CN105754899A - 一种n-脱氧核糖转移酶、编码基因及其高产菌株和应用 - Google Patents
一种n-脱氧核糖转移酶、编码基因及其高产菌株和应用 Download PDFInfo
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- CN105754899A CN105754899A CN201610218673.3A CN201610218673A CN105754899A CN 105754899 A CN105754899 A CN 105754899A CN 201610218673 A CN201610218673 A CN 201610218673A CN 105754899 A CN105754899 A CN 105754899A
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- deoxyribose transferase
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Abstract
本发明提供一种N?脱氧核糖转移酶、编码基因及其高产菌株和应用,涉及生物制药技术领域。N?脱氧核糖转移酶高产菌株,其分类命名为希氏乳杆菌(<i>Lactobacillus hilgardii</i>)ZJS01,保藏编号为CCTCC NO:M2015686。本发明还提供所述菌株产生的N?脱氧核糖转移酶及编码所述N?脱氧核糖转移酶的基因。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在合成脱氧核苷中的应用。
Description
技术领域
本发明涉及生物制药技术领域,具体涉及一种N-脱氧核糖转移酶、编码基因及其高产菌株和应用。
技术背景
肿瘤是一种严重威胁人类健康的疾病,尽管迄今尚未发现根治肿瘤的药物,但自1943年氮芥用于治疗恶性淋巴瘤后,几十年来抗癌化疗取得了相当大的进展。其中核苷类似物是一类重要的抗癌化疗剂,包括各种嘌呤和嘧啶核苷的衍生物。嘧啶核苷类似物中脱氧胞苷类似物是一类重要的抗癌化疗剂,其主要是嘧啶核苷的衍生物,如阿糖胞苷、吉西他滨、曲杀他滨、地西他滨等。
地西他滨是一类新型的核苷类似物抗癌化疗剂,是天然核苷2'-脱氧胞苷类似物。地西他滨是一类β-型异构体,它是经过在母体脱氧核糖支链的异头碳上引入嘧啶类碱基而形成的物质。该药物分别于2006年4月和5月由欧洲EMEA和美国FDA批准上市,用于治疗原发性和继发性骨髓增生异常综合症。地西他滨经磷酸化后转化为5'-单磷酸脱氧胞苷类似物,在DNA聚合酶作用下直接掺入至DNA甲基转移酶中,抑制DNA的合成及甲基化,从而抑制肿瘤细胞的生长。它在体外不能抑制DNA的合成,而在肿瘤细胞内能引发低甲基化,且有维持基因的相关细胞分化和增殖控制功能。由于特殊的药物作用机制,地西他滨引起了广大学者的研究兴趣。
核苷类化合物传统的合成方法多为化学合成法,步骤繁多,并且最终的转化率不高,产物成分复杂,分离纯化难度大。N-脱氧核糖转移酶催化脱氧核糖从嘌呤(或嘧啶)脱氧核苷直接转移到嘌呤(或嘧啶)核苷。利用N-脱氧核糖转移酶为催化剂催化合成脱氧核苷,例如地西他滨,整个反应只需一步即可完成,但是,现有N-脱氧核糖转移酶的催化效率较低,产物转化率较低,难以产业化。
发明内容
本发明的目的是提供一种N-脱氧核糖转移酶高产菌株,产生的N-脱氧核糖转移酶能够用于高效制备脱氧核苷。
本发明的另一目的是提供N-脱氧核糖转移酶及其编码基因,该酶能够用于高效制备脱氧核苷。
本发明的再一目的是提供所述N-脱氧核糖转移酶在合成脱氧核苷中的应用,产物的产率较高。
为了实现本发明的目的,本发明首先从本实验室菌库中获得一株N-脱氧核糖转移酶产生菌,分类命名为希氏乳杆菌(Lactobacillus hilgardii)ZJS01,其保藏编号为CCTCC NO:M 2015686。
本发明对希氏乳杆菌(Lactobacillus hilgardii)ZJS01的生物学特征进行鉴定,该菌株为革兰氏阳性菌株,无芽孢的杆菌,厌氧,最适生长温度为25~35℃。其生理生化特性表现在:发酵葡萄糖和果糖,通常不发酵其他碳水化合物。
