CN111487208A - 6-磷酸葡萄糖脱氢酶突变体及其在制备甲氨喋呤检测试剂中的用途 - Google Patents
6-磷酸葡萄糖脱氢酶突变体及其在制备甲氨喋呤检测试剂中的用途 Download PDFInfo
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- CN111487208A CN111487208A CN202010013644.XA CN202010013644A CN111487208A CN 111487208 A CN111487208 A CN 111487208A CN 202010013644 A CN202010013644 A CN 202010013644A CN 111487208 A CN111487208 A CN 111487208A
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Abstract
本申请涉及6‑磷酸葡萄糖脱氢酶突变体及其在制备甲氨喋呤检测试剂中的用途。具体而言,本申请的6‑磷酸葡萄糖脱氢酶突变体相较于野生型6‑磷酸葡萄糖脱氢酶包含选自以下的一个突变或其组合:D306C、D375C、G426C。使用本申请的6‑磷酸葡萄糖脱氢酶突变体所制备的检测试剂盒,其特异性强、灵敏度高、操作方便、检测时间短、定量准确,适合高通量检测。
Description
本申请要求2019年1月9日提交的申请号为201910017764.4和2019年5月21日提交的申请号为201910423122.4《6-磷酸葡萄糖脱氢酶突变体及其在制备检测试剂中的用途》的优先权,其通过引用并入此处。
技术领域
本申请涉及生物检测领域,特别是涉及一种突变的酶6-磷酸葡萄糖脱氢酶(简称G6PDH)及其在甲氨喋呤检测试剂盒中的应用。
背景技术
半抗原,某些小分子物质(分子量小于4000Da),其单独不能诱导免疫应答,即不具备免疫原性,但当其与大分子蛋白质或非抗原性的多聚赖氨酸等载体交联或结合后可获得免疫原性,诱导免疫应答。这些小分子物质可与应答效应产物结合,具备抗原性,它只有免疫反应性,不具免疫原性,又称不完全抗原。
半抗原能与对应抗体结合出现抗原-抗体反应,又不能单独激发人或动物体产生抗体的抗原。它只有免疫反应性,不具免疫原性,又称不完全抗原。大多数多糖、类脂、激素、小分子药物都属于半抗原。如果用化学方法把半抗原与某种蛋白分子(载体)结合,会获得新的免疫原性,并能刺激动物产生相应的抗体。
小分子抗原或半抗原因缺乏可作夹心法的两个以上的位点,因此不能用双抗体夹心法进行测定,多采用竞争模式。原理是标本中的抗原和一定量的酶标抗原竞争与固相抗体结合。标本中抗原量含量愈多,结合在固相上的酶标抗原愈少,显色愈浅。小分子激素、药物等ELISA测定多用此法。
甲氨喋呤(Methotrexate)结构式如下所示:
甲氨喋呤又名氨甲基叶酸、甲氨蝶呤、氨甲喋呤,橙黄色结晶性粉末,熔点185至204℃,常温下暴露于空气中性质稳定,易溶于稀碱、酸或碱金属的碳酸盐溶液,微溶于稀盐酸,几乎不溶于水、乙醇、氯仿、乙醚。
甲氨喋呤为抗叶酸类抗肿瘤药,主要通过对二氢叶酸还原酶的抑制而达到阻碍肿瘤细胞的合成,而抑制肿瘤细胞的生长与繁殖。主要功能全身用药治疗绒毛膜上皮癌、恶性葡萄胎、各类急性白血病、乳腺癌、肺癌、头颈部癌、消化道癌、宫颈癌及恶性淋巴瘤等。用动脉插管灌注对头颈部癌和肝癌也有较好疗效,而甲氨蝶呤与阿达木单抗等生物制剂联合用药,可以更有效地缓解关节风湿病患者的疾病症状,减缓关节损伤的进展,并且可以改善身体功能。还可在异位妊娠局部胎囊注射缓解出血。
甲氨喋呤是白血病的主要治疗药在使用,但1951年Gubner等人提出有关将氨基喋呤用于慢性RA是有效的报告后,作为RA的治疗药而受到注意,并主要在欧美使用。甲氨喋呤是处方药,必须由医生开置,高剂量可引起骨质疏松、关节痛、眼刺激以及糖尿病,还可能引起卵细胞缺乏和精子减少,生育能力减退。和其他叶酸拮抗剂一样,甲氨喋呤也有致畸作用,还可造成死胎。
