CN105670979B - 一种核苷磷酸化酶、编码基因及其高产菌株和应用 - Google Patents
一种核苷磷酸化酶、编码基因及其高产菌株和应用 Download PDFInfo
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- CN105670979B CN105670979B CN201610218675.2A CN201610218675A CN105670979B CN 105670979 B CN105670979 B CN 105670979B CN 201610218675 A CN201610218675 A CN 201610218675A CN 105670979 B CN105670979 B CN 105670979B
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- nucleoside phosphorylase
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Abstract
本发明提供一种核苷磷酸化酶、编码基因及其高产菌株和应用,涉及生物制药技术领域。核苷磷酸化酶高产菌株,其分类命名为波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01,保藏编号为CCTCC NO:M 2015685。本发明还提供由所述菌株产生的核苷磷酸化酶及其编码基因。本发明还提供含有所述基因的重组表达载体或重组菌及其应用。采用本发明核苷磷酸化酶制备核苷类药物,反应条件温和,产物成分单一,无副产物,底物转化率较高,产物易于纯化,成功克服了目前化学法糖基化修饰核苷类药物存在的问题,具有很好的应用潜力。
Description
技术领域
本发明涉及生物制药技术领域,具体涉及一种核苷磷酸化酶、编码基因及其高产菌株和应用。
背景技术
天然核苷是由核糖或脱氧核糖与天然碱基所形成,由于天然碱基的多样性,构成的核糖核苷或脱氧核糖核苷也是种类繁多。天然核苷中的碱基或糖基经过化学修饰形成新的核苷,这种经过人工合成的核苷即为核苷类似物。核苷类似物与核苷酸在化学结构上具有类似性,可以假乱真混入DNA合成的过程中,但因其不具备核苷酸的功能,所以导致合成的核酸链失去了正常的功能。通过氮杂、脱氮、取代等化学修饰方法,对正常碱基的嘧啶和嘌呤进行修饰就可得到核苷类药物。核苷类似物在病毒或肿瘤细胞的遗传物质表达的过程中,被整合到DNA链中,不仅大大降低了聚合酶的活性,也终止了DNA的合成,从而使病毒细胞无法增殖传代,导致病毒的灭亡。因此,核苷类药物在治疗艾滋病毒、乙肝病毒、肿瘤等领域表现出极大的优势。据统计,在目前使用的抗病毒药物中有将近50%是核苷类药物。
核苷类药物中的5-氟尿苷是一种重要的5-氟嘧啶类衍生物,可以制备多种抗癌抗病毒药物,特别是可以做为抗肿瘤新药氟铁龙(DFUR)的前体。氟铁龙(2'-deoxy-5-fluorouridine)为脲嘧啶类抗代谢物,属细胞周期特异性抗肿瘤药,可抑制胸腺嘧啶脱氧核苷酸合成酶,阻断脲嘧啶脱氧核苷酸转变成胸腺嘧啶核苷酸,从而影响DNA的合成,起到抗病毒和抗肿瘤的作用。该药对多种肿瘤细胞具有显著的抑制作用,临床上主要用于治疗肝癌、胃肠系癌、乳腺癌和头颈部肿瘤等,与常用抗肿瘤药物5-氟脲嘧啶(5-fluorouracil)相比,具有低细胞毒性和易于在体内被吸收的优点。
现有技术中,常采用化学法糖基化得到脱氧氟尿苷,产物中会出现多种同分异构体,并且合成步骤繁多,需要对碱基或核糖基上的活性基团进行修饰保护,且合成过程中大量使用有机溶剂,同时还要使用毒性较大的氟化物、溴化物、重金属等,对环境造成巨大的污染。核苷磷酸化酶可以催化核苷或脱氧核苷的糖苷键的可逆磷酸化反应,提供核糖-1-磷酸,并释放碱基,加入其它碱基可形成另一种新的核苷。因此,核苷磷酸化酶主要用作酶法合成核苷类似物的工具。但是,现有核苷磷酸化酶的转化率较低,制备核苷类药物的成本较高。
发明内容
本发明的目的是提供一种核苷磷酸化酶高产菌株,产生的核苷磷酸化酶能够高效制备核苷类似物。
本发明的另一目的是提供核苷磷酸化酶及其编码基因,该酶能够用于高效制备核苷类似物。