经16S rDNA序列分析,该菌株鉴定为希氏乳杆菌(Lactobacillus hilgardii)。
本发明进一步分离克隆到希氏乳杆菌(Lactobacillus hilgardii)ZJS01菌株所产N-脱氧核糖转移酶的编码基因,全长483个碱基,具体如SEQ ID NO:1所示。该酶具有SEQ ID NO:2所示的氨基酸序列,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。希氏乳杆菌(Lactobacillus hilgardii)ZJS01菌株所产N-脱氧核糖转移酶命名为N-脱氧核糖转移酶NDT。
本发明构建了N-脱氧核糖转移酶NDT基因的重组表达载体。它可以通过本领域常规方法将本发明N-脱氧核糖转移酶NDT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扩增所得的N-脱氧核糖转移酶NDT基因用限制性内切酶BamH Ⅰ和NheⅠ进行双酶切,同时将载体pET28a用限制性内切酶BamH Ⅰ和Nhe Ⅰ双酶切,经T4DNA连接酶连接,形成含有本发明N-脱氧核糖转移酶基因的重组表达载体pET-ndt。
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N-脱氧核糖转移酶NDT基因可被有效表达即可。本发明优选大肠杆菌,更优选大肠埃希氏菌E.coli BL21(DE3)。将前述重组表达质粒pET-ndt转化至E.coliBL21(DE3)中,即可得本发明优选的基因工程菌株,即大肠埃希氏菌E.coli BL21(DE3)/pET-ndt。
本发明还提供一种重组N-脱氧核糖转移酶的制备方法,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N-脱氧核糖转移酶。其中,所述的培养重组菌中采用的培养基可为本领域常规的任何可使重组菌生长并产生本发明N-脱氧核糖转移酶的培养基,优选LB培养基: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,只要重组菌能够生长并产生本发明所述N-脱氧核糖转移酶即可。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大肠杆菌E.coliBL21(DE3)/pET-ndt接种至含卡那霉素的LB培养基中培养,当培养液的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N-脱氧核糖转移酶。
在本发明中,N-脱氧核糖转移酶活力测定方法如下:用50mM磷酸缓冲液(pH7.0)配制含有25mmol/L 2'-脱氧尿苷的底物溶液A和含有25mmol/L腺嘌呤的底物溶液B。反应体系中先加入200μl适当稀释的酶液,再分别加入400μl底物溶液A和B,在振荡仪中进行反应,反应温度为37℃,反应时间为10min,反应结束后取样50ul加入到950ul的甲醇中终止反应。用高效液相法在254nm下检测反应结束时生成的2'-脱氧腺苷的量。以灭活酶液为空白对照。每1个单位(U)N-脱氧核糖转移酶定义为:在上述条件下,每分钟催化产生1μmol 2'-脱氧腺苷所需的酶量。
本发明还提供所述N-脱氧核糖转移酶、重组表达载体或重组菌在合成脱氧核苷中的应用。
采用含有所述N-脱氧核糖转移酶基因的重组菌或所述重组菌的培养物或所述N-脱氧核糖转移酶催化合成脱氧核苷。
优选的技术方案中,合成脱氧核苷反应中,核糖供体为2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,核糖受体为5-氮胞嘧啶、胞嘧啶、腺嘌呤、5-氟尿嘧啶、2,6-氨基嘌呤或尿嘧啶。