基于上述原因,在治疗过程中需及时进行甲氨喋呤血药浓度监测,是辅助临床治疗、改善治疗效果、降低毒性风险的有效方式。
目前已知的甲氨喋呤检测方法主要有:高效液相色谱法(HPLC)、发光免疫、酶联免疫吸附剂测定(ELISA)等方法。高效液相色谱法可将药物与代谢产物以及内源性物质分离,具有专一性强的特点,是检测MTX血浆浓度的金标准,但该法需复杂的前处理过程和较长的测定时间,不适合大样本的快速检测。免疫法一定程度上会受到交叉反应的影响,但是其凭借快速、易操作的优点,已逐渐成为治疗药物监测的主要方法。发光免疫法试剂成本昂贵,不适合常规治疗药物检测,不利于大范围推广。
现有技术CN104569373A描述了一种甲氨蝶呤均相酶免疫检测试剂及其制备和检测方法,其中公开了6-磷酸葡萄糖脱氢酶和甲氨喋呤偶联物的制备方法:
a)配置缓冲溶液称取1.09g/L磷酸二氢钾、1.70g/L磷酸氢二钠、8.5g/L氯化钠,pH调为7.4;
b)称取3mg 6-磷酸葡萄糖脱氢酶,室温溶解于3mL前述a步骤所得溶液中,配置成6-磷酸葡萄糖脱氢酶溶液;
c)称取3mg甲氨蝶呤衍生物,室温溶解于300μL前述a步骤所得溶液中,配置成甲氨蝶呤衍生物溶液:甲氨蝶呤衍生物如下所示:
d)将前述b和c步骤所得溶液混合在在2-8℃搅拌1小时,使6-磷酸葡萄糖脱氢酶与甲氨蝶呤的末端羧基连接;
e)将上述d步骤所得混合溶液放置于前述a步骤所得溶液中进行透析纯化,获得6-磷酸葡萄糖脱氢酶-甲氨喋呤偶联物。
上述现有技术中仅仅指出酶与甲氨蝶呤的末端羧基进行连接,然而酶上存在众多可以和羧基发生结合的基团(如氨基),那么将难以保证定向的偶联,因此仍存在所得偶联物一致性不强、试剂盒批间差异大的问题。
发明内容
鉴于本领域的需求,本申请提供了一种新型的6-磷酸葡萄糖脱氢酶突变体、及其在制备甲氨喋呤检测试剂盒中的用途。
根据一些实施方案,提供了一种6-磷酸葡萄糖脱氢酶突变体。区别于已有发表的专利US006090567A(Homogeneous immunoassays using mutant glucose-6-phosphatedehydrogenases)的6磷酸葡萄糖脱氢酶突变体,本申请的6-磷酸葡萄糖脱氢酶突变体,其包含选自以下的突变:D306C、D375C、G426C。
根据一些实施方案,提供了一种6-磷酸葡萄糖脱氢酶突变体,所述6-磷酸葡萄糖脱氢酶突变体是选自以下的序列所示:SEQ ID No.2、SEQ ID No.3、SEQ ID No.4。
根据一些实施方案,提供了一种多核苷酸,其编码本申请的6-磷酸葡萄糖脱氢酶突变体。
根据一些实施方案,提供了一种表达载体,其包含本申请的多核苷酸。
根据一些实施方案,提供了一种宿主细胞,其包含本申请的表达载体。宿主细胞可以是原核(如细菌)或真核(如酵母)。
根据一些实施方案,提供了一种偶联物,其是本申请的6-磷酸葡萄糖脱氢酶突变体与半抗原按照摩尔比1:1偶联而成。
在一些具体的实施方案中,半抗原的分子量为100Da至4000Da,例如:100、150、200、250、300、350、400、410、420、430、440、450、460、470、480、490、500、520、550、570、600、620、650、700、750、800、850、900、950、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、2600、2700、2800、2900、3000、3100、3200、3300、3400、3500、3600、3700、3800、3900、4000。
根据本申请,技术人员将理解,“半抗原”还包含其衍生物的形式。为了便于和6-磷酸葡萄糖脱氢酶进行偶联,对于那些自身不带有偶联基团(例如,与巯基反应的基团)的半抗原(例如甲氨喋呤),可以经改造而带有接头,以便和巯基共价结合。因此,在本申请中,半抗原衍生物是指,经改造而带有巯基反应基团的半抗原。