为了实现本发明的目的,本发明首先从本实验室耐有机溶剂菌库中筛选获得一株核苷磷酸化酶产生菌,分类命名为波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01,其保藏编号为CCTCC NO:M 2015685。
本发明对波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01的生物学特性进行鉴定,该菌株为革兰氏阳性菌,菌体的形态特征为杆状,细胞大小为(0.3~0.4μm)*(2~7μm),有明显的凹陷,周生鞭毛,中生芽孢,运动。菌落呈圆形,颜色为乳白色,表面光滑,不透明,不凹起。氧化酶反应呈阴性,明胶反应呈阳性。淀粉水解实验呈阴性,硝酸盐反应呈阳性,VP反应呈阴性,葡萄糖反应呈阳性,柠檬酸反应呈阳性,阿拉伯糖反应呈阴性,木糖反应呈阴性。
本发明筛选的LK01菌株通过16s rDNA基因序列对比分析,与波茨坦短芽孢杆菌(Brevibacillus borstelensis)的16s rDNA基因序列相似度最高,相似度为83.4%,结合菌体形态特征,生长条件,生理生化特性,确定为波茨坦短芽孢杆菌(Brevibacillusborstelensis)。波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01能够产生核苷磷酸化酶PyNP。
本发明分离克隆到核苷磷酸化酶PyNP的编码基因,它具有SEQ ID NO:1所示的核苷酸序列,全长为1308个核苷酸,编码435个氨基酸,其氨基酸序列如SEQ ID NO:2所示。为此基因的改造并在各种外源基因表达系统中高效表达提供了优良的基因材料。
本发明构建核苷磷酸化酶PyNP基因的表达载体。它可以通过本领域常规方法将本发明所述基因连接于各种载体上构建而成。所述的载体可为本领域常规的各种载体,如市售的质粒、粘粒、噬菌体或病毒载体等,优选pET28a。较佳的,可通过下述方法制得本发明的重组表达载体:将通过PCR扩增所得核苷磷酸化酶PyNP基因片段用限制性内切酶BamH Ⅰ和Nhe Ⅰ进行双酶切,同时将载体pET28a用限制性内切酶BamH Ⅰ和Nhe Ⅰ进行双酶切,然后采用T4DNA连接酶连接,形成含有本发明核苷磷酸化酶PyNP基因的重组表达载体pET-PyNP。
本发明将上述重组表达载体转化至宿主细胞制得重组菌。所述的宿主可为本领域常规的各种宿主,只要能满足重组表达载体可稳定地自行复制,且所携带的本发明的核苷磷酸化酶PyNP基因能被有效表达即可。本发明优选大肠杆菌,更优选大肠埃希氏菌,即E.coli BL21(DE3)。将前述重组表达载体pET-PyNP转化至E.coli BL21(DE3),即可得本发明优选的重组菌,即E.coli BL21(DE3)/pET-PyNP。
本发明还提供一种制备重组核苷磷酸化酶的方法,其包括如下步骤:培养本发明前述的重组菌,获得重组表达的核苷磷酸化酶PyNP。其中,所述的培养重组菌中所用的培养基可为本领域常规的任何可使转化体生长并产生本发明核苷磷酸化酶PyNP的培养基。优选LB培养基,含有蛋白胨10g/L,酵母膏5g/L,NaCl 10g/L,pH7.0。培养方法和培养条件没有特殊的限制,可以根据宿主类型和培养方法等因素的不同,按本领域普通知识进行适当的选择,只要是重组菌能够生长并产生本发明所述的核苷磷酸化酶PyNP即可。其他培养重组菌的具体操作均可按本领域常规操作进行,优选下述方法:将本发明涉及的重组菌E.coliBL21(DE3)/pET-PyNP接种至含卡那霉素的LB培养基中培养,当培养液OD600达到0.6-1.0时,在终浓度为0.1-1.0mmol/L的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)的诱导下,高效表达本发明的重组核苷磷酸化酶PyNP。
本发明还提供所述核苷磷酸化酶、重组表达载体或重组菌在合成核苷类似物中的应用。