在合成地西他滨时,反应体系含有5~40mM 5-氮胞嘧啶,5~40mM 2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,0.5~20U/mLN-脱氧核糖转移酶,溶剂为pH5.0~9.0的缓冲溶液,反应温度为20~55℃,反应时间为1~8h;优选地,核糖供体与核糖受体的摩尔比为1∶1~1∶4;最优选的,反应体系含有10mM 5-氮胞嘧啶,10mM 2'-脱氧尿苷或20mM胸苷,1U/mL N-脱氧核糖转移酶,溶剂为50mM、pH5.0的磷酸缓冲液,反应温度为35℃,反应时间为2h。
在合成5-氟尿苷时,反应体系含有5~40mM 5-氟尿嘧啶,5~40mM 2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,0.5~20U/mL N-脱氧核糖转移酶,溶剂为pH5.0~9.0的缓冲溶液,反应温度为20~55℃,反应时间为1~8h;优选的,反应体系含有10mM 5-氟尿嘧啶,10mM 2'-脱氧尿苷或20mM胸苷,1U/mL N-脱氧核糖转移酶,溶剂为50mM、pH5.0的磷酸缓冲液,反应温度为35℃,反应时间为2h。
本发明的有益效果在于:本发明针对目前传统化学法合成核苷类药物的研究中存在的步骤复杂、产率及转化率低等问题,提供一种新的N-脱氧核糖转移酶及利用重组N-脱氧核糖转移酶合成核苷类药物的方法。该N-脱氧核糖转移酶能以2'-脱氧尿苷等脱氧核苷为核糖供体酶法合成地西他滨等核苷类药物,产物转化率较高、易于纯化,大幅度降低了成本,具有很好的工业应用价值。
附图说明
图1为基因ndt的PCR扩增电泳图,其中:1.DNAMarker;4.基因ndt的PCR扩增产物。
图2为重组N-脱氧核糖转移酶NDT的聚丙烯酰胺凝胶电泳图,其中1:蛋白Marker;2:纯化后的N-脱氧核糖转移酶NDT;3:N-脱氧核糖转移酶NDT粗酶液。
图3为pH对酶法合成2’-脱氧腺苷的影响。
图4为温度对酶法合成2’-脱氧腺苷的影响。
图5为底物浓度对酶法合成2’-脱氧腺苷的影响。
希氏乳杆菌(Lactobacillus hilgardii)ZJS01,已经保藏。分类命名为Lactobacillus hilgardii ZJS01,保藏日期为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 2015686,保藏单位地址为:中国,武汉,武汉大学。
具体实施方式
实施例1
本实施例说明N-脱氧核糖转移酶产生菌ZJS01的生物学性质、鉴定。
从本实验室菌库中,获得一株N-脱氧核糖转移酶产生菌株ZJS01。菌株ZJS01的生物学性质:该菌株为革兰氏阳性菌株,无芽孢杆菌,厌氧,最适生长温度为25~35℃。其生理生化特性表现在:发酵葡萄糖和果糖,通常不发酵其他碳水化合物。
经16S rDNA序列分析,该菌株鉴定为希氏乳杆菌(Lactobacillus hilgardii),命名为希氏乳杆菌(Lactobacillus hilgardii)ZJS01。
实施例2
本实施例说明希氏乳杆菌(Lactobacillus hilgardii)ZJS01所产N-脱氧核糖转移酶编码基因的分离克隆程序。将希氏乳杆菌(Lactobacillus hilgardii)ZJS01所产N-脱氧核糖转移酶命名为N-脱氧核糖转移酶NDT。
采用酚-氯仿法抽提菌体总DNA。根据希氏乳杆菌(Lactobacillus hilgardii)N-脱氧核糖转移酶的基因,设计引物ZX-F和ZX-R。
ZX-F(SEQ ID NO:3)序列为:CGCGGCAGCCATATGGCTAGCATGGCTACTCATCAAAACTCTG。
ZX-R(SEQ ID NO:4)序列为:ACGGAGCTCGAATTCGGATCCTTACAATACCTTTTTGTCTGTG。
其中,引物ZXB-F下划线部分为NheI酶切位点,引物ZXB-R下划线部分为BamHI酶切位点。
以希氏乳杆菌(Lactobacillus hilgardii)ZJS01的总DNA为模板,进行PCR扩增。PCR体系为:2×Taq Plus Master Mix 10μl,引物ZX-F和ZX-R各1μl,DNA模板1μl和ddH2O 7μl。PCR扩增步骤为:(1)95℃,预变性5min;(2)95℃,变性30s;(3)55℃,退火30s;(4)72℃,延伸2min;步骤(2)~(4)重复30次;(5)72℃彻底延伸7min,冷却至4℃。PCR产物琼脂糖凝胶电泳图如图1,利用琼脂糖凝胶DNA回收试剂盒回收目的条带。获得一条完整的N-脱氧核糖转移酶NDT基因,命名为ndt,序列全长483bp,具体如SEQ ID NO:1。N-脱氧核糖转移酶NDT的氨基酸列如表中SEQ ID NO:2。