半抗原选自:小分子药物(如抗生素、精神药物)、激素、代谢物、糖、脂质、氨基酸。
半抗原例如但不限于:万古霉素、茶碱、苯妥英、维生素D、25羟维生素D、1,25双羟维生素D、叶酸、强心甙(包括地高辛、洋地黄毒苷)、酶酚酸、雷帕明、环孢霉素A、甲氨喋呤、乙胺碘复酮、甲胺喋呤、他克莫司、血清氨基酸、胆汁酸、甘胆酸、苯丙氨酸、乙醇、尿尼柯丁代谢产物柯替宁、尿吗啡、尿单羟酚衍生物、神经肽酪氨酸、血浆甘丙素、多胺、组织胺、促甲状腺激素、泌乳素、胎盘泌乳素、生长激素、促卵泡刺激素、促黄体生成素、促肾上腺皮质激素、抗利尿激素、降钙素、降钙素原、甲状旁腺激素、甲状腺素、三碘甲状原氨酸、反三碘甲状原氨酸、游离甲状腺素、游离三碘甲状原氨酸、皮质醇、尿17-羟皮质类固醇、尿17-酮类固醇、脱氢表雄酮及硫酸酯、醛固酮、尿香草苦杏仁酸、血浆肾素、血管紧张素、促红细胞生成素、睾酮、双氢睾酮、雄烯二酮、17α羟孕酮、雌酮、雌三醇、雌二醇、孕酮、人绒毛膜促性腺激素、胰岛素、胰岛素原、C肽、胃泌素、血浆前列腺素、血浆6-酮前列腺素F1α、前列环素、肾上腺素、儿茶酚胺、去甲肾上腺素、胆囊收缩素、纳素、环磷酸腺苷、环磷酸鸟苷、血管活性肽、生长抑素、促胰液素、P-物质、神经降压素、血栓素A2、血栓素B2、5羟色胺、神经肽Y、骨钙素。
在具体的实施方案中,半抗原是甲氨喋呤或其衍生物。
在具体的实施方案中,半抗原是甲氨喋呤衍生物,其带有巯基反应基团,例如来酰亚胺、溴乙酰基、乙烯基砜或氮丙啶。
在具体的实施方案中,半抗原是甲氨喋呤衍生物,如式I所示:
在一些实施方案中,m为1至10的整数,优选1至5的整数,例如1、2、3、4、5。
在具体的实施方案中,甲氨喋呤衍生物如式II所示:
根据一些实施方案,提供了一种试剂,其包含本申请的偶联物。
根据一些实施方案,提供了本申请的6-磷酸葡萄糖脱氢酶突变体在制备甲氨喋呤检测试剂中的用途。
根据一些实施方案,提供了本申请的偶联物在制备甲氨喋呤检测试剂中的用途。
在具体的实施方案中,所述的检测试剂选自:酶联免疫法检测试剂、化学发光免疫法检测试剂、均相酶免疫法检测试剂、胶乳增强免疫比浊法检测试剂。
在具体的实施方案中,所述的检测试剂优选是基于竞争法检测的试剂。
根据一些实施方案,提供了本申请的偶联物在制备甲氨喋呤检测装置中的用途。
在具体的实施方案中,所述的检测装置可以制备成孔板(例如96-孔板)的形式,比如板上包被根据本申请的试剂。
在具体的实施方案中,所述的检测装置可以制备成颗粒(例如胶乳、磁珠)的形式,比如颗粒上包被根据本申请的试剂。
根据一些实施方案,提供了一种甲氨喋呤检测试剂盒,其包含:
-第一试剂,所述第一试剂包含底物、缓冲液和甲氨喋呤抗体;所述底物是6-磷酸葡萄糖脱氢酶的底物;
-第二试剂,所述第二试剂包含本申请的偶联物和缓冲液;
-任选地,校准品,所述校准品包含10mM至500mM缓冲液、0μM至2.0μM甲氨喋呤;以及
-任选地,质控品,所述质控品包含10mM至500mM缓冲液、0.2μM至2.0μM甲氨喋呤。
根据一个实施方案,提供了一种甲氨喋呤检测试剂盒,其包含:
第一试剂,其包含:
10mM至500mM缓冲液、
5mM至50mM底物、
0.05%至0.5%w/v,优选0.2%至0.5%w/v甲氨喋呤抗体、0.05%至0.5%w/v,优选0.05%至0.1%w/v稳定剂、
0.05%至1%w/v,优选0.5%至1%w/v氯化钠、
0.05%至0.5%w/v,优选0.05%至0.1%w/v防腐剂;
第二试剂,其包含:
10mM至500mM缓冲液、
0.1%至0.5%w/v根据本申请的偶联物、
0.05%至0.5%w/v,优选0.05%至0.1%w/v稳定剂、
0.05%至1%w/v,优选0.5%至1%w/v氯化钠、
0.05%至0.5%w/v,优选0.05%至0.1%w/v防腐剂;