本发明提供采用含有所述核苷磷酸化酶基因的重组菌或所述重组菌的培养物或所述核苷磷酸化酶催化合成核苷类似物。优选的技术方案中,催化合成核苷类似物反应中,核糖供体为尿苷、2’-脱氧尿苷、胸苷、2’,3’-双脱氧胸苷或阿糖尿苷,碱基受体为5-甲基脲嘧啶、胸腺嘧啶、5-氟脲嘧啶、5-氮胞嘧啶或5-氟胞嘧啶。所述核苷类似物为5-甲基尿苷、5-氟尿苷、5-氮胞苷、5-氟胞苷、胸苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氮胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-甲基尿苷、阿糖-5-氟尿苷、阿糖-5-氮胞苷或阿糖-5-氟胞苷。
优选地,反应体系含有10~50mM的碱基受体,20~100mM的核糖供体,0.5~20U/ml核苷磷酸化酶,溶剂为20~200mM、pH4.9~9.2的磷酸缓冲液,反应温度为30~65℃,反应时间为1~5h;优选地,碱基受体与核糖供体的浓度为1:2~5;优选的,合成5-氟尿苷时,反应体系含有30mM的5-氟脲嘧啶,60mM的尿苷,1.5U/mL核苷磷酸化酶,溶剂为80mM、pH8.0的磷酸缓冲液,反应温度为50℃,反应时间为3h。优选地,合成2’-脱氧5-氟尿苷时,反应体系含有30mM的5-氟脲嘧啶,60mM的2’-脱氧尿苷或者胸苷,1.5U/mL核苷磷酸化酶,溶剂为80mM、pH8.0的磷酸缓冲液,反应温度为50℃,反应时间为3h。
优选的技术方案中,反应体系中还含有0.5-2mM EDTA,作为过渡态中间体稳定剂。
本发明的有益效果在于:目前采用化学法糖基化修饰制备核苷类药物时,转化率低,会得到α,β同分异构体,并且合成步骤繁多,需要对碱基或核糖基上的活性基团进行修饰保护,且合成过程中大量使用有机溶剂,同时还要使用毒性较大的氟化物、溴化物、重金属等,对环境造成巨大的污染。本发明提供一种新的核苷磷酸化酶及利用重组核苷磷酸化酶在水相中修饰得到核苷类药物的方法。反应以廉价易得的(2’-脱氧)尿苷为核糖供体,在温和条件下进行转糖基反应,产物成分单一,无副产物,底物转化率较高,产物易于纯化,成功克服了目前化学法糖基化修饰核苷类药物存在的问题,具有很好的应用潜力。
附图说明
图1为核苷磷酸化酶PyNP基因片段的PCR扩增电泳图,其中:泳道1为DNA Marker;泳道2~5为核苷磷酸化酶PyNP基因片段的PCR扩增产物。
图2为核苷磷酸化酶PyNP的聚丙烯酰胺凝胶电泳图,其中泳道1为蛋白Marker,泳道2是纯化后的核苷磷酸化酶PyNP。
图3为pH对核苷磷酸化酶PyNP活力的影响。
图4为温度对核苷磷酸化酶PyNP活力的影响。
波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01已被保藏,分类命名为Brevibacillus borstelensis LK01,保藏时间为2015年11月19日,保藏单位全称为中国典型培养物保藏中心,简称CCTCC,保藏编号为CCTCC NO:M 2015685,保藏单位地址:中国,武汉,武汉大学。
具体实施方式
实施例1
本实施例说明产核苷磷酸化酶的波茨坦短芽孢杆菌(Brevibacillusborstelensis)LK01的生物学性质及鉴定。
从本实验室耐有机溶剂菌库中,获得一株核苷磷酸化酶产生菌株LK01。
菌株LK01的生物学性质:菌株为革兰氏阳性菌,菌体的形态特征为杆状,细胞大小为(0.3~0.4)μm*(2~7)μm,有明显的凹陷,周生鞭毛,中生芽孢,运动。菌落呈圆形,颜色为乳白色,表面光滑,不透明,不凹起。氧化酶反应呈阴性,明胶反应呈阳性。淀粉水解实验呈阴性,硝酸盐反应呈阳性,VP反应呈阴性,葡萄糖反应呈阳性,柠檬酸反应呈阳性,阿拉伯糖反应呈阴性,木糖反应呈阴性。
经16S rDNA序列分析,菌株LK01被鉴定为波茨坦短芽孢杆菌(Brevibacillusborstelensis),命名为波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01。
实施例2
波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01所产核苷磷酸化酶命名为核苷磷酸化酶PyNP。
本实施例说明核苷磷酸化酶PyNP编码基因的分离克隆程序。