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N-脱氧核糖转移酶基因片段在37℃用限制性内切酶BamH Ⅰ和Nhe I酶切3h,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NheI酶切后的质粒pET28a,在16℃下连接过夜得到重组表达质粒pET-ndt。
将重组表达质粒pET-ndt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,获得阳性重组菌,命名为E.coli BL21(DE3)/pET-ndt。
实施例4
本实施例说明重组N-脱氧核糖转移酶NDT的诱导表达与纯化过程。
将实施例3所得重组菌E.coli BL21(DE3)/pET-ndt,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,30℃诱导6h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N-脱氧核糖转移酶的粗酶液。
粗酶液用Ni-NTAAgarose亲和柱(General Electric Company)层析除去杂蛋白,得到了纯化后的N-脱氧核糖转移酶NDT。从图2可以看到粗酶液在21kDa位置处有目标条带,纯化后的N-脱氧核糖转移酶NDT达到了电泳纯、且在21kDa位置处有一条目标条带。
其中,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培养基中含有浓度为50μg/mL卡那霉素。
实施例5
本实施例说明不同因素对N-脱氧核糖转移酶NDT催化合成脱氧核苷的影响。
以缓冲液为反应介质,以终浓度为10mM的2'-脱氧尿苷和腺嘌呤为底物,加入终浓度为1U/mL的N-脱氧核糖转移酶NDT粗酶液,放入转速180rpm的摇床中反应,反应结束后取样50ul加入到950ul的甲醇中终止反应。考察缓冲液pH、反应温度及反应时间对转化率的影响。从图3、4中可以看出最适反应pH为4.9左右,最适反应温度为50℃。反应时间为2h达到最大转化率。
在最适反应pH、最适反应温度、反应体系中N-脱氧核糖转移酶NDT粗酶液浓度为1U/mL条件下,反应时间为2h,考察反应物的最佳浓度(2'-脱氧尿苷和腺嘌呤的摩尔比为1:1)对转化率的影响,结果如图5,可以看出最适底物浓度为10mM。
产物检测方法:反应结束后取样50μl加入到950μl的甲醇中终止反应。采用HPLC检测产物浓度,计算产物2'-脱氧腺苷的产率。
实施例6-11
本实施例说明N-脱氧核糖转移酶NDT在酶法合成脱氧核苷中的应用。
在浓度为50mM、pH为5.0的磷酸缓冲液中加入终浓度均为10mmol/L的核糖供体和核糖受体,加入终浓度为1U/mL的N-脱氧核糖转移酶NDT粗酶液,放入35℃转速180rpm的摇床中反应2h,反应结束后取样50ul加入到950ul的甲醇中终止反应。采用HPLC进行检测产物浓度,计算产物的产率,反应底物、产物及产率见表1。
表1.NDT酶法合成核苷类化合物结果
结果表明N-脱氧核糖转移酶NDT以2'-脱氧尿苷、2'-脱氧胞苷等脱氧核苷为核糖供体,具有良好的酶法合成核苷类化合物的能力,产率较高,表明该酶具有广阔的应用前景。
SEQUENCE
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<110> 南京工业大学
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Claims (10)
1.一种N-脱氧核糖转移酶高产菌株,其分类命名为希氏乳杆菌(Lactobacillus hilgardii)ZJS01,保藏编号为CCTCC NO :M2015686。