在一些实施方案中,所述缓冲液选自以下的一种或组合:TAPS、氨基丁三醇缓冲液、磷酸盐缓冲液、Tris-HCl缓冲液、柠檬酸-柠檬酸钠缓冲液、巴比妥缓冲液、甘氨酸缓冲液、硼酸盐缓冲液、三羟甲基甲烷缓冲液;优选地,磷酸盐缓冲液;所述缓冲液的浓度为10mmol/L至500mmol/L,优选50至200mM;所述缓冲液的pH为7至8.4。
在一些实施方案中,所述稳定剂选自以下的一种或组合:牛血清白蛋白、海藻糖、甘油、蔗糖、甘露醇、甘氨酸、精氨酸、聚乙二醇6000、聚乙二醇8000;优选牛血清白蛋白。
在一些实施方案中,所述的防腐剂选自以下的一种或组合:叠氮化物、MIT、生物防腐剂PC(如PC-300)、硫柳汞;所述叠氮化物选自:叠氮钠、叠氮锂。
在一些实施方案中,所述底物包含:6-磷酸葡糖糖、β-烟酰胺腺嘌呤二核苷酸。
在一些具体的实施方案中,所述甲氨喋呤抗体源自:兔、小鼠、大鼠、山羊、绵羊、猫、豚鼠、犬、灵长类、牛、马、骆驼科、禽、人。
在一些具体的实施方案中,所述甲氨喋呤抗体选自:单抗、多抗、重组抗体、嵌合抗体、抗原结合片段。
根据一些实施方案,提供了一种偶联物的制备方法,包括步骤:
1)提供根据本申请的甲氨喋呤衍生物,尤其是在非质子性溶剂(例如但不限于乙腈、二甲基甲酰胺、二甲基亚砜)中提供根据本申请的甲氨喋呤衍生物;
2)提供6-磷酸葡萄糖脱氢酶突变体,优选在缓冲液(其提供反应环境,例如但不限于PBS、Tris、TAPS、TAPSO,所述缓冲液pH为6.0至8.0)中提供6-磷酸葡萄糖脱氢酶突变体;
3)在18℃至28℃,将所述甲氨喋呤衍生物和所述6-磷酸葡萄糖脱氢酶突变体按照摩尔比n:1接触1小时至4小时(优选2小时至3小时)使得所述甲氨喋呤衍生物和所述6-磷酸葡萄糖脱氢酶突变体发生偶联,得到所述种偶联物;
4)根据需要,任选对所述种偶联物进行纯化,例如脱盐处理等。
在一些实施方案中,反应体系中酶和半抗原的接触摩尔比1:n,其中n是0.1至500,例如,0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、100、200、300、400、500及其任意上述数值之间的范围。在一个具体的实施方案中,反应体系中酶和半抗原的接触摩尔比为5:1至1:5。
在一些具体的实施方案中,步骤1)和2)能够互换或并行。
在一些具体的实施方案中,在偶联之前,所述6-磷酸葡萄糖脱氢酶包含一个或多个游离的巯基,从而允许和甲氨喋呤实现定向反应。
野生型6-磷酸葡萄糖脱氢酶不含游离的巯基,因此在一些具体的实施方案中,6-磷酸葡萄糖脱氢酶是经过基因工程改造的,使其在特定位点(306、375或426位)上的氨基酸突变为半胱氨酸,从而带有一个游离巯基。
附图说明
图1.G6PDH(野生型)氨基酸序列(SEQ ID No.1);源自假肠膜明串珠菌Leuconostoc pseudomesenteroides。
图2.G6PDH(D306C)氨基酸序列(SEQ ID No.2)。
图3.G6PDH(D375C)氨基酸序列(SEQ ID No.3)。
图4.G6PDH(G426C)氨基酸序列(SEQ ID No.4)。
具体实施方式
实施例
实施例1.甲氨喋呤衍生物的合成
向圆底烧瓶中加入甲氨蝶呤(100mg,0.22mmol),溶于干燥的N,N-二甲基甲酰胺(10mL)中,向其中加入4-马来酰亚胺丁酸(39mg,0.22mmol),加入三乙胺(44mg,0.44mmol),氮气保护下,搅拌至全部溶解。
向反应体系中加入HOAt(45mg,0.33mmol),搅拌至全部溶解,加入HATU(125mg,0.33mmol),室温(18-28℃,优选20至25℃)搅拌约4h,TLC检测。反应完毕直接用制备板纯化(MeOH/DCM=5:3),最终得到甲氨蝶呤衍生物(45mg,产率35%)。
按常规方法确认产物结构。本实施例使得甲氨喋呤带有一个可以和酶结合的基团。
