CN117568322A - 一种产量高、稳定性好的重组肌酐酶及其制备方法和应用 - Google Patents
一种产量高、稳定性好的重组肌酐酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种产量高、稳定性好的重组肌酐酶及其制备方法和应用,重组肌酐酶,其氨基酸序列如SEQ ID NO:1所示,其核苷酸序列如SEQ ID NO:3所示。其制备方法如下:以SEQ ID NO:3为模板,以SEQ ID NO:4和5为引物扩增核苷酸序列,S1:将扩增的核苷酸序列连接到质粒载体,形成重组质粒;S2:将重组质粒转化入大肠杆菌感受态细胞,进行重组蛋白表达;S3:通过添加IPTG诱导重组蛋白表达;S4:通过高温孵育除杂、Ni离子亲和层析等方法分离纯化重组蛋白。所述重组肌酐酶具有产量高、热稳定性好、纯度高、酶活高的特点。由该抗原制备的试剂盒,能快速定量检测人体血清中肌酐酶含量,准确度高、精密度高、表达高效,操作简单。
Description
技术领域
本发明涉及体外诊断技术领域和基因工程技术领域,特别涉及一种产量高、稳定性好的重组肌酐酶及其制备方法和应用。
背景技术
肌酐是人体肌肉中的磷酸肌酸通过自发的且不可逆转的过程形成的,在肾脏功能正常情况下,肌酐在血液中的浓度维持在一个较低的水平,而肾脏功能不全时,会导致肌酐在血清中的积累,血清中肌酐含量是反映肾脏功能的重要指标。对于肌酐的检测,应用最为广泛的是碱性苦味酸法,但存在的着反应特异性差与灵敏度有限的问题,目前正逐渐被酶法(肌酐酶酶偶联肌氨酸氧化酶法)检测所替代,其中肌酐酶、肌酸酶和肌氨酸氧化酶是三个关键性的酶,肌酐酶可逆地水解肌酐生成肌酸,肌酸酶催化肌酸水解产生肌氨酸和尿素,肌氨酸在肌氨酸氧化酶作用下水解生成甲醛、甘氨酸和H2O2,偶联Trinder氏反应,通过比色测定,计算出样品中肌酐的含量。酶法具有抗干扰能力强、特异性好、线性范围宽、灵敏度高等特点,商品化的试剂盒大多采用肌酐酶偶联肌氨酸氧化酶法,在临床上得到了越来越多的使用。
作为酶法检测肌酐的关键酶,肌酐酶(Creatininase,CRN)催化肌酐水解为肌酸的可逆反应。在自然环境中,恶臭假单胞菌(Pseudomonas putida)、产脲节杆菌(Arthrobacter ureafaciens)、烟草节杆菌(Arthrobacter nicotianae)等微生物均可产生一定的肌酐酶。但野生菌株存在着肌酐酶产量低、工业化放大困难的问题,这很大程度上导致肌酐酶生产成本过高,如何进一步实现肌酐酶的高效表达对于促进其在肌酐酶法检测中的应用尤为重要。
文献中已有报道分别实现了假单胞菌(Pseudomonas sp.)来源和Pseudomonasputida来源的肌酐酶在大肠杆菌(Escherichia coli)的表达,但表达的肌酐酶比酶活不高,且存在纯化方法复杂、酶蛋白回收率低等问题。
因此,现有技术有待进一步改进。
发明内容
针对上述问题,本发明提供了一种产量高、稳定性好的重组肌酐酶及其制备方法和应用,所述重组肌酐酶在肌酐酶氨基酸N端添加一段多肽,该多肽片段有助于提高肌酐酶在大肠杆菌中的表达量,同时也有助于提高重组肌酐酶的热稳定性,本发明所得的重组肌酐酶具有产量高、纯度高、酶活高、热稳定性好的特点,能够克服现有技术中表达的肌酐酶比酶活不高,且存在纯化方法复杂、酶蛋白回收率低等问题。另外本发明的重组肌酐酶可借助现有的原核表达体系或真核表达体系进行工业化生产,极大提高其生产效率、批间差及产量。
具体的,本申请提供的技术方案如下:
第一方面,本申请提供一种重组肌酐酶,其氨基酸序列如SEQ ID NO:1所示,优选的,该序列中包含多肽片段,其氨基酸序列如SEQ ID NO:2所示。
第二方面,本申请还提供一种上述的重组肌酐酶的编码基因,其核苷酸序列如SEQID NO:3所示。该序列是基于大肠杆菌表达体系进行密码子优化后的序列,不含有大肠杆菌的稀有密码子,适合在大肠杆菌表达系统中进行表达。