2.一种由权利要求1所述菌株产生的N-脱氧核糖转移酶,其氨基酸序列如SEQ ID NO :2所示。
3.编码权利要求2所述N-脱氧核糖转移酶的基因,所述N-脱氧核糖转移酶编码基因的序列如SEQ ID NO :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N-脱氧核糖转移酶、权利要求4所述重组表达载体或重组菌在合成脱氧核苷中的应用。
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N-脱氧核糖转移酶催化合成脱氧核苷。
7.根据权利要求6所述应用,其特征在于,合成脱氧核苷反应中,核糖供体为2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,核糖受体为5-氮胞嘧啶、胞嘧啶、腺嘌呤、5-氟尿嘧啶、2,6-氨基嘌呤或尿嘧啶。
8.根据权利要求7所述应用,其特征在于,合成地西他滨时,反应体系含有5~40mM 5-氮胞嘧啶,5~40mM 2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,0.5~20 U/mLN-脱氧核糖转移酶,溶剂为pH5.0~9.0的缓冲溶液,反应温度为20~55 ℃,反应时间为1~8 h;优选的,核糖供体与核糖受体的摩尔比为1∶1~1∶4;优选的,反应体系含有10
mM 5-氮胞嘧啶,10 mM 2'-脱氧尿苷或10 mM胸苷,1 U/mL N-脱氧核糖转移酶,溶剂为50 mM、 pH5.0的磷酸缓冲液,反应温度为35 ℃,反应时间为2 h。
9.根据权利要求7所述应用,其特征在于,合成5-氟尿苷时,反应体系含有5~40mM 5-氟尿嘧啶,5~40mM 2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,0.5~20 U/mLN-脱氧核糖转移酶,溶剂为pH5.0~9.0的缓冲溶液,反应温度为20~55 ℃,反应时间为1~8 h;优选的,反应体系含有10 mM 5-氟尿嘧啶,10 mM 2'-脱氧尿苷或10 mM胸苷,1 U/mL N-脱氧核糖转移酶,溶剂为50 mM、pH5.0的磷酸缓冲液,反应温度为35 ℃,反应时间为2 h。
10.根据权利要求7所述应用,其特征在于,合成2’-脱氧胞苷时,反应体系含有5~40mM 胞嘧啶,5~40mM 2'-脱氧尿苷、胸苷、2'-脱氧胞苷或2'-脱氧腺苷,0.5~20 U/mLN-脱氧核糖转移酶,溶剂为pH5.0~9.0的缓冲溶液,反应温度为20~55 ℃,反应时间为1~8 h;优选的,反应体系含有10 mM 胞嘧啶,10 mM 2'-脱氧尿苷或10 mM胸苷,1 U/mL N-脱氧核糖转移酶,溶剂为50 mM、pH5.0的磷酸缓冲液,反应温度为35 ℃,反应时间为2 h。
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WO2019066172A1 (ko) * | 2017-09-29 | 2019-04-04 | 에스티팜 주식회사 | N-데옥시리보실 트랜스퍼라아제 변이체 및 이를 이용한 뉴클레오시드의 제조방법 |
CN110819604A (zh) * | 2019-12-05 | 2020-02-21 | 上海兆维科技发展有限公司 | 脱氧核糖转移酶突变体及其应用 |
CN114231522A (zh) * | 2021-12-27 | 2022-03-25 | 上海合全药物研发有限公司 | 固定化的n-脱氧核糖转移酶和脱氧核苷的制备方法 |
CN114231522B (zh) * | 2021-12-27 | 2024-04-26 | 上海合全药物研发有限公司 | 固定化的n-脱氧核糖转移酶和脱氧核苷的制备方法 |
CN115725535A (zh) * | 2022-11-30 | 2023-03-03 | 杭州珲益生物科技有限公司 | 一种n-脱氧核糖转移酶及其在脱氧核苷制备中的应用 |
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