实施例2.甲氨喋呤衍生物与G6PDH分子的偶联
一、本申请的偶联方法
根据本申请的G6PDH-甲氨喋呤偶联物,按照以下方式进行偶联:甲氨喋呤衍生物分子上的巯基反应性基团(如但不限于马来酰亚胺基团)与G6PDH分子上的巯基共价结合。
1.将实施例1制备的甲氨喋呤衍生物溶于N,N-二甲基甲酰胺中(10mM);
2.G6PDH溶液:G6PDH(本申请的突变体或现有技术突变体)溶于0.2M磷酸盐缓冲液,pH 8.0(6.4mM酶);
3.将200μl葡萄糖6磷酸脱氢酶突变体溶液加入至750μL缓冲溶液(0.05mol/LNa2HPO4、150mM NaCl、10mM EDTA、0.1%NaN3、pH=7.2)中,再加入50μl甲氨喋呤衍生物溶液;
4.上述混合溶液,在室温(18-28℃,优选20至25℃)下充分震荡2-3小时,脱盐处理,所得产物即G6PDH-甲氨喋呤偶联物(浓度0.1mM-2.0mM)。
二、对照偶联方法A
参照CN104569373A实施例四中公开的方法制备G6PDH-甲氨喋呤偶联物。
三、对照偶联方法B(依靠对甲氨喋呤自带基团的激活)
1.将2.38mL葡萄糖六磷酸脱氢酶溶解于12mL Tris缓冲液中,然后依次加入225mg还原态的烟酰胺腺嘌呤二核苷酸NADH,135mg葡萄糖-6-磷酸和2.25mL二甲基亚砜;所述Tris缓冲液pH为9.0,各组分浓度为:0.05mol/L Tris、3.3mM氯化镁、145.4mM氯化钠。
2.甲氨喋呤衍生物的活化:将10mg甲氨喋呤衍生物溶解于420μL二甲基亚砜和180μL二甲基甲酰胺中,加入6μL三丁胺和350μL氯甲酸异丁酯在2-8℃条件下搅拌30分钟;
3.将所述步骤1和2所得的溶液混合,在2-8℃条件下搅拌12-16小时,并将偶联的酶标抗原进行G-25凝胶层析柱纯化,得到葡萄糖-6-磷酸脱氢酶标记的甲氨喋呤衍生物。
实施例3.试剂盒的制备
制备以下检测甲氨喋呤的试剂盒,其包含:
1.第一试剂的制备:
2.第二试剂的制备:
3.校准品:20mM HEPES缓冲液,以及0.2-0.8μM、0.9-1.2μM、1.3-2.0μM甲氨喋呤(或按需加入);
4.质控品:20mM HEPES缓冲液,以及0μM、0.13μM、0.25μM、0.50μM、1.00μM、2.00μM甲氨喋呤(或按需加入)。
5.将上述试剂(任选包含质控品、校准品),组装成检测试剂盒。
检测例
在均相反应体系中,样本中的甲氨喋呤和G6PDH-甲氨喋呤偶联物同时竞争结合抗甲氨喋呤抗体位点,由于抗体与偶联物结合后酶活性下降,样品中游离的甲氨喋呤越多,竞争结合的抗体位点越多,抗体与酶偶联物的结合就越少,未结合抗体的酶偶联物催化β-烟酰胺腺嘌呤二核苷酸氧化型(NAD+)转化为β-烟酰胺腺嘌呤二核苷酸还原型(NADH),样品中的甲氨喋呤浓度与NADH的生成量成正比,通过吸光度的变化即可得到样品中甲氨喋呤的浓度。
表1.全自动生化仪参数
检测例1.本申请试剂盒的性能
1.定标吸光度
甲氨喋呤校准品溶于缓冲溶液中(0.9%NaCl,0.1%NaN3),配制6种浓度的校准品。定标工作体积为2-10μL,然后加入100-200μL的第一试剂和50-100μL的第二试剂,速率法,检测主波长340nm,副波长405nm,特定读点的吸光度变化率,绘制成定标曲线。本申请中定标曲线的建立和优化均是在日立7180上完成,但其他主流机型(AU680、雅培C16000等)经检测可用。
2.精密度实验
利用如上建立起的定标曲线,测定高、中、低质控品、临床样本。
表2.精密度
3.线性
表3.线性
4.机载稳定性(D375C突变体)
选择高、中、低三个浓度的质控品,每天或者隔天检测三次,数据显示本申请的试剂盒定标周期可以稳定两周以上;试剂开封之后放置于机器上,检测显示机载稳定性超过40天,数据参见表6(其中,40天*表示机载40天重新定标之后检测数据)。
5.药物干扰实验