采用酚-氯仿法抽提菌体总DNA。根据对波茨坦短芽孢杆菌(Brevibacillusborstelensis)(NCBI Reference Sequence:I532_RS05705)的全基因测序结果进行分析,获得一个编码嘧啶核苷磷酸化酶的基因,根据该基因序列设计引物SF和SR。
SF(SEQ ID NO:3)序列为:ACGGAGCTCGAATTCG GATCCATGCGCATGGTCGATATCATTG。
SR(SEQ ID NO:4)序列为:CGCGGCAGCCATATGG CTAGCCTATTCGGTTATGATTTTGTAAATTAATGG。
其中,引物SF下划线部分为Nhe Ⅰ酶切位点,引物SR下划线部分为Bamh Ⅰ酶切位点。
以波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01的基因组为模板,进行PCR扩增。PCR体系为:2×EVO Master Mix(从Takara购买)25μl,引物SF和SR各2μl,DNA模板2μl和ddH2O 19μl。PCR扩增步骤为:(1)94℃,预变性3min;(2)94℃,变性15s;(3)60℃,退火15s;(4)72℃,延伸30s;步骤(2)~(4)重复30次;(5)72℃彻底延伸7min,冷却至4℃。PCR产物经琼脂糖凝胶电泳纯化,利用琼脂糖凝胶DNA回收试剂盒回收目的条带(图1)。获得完整的核苷磷酸化酶PyNP基因序列,全长1308bp,其碱基序列如表中SEQ ID NO:1。核苷磷酸化酶PyNP的氨基酸序列如SEQ ID NO:2所示。
实施例3
本实施例说明重组表达载体和重组菌的制备。
将实施例2所得核苷磷酸化酶PyNP基因片段在37℃用限制性内切酶BamH Ⅰ和NheⅠ双酶切12h,经琼脂糖凝胶电泳纯化,利用琼脂糖凝胶DNA回收试剂盒回收目标片段。将目标片段在T4DNA连接酶的作用下,与同样经BamH Ⅰ和Nhe Ⅰ酶切后的质粒pET28a,在16℃下过夜连接得到重组表达质粒pET-PyNP。
将重组表达质粒pET-PyNP转化到大肠埃希氏菌E.coli BL21(DE3)感受态细胞中,在含有卡那霉素的抗性平板上对阳性重组体进行筛选,挑选单克隆,菌落PCR验证阳性克隆,即获得阳性重组菌大肠埃希氏菌E.coli BL21(DE3)/pET-PyNP。
将重组菌E.coli BL21(DE3)/pET-PyNP,接种至含卡那霉素的LB培养基中,37℃振荡培养过夜,按2%(v/v)的接种量接入装有40ml LB培养基(含卡那霉素)的250ml三角瓶中,置37℃、180rpm摇床培养,当培养液OD600达到0.6时,加入终浓度为0.5mmol/L的IPTG作为诱导剂,25℃诱导8h后,将培养液离心,收集细胞,并用生理盐水洗涤两次,得静息细胞。将所得的静息细胞悬浮于pH7.0的缓冲液中,在冰浴中超声破碎,离心收集上清液,即为重组核苷磷酸化酶的粗酶液。
粗酶液用Ni-NTA Agarose亲和柱(从GE公司购买)层析,除去不带6His标记的杂蛋白,得到了纯化后的核苷磷酸化酶。从图2可以看出,纯化后的核苷磷酸化酶纯度达到电泳纯。
LB培养基:含有蛋白胨10g/L,酵母膏5g/L,NaCl 10g/L,pH7.0。
含卡那霉素的LB培养基:LB培养基中添加有终浓度为50μl/ml的卡那霉素。
实施例4
本实施例说明核苷磷酸化酶PyNP活力测定过程。
配制反应液:将KH2PO4和K2HPO4配制成50mmol/L、pH8.0的磷酸盐缓冲液。以磷酸盐缓冲液为溶剂,配制含有30mmol/L尿苷、1mmol/L EDTA(乙二胺四乙酸)的反应液。
酶活力测定过程:取0.95ml反应液在50℃水浴预热3min,加入50μl核苷磷酸化酶(粗酶液或纯化后的酶)启动反应,反应30min后,用1ml浓度为0.1mol/L的NaOH水溶液终止反应,测定OD290nm。
酶活单位定义:在上述测定条件下,1min内,OD290nm每变化0.01所需要的酶量定义为1个酶活单位。
实施例3制备的粗酶液酶活为0.92U/mg湿菌体。
考察了反应体系的pH值、反应温度对酶活力的影响,结果如图3和4。该酶的最适反应pH为8.0,最适反应温度为70℃。
实施例5-19