其他情况下,为了将上述氨基酸序列如SEQ ID NO:1所示的重组肌酐酶在其他原核或真核体系中进行表达,可基于本领域常规技术手段对其基因序列进行对应的优化,从而得到对应的编码序列。
因此,本申请保护的重组肌酐酶不限于编码基因序列如SEQ ID NO:3所示的序列。
第三方面,本申请还提供一种重组表达载体,由表达载体和前述的重组肌酐酶的编码基因添加酶切位点重组而成。
优选地,所述表达载体为pET系列表达载体,如pET-32a质粒、pET-28a或者pET-41a等。
进一步的,以第二方面所述的核苷酸序列SEQ ID NO:3为模板,以SEQ ID NO:4,SEQ ID NO:5为引物,在N,C末端引入Nco I,Xho I酶切位点,通过酶切连接将目的基因连接到原核表达载体pET28a中,制备得到重组表达载体pET28a-rCRN。
其他情况下,基于本领域常规技术手段对其基因序列进行对应的优化,从而得到对应的编码序列,表达载体可采用适合转化其他原核或真核表达系统的表达载体。
第四方面,本申请还提供一种重组工程菌株,是向宿主菌种转化如第三方面所述的重组表达载体制备而成。
优选地,所述宿主菌为大肠杆菌,如大肠杆菌BL21(DE3)、大肠杆菌Rosseta或者其他大肠杆菌突变表达菌株等。
其他情况下,基于本领域常规技术手段对其基因序列进行对应的优化,从而得到对应的编码序列,表达载体采用适合转化其他原核或真核表达系统的表达载体,宿主菌可采用与其匹配的大肠杆菌Rosseta或者其他大肠杆菌突变表达菌株,毕赤酵母等。
第五方面,本申请还提供一种前述的重组肌酐酶的制备方法,包括以下步骤:以合成基因SEQ ID NO.3为模板,以SEQ ID NO.4、SEQ ID NO.5为引物扩增所述核苷酸序列,
S1:将扩增的核苷酸序列连接到质粒载体pET28a中,形成重组质粒;
S2:将步骤1中的重组质粒转化入大肠杆菌感受态细胞,进行重组蛋白表达;
S3:通过添加IPTG诱导重组蛋白表达;
S4:通过高温孵育除杂、Ni离子亲和层析等方法分离纯化重组蛋白。
第六方面,本申请还提供一种肌酐酶检测试剂盒,所述试剂盒中所用肌酐酶为权利要求1所述的重组肌酐酶。
进一步的,所述肌酐酶试剂盒由试剂R1和试剂R2两部分组成:
试剂R1:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20-50mmol/L
N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺0.4-1mmol/L
肌酸酶5-10ku/L
肌氨酸氧化酶2-4ku/L
抗坏血酸氧化酶0.5-1ku/L
试剂R2:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20-50mmol/L
4-氨基安替比林2-5mmol/L
肌酐酶100-150ku/L
过氧化物酶2-5ku/L
优选的最佳配方:所述肌酐酶试剂盒由试剂R1和试剂R2两部分组成:
试剂R1:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20mmol/L
N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺0.4mmol/L
肌酸酶5ku/L
肌氨酸氧化酶2ku/L
抗坏血酸氧化酶0.5ku/L
试剂R2:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20mmol/L
4-氨基安替比林2mmol/L
重组肌酐酶100ku/L
过氧化物酶2ku/L
上述肌酐酶检测试剂盒可应用于检测人体血清中肌酐含量。
本发明具有如下有益效果:
1、本发明提供的重组肌酐酶,首次创造性的采用了多肽片段与肌酐酶序列重组,所得重组肌酐酶具有产量高、热稳定性好、纯度高、酶活高的特点。
2、由该抗原制备的肌酐酶检测试剂盒,能快速定量检测人体血清中肌酐酶含量,准确度高、精密度高、线性范围宽,表达高效,操作简单,为人体血清肌酐酶测定提供了技术支持。