选取常用的20种化合物和药物,在甲氨喋呤浓度为10μM,如下浓度的化合物对本申请试剂盒的检测(D375C突变体)无明显干扰。
表4.药物干扰实验
检测例2.偶联物中的抗体抑制率
1.抗体抑制率的检测原理
当抗体与G6PDH-甲氨喋呤偶联物结合时,由于空间位阻导致G6PDH酶活性受到影响,从而使得其催化NAD转化为NADH的效率降低,通过检测NADH量的变化,从而比较加入抗体与未加入抗体的实验组的差异,这种差异即体现为抗体对G6PDH的抑制能力。
2.反应体系
表5.抗体抑制率的检测试剂制备
3.结果
比较加入抗体与未加入抗体时,分别检测G6PDH-甲氨喋呤偶联物吸光度测值,即可得到抗体对G6PDH的抑制情况。
相对于已发表的突变位点(A45C、K55C)所制备的偶联物相比,本申请的酶突变体在抗体抑制率上有明显提高,能达到44.6%以上(G426C:44.6%;D375C:51%),最高达60.3%(D306C)。而之前已发表的突变位点(例如A45C、K55C)的抑制率为39.7%至42.2%。
虽然不限于具体理论,但是可以部分地解释为:和现有技术中的G6PDH突变体(A45C、K55C)相比,本申请酶突变体中突变位点(即引入游离巯基的位点)是和半抗原(比如激素、小分子药物等)发生偶联的位置所在。半抗原在这个位置上与半抗原特异性抗体结合时,所构成的空间位阻对G6PDH酶的活性影响大,同时在引入突变后,还不能实质上影响分子的空间折叠。因此,这个突变位点的位置非常重要,需要同时兼顾G6PDH酶的活性、偶联分子的空间折叠、以及半抗原表位的充分暴露。
由于酶的突变体在抗体抑制率上有明显的提高。将酶的突变体与甲氨喋呤偶联后的偶联物配制成试剂盒后,试剂在批间变异系数、线性、特异性等性能方面有明显的性能提升。
序列表
<110> 北京九强生物技术股份有限公司
<120> 6-磷酸葡萄糖脱氢酶突变体及其在制备甲氨喋呤检测试剂中的用途
<130> 300004CG
<150> 201910017764.4
<151> 2019-01-09
<150> 201910423122.4
<151> 2019-05-21
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<211> 486
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> VARIANT
<222> (426)..(426)
<223> G6PDH突变体,相较于野生型,426位的G替换为C
<400> 5
Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly
1 5 10 15
Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
20 25 30
Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln
35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe
65 70 75 80
Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu
85 90 95
Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn
100 105 110
Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala
115 120 125
Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
130 135 140
Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu
145 150 155 160
Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg
165 170 175
Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu
180 185 190
Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile
195 200 205
Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg
210 215 220
Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn
225 230 235 240
His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser
245 250 255
Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala
260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu
290 295 300
Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg
325 330 335
Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe
340 345 350
Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala
355 360 365
Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu
370 375 380
Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu
385 390 395 400
Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr
405 410 415
Glu Arg Met Ile His Asp Thr Met Asn Cys Asp Gly Ser Asn Phe Ala
420 425 430
Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser
435 440 445
Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly
450 455 460
Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp
465 470 475 480
Ala Trp Val Phe Lys Gly
485
Claims (10)
2.根据权利要求1所述的偶联物,其中:
所述6-磷酸葡萄糖脱氢酶突变体,相较于野生型6-磷酸葡萄糖脱氢酶,包含选自以下的一个突变:D306C、D375C、G426C;
优选地,所述6-磷酸葡萄糖脱氢酶突变体是选自以下的序列所示:SEQ ID No.2、SEQID No.3、SEQ ID No.4。
3.一种试剂,其包含权利要求1或2所述的偶联物。
4.权利要求1或2所述的偶联物在制备检测试剂中的用途,其中:
所述检测试剂是甲氨喋呤的检测试剂;
优选地,所述检测试剂选自:酶联免疫法检测试剂、化学发光免疫法检测试剂、均相酶免疫法检测试剂、胶乳增强免疫比浊法检测试剂。
5.权利要求1或2所述的偶联物在制备检测装置中的用途:
所述检测装置是针对甲氨喋呤的检测装置;
所述检测装置选自以下任一的形式:孔板、颗粒、芯片、试纸。
6.一种甲氨喋呤检测试剂盒,其包含:
