L NaCl，250mmol/L Imidazole，100μmol/LPLP，pH7.9)洗脱纯化后的重组CBL。纯化后的重组 CBL溶液加入5％甘油-20度冻存。
CBL纯化前后全过程如图1所示，重组CBL菌体的全细菌裂解液中，即通过His亲和柱之前CBL纯度只有60％左右(图1中的1泳道)，然后经过 His亲和柱亲和纯化，第一次亲和后流出液的CBL含量明显减少70％(图1 中的2泳道)，第二至五次亲和纯化后流出液中的CBL含量较全细裂解液减少约95％(图1中的3至6泳道)，最后用洗脱液将亲和柱上的CBL纯化蛋白洗脱下来，其回收率高达95％以上(图1中的7泳道)，纯化蛋白的可溶表达量为350mg/L(以LB培养基培养的可溶表达量为131.4mg/L，而将LB培养基更换成TB培养基，表达量提高至350mg/L)。从图1结果所示，自制CBL 纯化蛋白纯化量很高，另经检测，所得CBL的单位酶活为154.9U/mg。说明其可为后续同型半胱氨酸检测试剂盒的开发提供充足的的原材料来源。
实施例2HCY循环酶法试剂盒
使用高纯度和高活性的CBL(实施例1制得)和CBS重组蛋白配制同型半胱氨酸检测试剂盒(三酶法)，主要成分为试剂A(ReagentA):500mmol/L L-Ser、70mmol/LNADH、2％BSA、50mmol/LTris-HCl)和试剂B(Reagent B): 5U/mgCBS、60U/mg CBL、5U/μl LDH、2％BSA、50mmol/LTris-HCl、100 μmol/LPLP。
实施例3试剂盒检测效果
1、线性关系
以不含Hcy的溶液为空白对照，以市售重组CBL作为阳性对照，分别取 8.6μmol/L、20.5μmol/L、38.3μmol/L的Hcy标准品作为待测样品，通过 HCY循环酶法试剂盒进行循环酶法检测，根据340nm波长下吸光度的变化速率绘制标准曲线(图2)，结果表明，以本发明实施例1制备的重组CBL检测 HCY，具有良好的线性关系。
2、HCY试剂盒准确度
自制重组蛋白CBL用于HCY检测试剂盒，检测2个水平的临床血清样本，实际检测平均浓度与目标靶值的相对偏差<10％，证明准确度高。
表1.准确度检测结果
| 血清样本(mM) | 检测浓度(mM) | 平均浓度(mM) | 相对偏差 | ||
| 11.5 | 11.52 | 12.01 | 11.49 | 11.67 | 1.49％ |
| 28.08 | 29.12 | 30.47 | 29.4 | 29.67 | 5.65％ |
3、HCY试剂盒精密度
将Hcy低值和高值血清样本，分别用自制的HCY检测试剂盒平行测10 次，变异系数CV均在5％以内，证明自制的HCY检测试剂盒合格。
表2.精密度检测结果
| 质控点 | n | 平均浓度(mM) | 标准差 | 变异系数 |
| 低值 | 10 | 10.02 | 0.23 | 2.25％ |
| 高值 | 10 | 19.9 | 0.39 | 1.97％ |
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 三诺生物传感股份有限公司
<120> 重组胱硫醚β-裂解酶的制备方法及其应用
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Arg Asn Arg Ala Asn Gly Glu Leu Phe Tyr Gly Arg Arg Gly Thr Leu
85 90 95
Thr His Phe Ser Leu Gln Gln Ala Met Cys Glu Leu Glu Gly Gly Ala
100 105 110
Gly Cys Val Leu Phe Pro Cys Gly Ala Ala Ala Val Ala Asn Ser Ile
115 120 125
Leu Ala Phe Ile Glu Gln Gly Asp His Val Leu Met Thr Asn Thr Ala
130 135 140
Tyr Glu Pro Ser Gln Asp Phe Cys Ser Lys Ile Leu Ser Lys Leu Gly
145 150 155 160
Val Thr Thr Ser Trp Phe Asp Pro Leu Ile Gly Ala Asp Ile Val Lys
165 170 175
His Leu Gln Pro Asn Thr Lys Ile Val Phe Leu Glu Ser Pro Gly Ser
180 185 190
Ile Thr Met Glu Val His Asp Val Pro Ala Ile Val Ala Ala Val Arg
195 200 205
Ser Val Val Pro Asp Ala Ile Ile Met Ile Asp Asn Thr Trp Ala Ala