本实施例说明以核苷磷酸化酶PyNP为催化剂,采用不同的核糖供体和碱基受体,制备5-甲基尿苷、5-氟尿苷、5-氮胞苷、5-氟胞苷、胸苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氮胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-甲基尿苷、阿糖-5-氟尿苷、阿糖-5-氮胞苷和阿糖-5-氟胞苷。
在1ml的pH8.0、80mM的磷酸缓冲液中(含有1mM的EDTA),分别加入终浓度为60mM的核糖供体和终浓度为30mM的碱基受体,加入终浓度为1.5U/mL纯化后的核苷磷酸化酶,在50℃、转速为900rpm的恒温振荡反应器中反应。反应3h后,取50μl样品加入到950μl甲醇中以终止反应,利用高效液相色谱HPLC检测产物。检测条件:利用Agilent TC-C18的检测柱,流动相为甲醇:水=95:5,检测波长为254nm,柱温为30℃,流速为1ml/min。根据峰面积计算底物的转化率。
其中核糖供体为尿苷、2’-脱氧尿苷、胸苷、2’,3’-双脱氧胸苷或阿糖尿苷。
碱基受体为5-甲基脲嘧啶、胸腺嘧啶、5-氟脲嘧啶、5-氮胞嘧啶或5-氟胞嘧啶。
各反应中的核糖供体和碱基受体具体如表1所示。从表1可以看到,本发明中核苷磷酸化酶PyNP底物范围广,具有合成多种核苷类药物的能力,转化率较高,表明该酶将来在修饰新型核苷类药物中具有巨大潜力,具有广阔的应用前景。
表1.利用核苷磷酸化酶PyNP合成核苷类似物的反应物、产物即转化率
SEQUENCE LISTING
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<223> SR
<400> 4
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Claims (11)
1.一种核苷磷酸化酶高产菌株,其分类命名为波茨坦短芽孢杆菌(Brevibacillus borstelensis)LK01,保藏编号为CCTCC NO: M 2015685。
2.一种由权利要求1所述菌株产生的核苷磷酸化酶,其氨基酸序列如SEQ ID No:2所示。
3.编码权利要求2所述核苷磷酸化酶的基因,所述核苷磷酸化酶基因的序列如SEQ IDNO:1所示。
4.含有权利要求3所述基因的重组表达载体或重组菌。
5.权利要求2所述核苷磷酸化酶、权利要求4所述重组表达载体或重组菌在合成核苷类似物方面的应用。
6.根据权利要求5所述应用,其特征在于:采用含有权利要求3所述基因的重组菌或所述重组菌的培养物或所述核苷磷酸化酶合成核苷类似物。
7.根据权利要求6所述应用,其特征在于,合成核苷类似物反应中,核糖供体为尿苷、2’-脱氧尿苷、胸苷或阿糖尿苷;碱基受体为5-甲基脲嘧啶、胸腺嘧啶、5-氟脲嘧啶、5-氮胞嘧啶或5-氟胞嘧啶;所述核苷类似物为5-甲基尿苷、5-氟尿苷、5-氮胞苷、5-氟胞苷、胸苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氮胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-氟尿苷、2’-脱氧-5-氮胞苷、2’-脱氧-5-氟胞苷、2’-脱氧-5-甲基尿苷、阿糖-5-氟尿苷、阿糖-5-氮胞苷或阿糖-5-氟胞苷。
8.根据权利要求7所述应用,其特征在于,反应体系含有10~50mM的碱基受体, 20~100mM的核糖供体,0.5~20U/ml核苷磷酸化酶,溶剂为pH4.9~9.2的磷酸缓冲液,反应温度为30~65℃,反应时间为1~5h 。
9.根据权利要求8所述应用,其特征在于所述碱基受体与核糖供体的浓度为1:2~5。
10.根据权利要求9所述应用,其特征在于合成5-氟尿苷时,反应体系含有30mM的5-氟脲嘧啶,60mM的尿苷,1.5U/mL核苷磷酸化酶,溶剂为80mM、pH8.0的磷酸缓冲液,反应温度为50℃,反应时间为3h。
11.根据权利要求10所述应用,其特征在于,反应体系中还含有0.5-2 mM EDTA。
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