第一试剂,所述第一试剂包含底物、抗甲氨喋呤抗体、缓冲液;
第二试剂,所述第二试剂包含权利要求1或2所述的偶联物、缓冲液;
任选地,校准品,所述校准品包含10mM至500mM缓冲液、0μM至2.0μM;以及
任选地,质控品,所述质控品包含10mM至500mM缓冲液、0.2μM至2.0μM甲氨喋呤。
7.根据权利要求6所述的甲氨喋呤检测试剂盒,其包含:
第一试剂,其包含:
10mM至500mM,优选50mM至200mM缓冲液、
5mM至50mM,优选10mM至20mM葡萄糖-6-磷酸、
5mM至50mM,优选10mM至20mM氧化型β-烟酰胺腺嘌呤二核苷酸、
0.05%至0.5%w/v,优选0.2%至0.5%w/v甲氨喋呤抗体、
0.05%至0.5%w/v,优选0.05%至0.1%w/v稳定剂、
0.05%至1%w/v,优选0.5%至1%w/v氯化钠、
0.05%至0.5%w/v,优选0.05%至0.1%w/v防腐剂;
第二试剂,其包含:
10mM至500mM,优选50mM至200mM缓冲液、
0.05%至0.5%w/v,优选0.1%至0.5%w/v权利要求1或2所述的偶联物、
0.05%至0.5%w/v,优选0.05%至0.1%w/v稳定剂、
0.05%至1%w/v,优选0.5%至1%w/v氯化钠、
0.05%至0.5%w/v,优选0.05%至0.1%w/v防腐剂;
所述缓冲液选自以下的一种或组合:TAPS缓冲液、磷酸缓冲液、甘氨酸缓冲液、Tris缓冲液、硼酸缓冲液、MOPS缓冲液、HEPES缓冲液;
所述缓冲液的pH为7至8.4;
所述稳定剂选自以下的一种或组合:牛血清白蛋白、海藻糖、甘油、蔗糖、甘露醇、甘氨酸、精氨酸、聚乙二醇6000、聚乙二醇8000;优选牛血清白蛋白;
所述防腐剂选自以下的一种或组合:叠氮化物、MIT、生物防腐剂PC、硫柳汞;
优选地,所述防腐剂选自:叠氮钠、叠氮锂、PC-300。
8.根据权利要求6所述的甲氨喋呤检测试剂盒,其包含:
第一试剂,其包含:
Tris缓冲液55mM,PH8.0、
10mM葡萄糖-6-磷酸、
10mM氧化型β-烟酰胺腺嘌呤二核苷酸、
0.2%w/v甲氨喋呤抗体、
0.1%w/v牛血清白蛋白、
0.85%w/v NaCl、
0.05%w/v叠氮钠;
第二试剂,其包含:
120mM Tris缓冲液,pH8.2、
0.1%w/v权利要求1或2所述的偶联物、
0.1%w/v牛血清白蛋白、
0.85%w/v NaCl、
0.05%w/v叠氮钠。
10.根据权利要求9所述的偶联物的制备方法,其包括步骤:
1)提供甲氨喋呤衍生物,优选在非质子性溶剂中提供甲氨喋呤衍生物;
2)提供权利要求2所限定的6-磷酸葡萄糖脱氢酶突变体,优选在缓冲液中提供所述6-磷酸葡萄糖脱氢酶突变体;
3)在18℃至28℃优选20℃至25℃,将所述6-磷酸葡萄糖脱氢酶突变体和所述甲氨喋呤衍生物接触1小时至4小时,优选2小时至3小时,使得所述甲氨喋呤衍生物和所述6-磷酸葡萄糖脱氢酶突变体发生偶联,得到所述偶联物;
4)任选,对所述偶联物进行纯化,优选对所述偶联物进行脱盐纯化;
步骤1)和步骤2)能够互换或并行;
所述缓冲液选自:磷酸缓冲液、Tris缓冲液、Hepes缓冲液、PBS缓冲液、TAPSO缓冲液,
所述缓冲液的pH为6.0至8.0;
所述非质子性溶剂选自以下一种或组合:乙腈、二甲基甲酰胺、二甲基亚砜;
优选地,在步骤3)之前,所述6-磷酸葡萄糖脱氢酶突变体包含一个游离巯基;更优选地,所述6-磷酸葡萄糖脱氢酶突变体在第306、375或426位有游离巯基;
优选地,所述甲氨喋呤衍生物和所述6-磷酸葡萄糖脱氢酶突变体接触的摩尔比为n:1,n是0.5至120,优选为0.5、0.6、0.7、0.8、0.9、1、2、5、10、15、20、25、30、35、40、45、50、60。
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