210 215 220
Gly Val Leu Phe Lys Ala Leu Asp Phe Gly Ile Asp Val Ser Ile Gln
225 230 235 240
Ala Ala Thr Lys Tyr Leu Val Gly His Ser Asp Ala Met Ile Gly Thr
245 250 255
Ala Val Cys Asn Ala Arg Cys Trp Glu Gln Leu Arg Glu Asn Ala Tyr
260 265 270
Leu Met Gly Gln Met Val Asp Ala Asp Thr Ala Tyr Ile Thr Ser Arg
275 280 285
Gly Leu Arg Thr Leu Gly Val Arg Leu Arg Gln His His Glu Ser Ser
290 295 300
Leu Lys Val Ala Glu Trp Leu Ala Glu His Pro Gln Val Ala Arg Val
305 310 315 320
Asn His Pro Ala Leu Pro Gly Ser Lys Gly His Glu Phe Trp Lys Arg
325 330 335
Asp Phe Thr Gly Ser Ser Gly Leu Phe Ser Phe Val Leu Lys Lys Lys
340 345 350
Leu Asn Asn Glu Glu Leu Ala Asn Tyr Leu Asp Asn Phe Ser Leu Phe
355 360 365
Ser Met Ala Tyr Ser Trp Gly Gly Tyr Glu Ser Leu Ile Leu Ala Asn
370 375 380
Gln Pro Glu His Ile Ala Ala Ile Arg Pro Gln Gly Glu Ile Asp Phe
385 390 395 400
Ser Gly Thr Leu Ile Arg Leu His Ile Gly Leu Glu Asp Val Asp Asp
405 410 415
Leu Ile Ala Asp Leu Asp Ala Gly Phe Ala Arg Ile Val
420 425
Claims (10)
1.胱硫醚β-裂解酶的表达载体，其特征在于，以pET-28a(+)为骨架载体，其中含有SEQID NO:1所示序列的核酸。
2.根据权利要求1所述的表达载体，其特征在于，SEQ ID NO:1所示序列的核酸的插入位点为BamHI和NotI。
3.重组大肠杆菌，其特征在于，为转化权利要求1或2所述的质粒载体的大肠杆菌E.coli BL21(DE3)。
4.胱硫醚β-裂解酶的制备方法，其特征在于，包括：诱导培养权利要求3所述的重组大肠杆菌，收集菌体经纯化获得胱硫醚β-裂解酶。
5.根据权利要求4所述的制备方法，其特征在于，所述诱导培养的培养基为TB培养基；所述诱导的诱导剂为IPTG，诱导剂的剂量为1mmol/L。
6.根据权利要求4所述的制备方法，其特征在于，所述纯化包括：
超声破碎菌体后，离心取上清液；所述上清液过镍琼脂糖凝胶柱，以清洗缓冲液清洗然后用洗脱缓冲液洗脱；
所述清洗缓冲液包括水和20mmol/L Tris-HCl，250mmol/L NaCl，50mmol/LImidazole，100μmol/L PLP；
所述洗脱缓冲液包括水和20mmol/L Tris-HCl，250mmol/L NaCl，250mmol/LImidazole，100μmol/L PLP。
7.根据权利要求6所述的制备方法，其特征在于，所述超声破碎前，以平衡缓冲液重悬菌体；所述重悬至重悬液中菌体浓度为0.05g/mL；所述平衡缓冲液包括水和20mmol/LTris-HCl，250mmol/L NaCl，25mmol/L Imidazole，100μmol/L PLP。
8.根据权利要求6或7所述的制备方法，其特征在于，所述超声破碎的条件包括：-4～4℃，25％功率，工作3s停3s。
9.权利要求1～8任一项所述制备方法制得的胱硫醚β-裂解酶。
10.HCY循环酶法检测试剂盒，其特征在于，包括试剂A和试剂B；
所述试剂A包括水和500mmol/L L-Ser、70mmol/L NADH、2％BSA和50mmol/L Tris-HCl；
所述试剂B包括水和5U/mg CBS、60U/mg CBL、5U/μL LDH、2％BSA、50mmol/LTris-HCl和100μmol/L PLP；
其中，CBL为权利要求1～8任一项所述制备方法制得的胱硫醚β-裂解酶。
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