3、本申请提供的重组肌酐酶可通过大肠杆菌表达体系进行工业化生产,不仅产量高,且条件可控,极大的降低了重组肌酐酶的生产成本。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是本发明的一种实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1:本发明实施例2重组肌酐酶诱导表达全菌图;
其中,诱导前:未添加诱导剂全菌蛋白;诱导后:添加诱导剂诱导后全菌蛋白;M:蛋白质Marker
图2:本发明实施例2重组肌酐酶纯化后SDS-PAGE图;
图3:本发明实施例3的37℃孵育14d酶活变化;
图4:本发明实施例4线性质控检测结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例,以下实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。基于本发明中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。本发明所使用的实验方法如无特殊说明,均为常规方法。本发明所用的材料、试剂等,如无特殊说明,均可从商业途径得到。此外,本发明中所使用的其它术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。
本发明所制备或者所述的重组蛋白,即为重组肌酐酶。
实验材料及方法如下:
菌种与质粒:大肠杆菌表达菌株Rosetta(DE3),质粒PET28a,购自宝生物工程(大连)有限公司。
分子生物学试剂:
Nco I和XhoI双酶切等本发明所采用的各种分子生物学用酶均购自大连宝生物工程有限公司。
基因合成和DNA序列测序由上海生工公司完成。
基因克隆方法采用常规的DNA酶切、连接、电泳;质粒的提取纯化;蛋白的SDS-PAGE分析等一般的分子克隆方法按常规方法进行。其他试剂盒按说明书进行。
实施例1:重组质粒构建
(1)重组质粒构建
参考GenBank:1Q3K_A恶臭假单胞菌肌酐酶氨基酸序列,N端添加SEQ ID NO.2所示氨基酸序列,交合成公司,经密码子优化合成基因pUC19-rCRN;以pUC19-rCRN模板,扩增rCRN核酸序列。利用primer5引物设计软件,根据引物设计及酶切位点设计原则,结合pET28a载体酶切位点图谱,分别在上下游引物前添加酶切位点及保护碱基,设计引物如下:
P1:5’-catgccatggcgatgagcgataaaattattca-3’(Nco I)
P2:5’-ccctcgagagtcggcggaaattcttga-3’(Xho I)
引物P1为序列表SEQ ID NO.4所示,引物P2为序列表SEQ ID NO.5所示。
按表1添加反应体系,通过表2扩增程序进行扩增。
表1PCR反应体系
表2扩增程序
将PCR扩增产物回收后,通过Nco I和XhoI双酶切,之后回收酶切产物,连接到NcoI和Xho I酶切之后的载体pET28a上,经过转化DH5α、PCR鉴定重组质粒、得到阳性转化子,提取重组质粒,转化大肠杆菌表达菌株Rosetta(DE3),获取重组蛋白表达菌株:R/pET28a-rCRN。
同时制备对照用重组肌酐酶R/pET28a-CRN,对照重组肌酐酶氨基酸序列为去掉N端多肽序列,氨基酸序列如SEQ ID NO.6所示。
实施例2:重组肌酐酶表达及纯化
2.1诱导表达:
取10μl重组质粒表达菌种接种于含100ug/ml卡那霉素(Kan)(购自索莱宝)的5mlLB培养基中,37℃,200rpm振荡培养过夜(约16h),从中取200μl菌液加入到含有相同浓度Kan的60ml LB培养三基中,37℃,200rpm振荡培养过夜,将全部菌液接种至含有相同浓度Kan的1L LB培养基中,28℃,150rpm震荡培养2-2.5h后,测定OD600在0.5-0.8之间,加入终浓度为0.5mM的诱导剂IPTG(购自AMRESCO),继续以28℃,150rpm振荡培养4.5h后以6000rpm离心5min,收集菌沉淀;菌沉淀用50ml 20mM Tris-HCl(pH 8.0)重新悬起,6000rpm离心15min,收集沉淀。对照菌株按相同方式发酵,诱导表达产物经SDS-PAGE检测,结果如图1,两者分子量均在30kD附近,从表达结果看,本专利所发明重组肌酐酶表达量优于对照菌株。
2.2高压破碎:
菌沉淀用30ml 20mM Tris-HCl(pH 8.0)重新悬起,进行低温高压破碎,参数设置:压力值910-980bar,循环3次,温度设置4℃,实际运行温度不得高于13℃。破碎结束后,将破碎产物以9000rpm,4℃离心30min,收集上清。
2.3蛋白粗纯:
将高压破碎收集上清进行60℃温育30min处理,杂蛋白发生沉淀,经9000rpm 4℃离心30min,去除沉淀,收集上清。
2.4蛋白精纯:
取镍离子亲和层析柱:IMAC Bestarose-FF(购自博格隆)进行亲和层析,用10倍柱体积超纯水平衡层析柱;再用10倍柱体积平衡缓冲液(20mM Tris-HCl pH 8.0)平衡层析柱后加入蛋白样;上样结束后,用10倍柱体积平衡缓冲液(20mM Tris-HCl pH 8.0)洗去未结合的杂蛋白;再用10倍柱体积洗涤缓冲液(20mM Tris-HCl-50mM IM pH 8.0)洗去杂蛋白,最后用洗脱缓冲液(20mM Tris-HCl-250mM IM pH8.0)洗脱目的蛋白。
将收集的蛋白样品装入透析袋,放入装有透析液(20mM Tris-HCl pH8.0)的烧杯中,4℃透析过夜。将纯化后蛋白样品进行SDS-PAGE检测纯化情况,结果如图2,蛋白目的大小在30kD大小附近,纯度高,纯度≥95%。(对照菌株按相同方式纯化),重组肌酐酶纯化后浓度5.8mg/ml,共获得18ml,通过计算得纯化后产量104.4mg/L,对照菌株纯化后浓度为3.6mg/ml,共获得12ml,通过计算得纯化后产量在43.2mg/L,本专利中重组肌酐酶产量是对照菌株肌酐酶产量的2.4倍。
实施例3纯化后的重组肌酐酶热稳定性实验
将实施例2获得的重组肌酐酶溶液进行热稳定性实验。
将酶溶液各取1mg经75℃水浴处理30min然后在未处理和热处理后的酶溶液取0.2mg,加入1ml的酶活反应液,37℃孵育5min,加入0.1倍体积的100%TCA终止反应,在546nm下测定吸光度变化。吸光度变化见表3。热稳定性数据结果表明重组肌酐酶具有较强热稳定性,经75℃处理30min酶活依然可维持在75.8%,对照酶活维持在64.7%,较对照提高了17.16%。
将酶溶液各取2mg分别经37℃水浴处理14d,分别在热处理0d、3d、5d、7d、10d、12d、14d各取0.2mg,加入1ml的酶活反应液,37℃孵育5min,加入0.1倍体积的100%TCA终止反应,在546nm下测定吸光度变化。酶活变化见图3。图3数据表明重组肌酐酶(rCRN)37℃热处理7d酶活仍在98.6%,热处理14d酶活仍可维持在80%以上,对照酶(CRN)37℃热处理7d酶活维持在93.4%,热处理10d酶活可维持在80%以上,热处理14d酶活降至69.3%。
酶活反应液为:肌酐2mM、三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20mmol/L、N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺0.4mmol/L、肌酸酶5ku/L、肌氨酸氧化酶2ku/L、抗坏血酸氧化酶0.5ku/L、4-氨基安替比林2mmol/L、过氧化物酶2ku/L。
表3重组肌酐酶与对照75℃处理30min热稳定性数据
实施例4:重组肌酐酶在肌酐检测试剂盒中应用
将实施例2制备的重组肌酐酶溶液添加到肌酐检测试剂盒中,并通过生化仪分析试剂盒应用过程中的准确度,精密度,线性关系等指标;同时将试剂盒进行37℃热破坏7d,检测准确度、精密度、线性关系等指标。
通过数据分析重组肌酐酶应该用到试剂盒中以及进行37℃热破坏7d,检测准确度、精密度、线性关系等指标,均符合要求。
试剂盒中由R1和R2两组分组成:
试剂R1:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20mmol/L
N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺0.4mmol/L
肌酸酶5ku/L
肌氨酸氧化酶2ku/L
抗坏血酸氧化酶0.5ku/L
试剂R2:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20mmol/L
4-氨基安替比林2mmol/L
重组肌酐酶100ku/L
过氧化物酶2ku/L
(1)试剂盒准确性测定
试剂盒准确性测定是在试剂盒正常校准情况下,测定赋值质控,将测定值与质控进行比较,偏差(CV)±5%范围内视为准确度合格。
检测结果如表4,未破坏以及进行37℃7d热破坏后偏差均在±5%以内。
表3准确性测定结果
(2)试剂盒精密度测定
精密度测定是在试剂盒正常校准情况下,分别连续测定精密性质控品10次,经统计学分析后计算CV,CV≤5%视为精密度合格。
检测结果见表4,未进行热处理与37℃热处理7d检测精密性,偏差均在5%以内。
表4精密度测定结果
(3)试剂盒线性质控品检测
检测23.0μmol/L、344.2μmol/L、986.5μmol/L、1628.8μmol/L、1950.0μmol/L线性质控品,将测定值与理论值进行回归分析,R2≥0.995视为合格。
线性关系测定结果见图4,未处理R2=0.9999、37℃热处理7dR2=0.9999,均符合要求。
可以理解的是,对本领域普通技术人员来说,可以根据本发明的技术方案及本发明构思加以等同替换或改变,而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。
Claims (7)
1.一种重组肌酐酶,其特征在于,其氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的一种重组肌酐酶,其特征在于,其核苷酸序列如SEQ ID NO:3所示。
3.一种重组表达载体,其特征在于,由表达载体和如权利要求2所述的重组肌酐酶的编码基因添加酶切位点重组而成。
4.一种重组工程菌株,其特征在于,是向宿主菌种转化如权利要求3所述的重组表达载体制备而成。
5.一种如权利要求1所述的重组肌酐酶的制备方法,其特征在于,包括以下步骤:以SEQID NO:3为模板,以SEQ ID NO:4、SEQ ID NO:5为引物扩增所述核苷酸序列,
S1:将扩增的核苷酸序列连接到质粒载体中,形成重组质粒;
S2:将步骤1中的重组质粒转化入大肠杆菌感受态细胞,进行重组蛋白表达;
S3:通过添加IPTG诱导重组蛋白表达;
S4:通过高温孵育除杂、Ni离子亲和层析等方法分离纯化重组蛋白。
6.一种肌酐酶检测试剂盒,其特征在于,所述试剂盒中所用肌酐酶为权利要求1所述的重组肌酐酶。
7.根据权利要求6所述的一种肌酐酶检测试剂盒,其特征在于,所述肌酐酶试剂盒由试剂R1和试剂R2两部分组成:
试剂R1:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20-50mmol/L
N-乙基-N-(2-羟基-3-磺丙基)-3-甲基苯胺0.4-1mmol/L
肌酸酶5-10ku/L
肌氨酸氧化酶2-4ku/L
抗坏血酸氧化酶0.5-1ku/L
试剂R2:三羟甲基氨基甲烷-盐酸盐(Tris-HCl)20-50mmol/L4-氨基安替比林2-5mmol/L
重组肌酐酶100-150ku/L
过氧化物酶2-5ku/L。
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