CN116082362A - 一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用 - Google Patents
一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用 Download PDFInfo
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- CN116082362A CN116082362A CN202310007907.XA CN202310007907A CN116082362A CN 116082362 A CN116082362 A CN 116082362A CN 202310007907 A CN202310007907 A CN 202310007907A CN 116082362 A CN116082362 A CN 116082362A
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Abstract
本发明涉及一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用。本发明构建了细菌美登木素基因簇asc的途径特异性正调控因子基因ascR4和氨甲酰基转移酶基因asc21b的共表达载体,实现了在珍贵橙色束丝放线菌突变株中异源共表达,进而制备氨甲酰美登醇的目的,并且使得氨甲酰美登醇的产量达到了8.3mg/L,提高了30.7倍,有效克服了现有技术中存在的产量低的问题,可以降低美登木素抗体偶联物药物的生产成本,并促进这类药物的研究、生产和临床应用。
Description
技术领域
本发明涉及一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用,属于天然药物化学与医药应用技术领域。
背景技术
癌症是全球性的公共健康问题,因此长期以来抗肿瘤药物都是创新药物研发关注的一个热点领域。美登木素属于安莎类抗生素,是已知天然产物中活性最强的微管蛋白抑制剂,对多种肿瘤细胞和实体瘤系统都有很强的抑制活性,是著名的药物先导,在抗肿瘤药物的研发中备受关注。
2013年,FDA批准了美登木素抗体药物偶联物ado-trastuzumab emtansine(T-DM1)的上市,应用于人表皮生长因子受体2(HER2)阳性乳腺癌的临床治疗。2020年1月,T-DM1(商品名赫赛莱,通用名注射用恩美曲妥珠单抗)在我国获批上市,用于治疗接受新辅助治疗后仍有残存病灶的HER-2阳性乳腺癌。抗体药物偶联物(antibody drug conjugates,ADC)由抗体或抗体片段通过连接体(linker)与“弹头”药物连接而成,可以特异性识别肿瘤细胞表面的抗原表位,避免对正常细胞的杀伤。T-DM1可以避免美登木素产生的神经毒性,使其具有更优越的安全性和耐受性。
根据来源不同,美登木素可以分为植物美登木素和细菌美登木素两大类。植物来源美登木素的产量极低,无法满足大规模生产应用的需求。微生物来源的细菌美登木素,即安丝菌素,与植物美登木素具有相似的化学结构,且产量相对较高,为解决这一问题提供了契机。然而,安丝菌素的C-3位一般为酰基,无法与抗体直接进行偶联,需要通过多个步骤的半合成才能得到具有偶联基团的“弹头”。这些反应步骤的副反应复杂、产率较低,造成昂贵原料的浪费,是导致T-DM1价格高昂的重要因素之一。因此,如何应用微生物高效制造可用于合成靶向抗肿瘤药物的美登木素类化合物是抗肿瘤药物研究领域一个亟待解决的问题。微生物中次级代谢产物的产生受到复杂而严谨的调控,为了提高目标化合物的产量,需要寻找途径特异性调控因子。
发明内容
基于前期对氨甲酰基转移酶Asc21B的研究发现,Asc21B可以在安丝菌素产生菌橙色珍贵束丝放线菌Actinosynnema pretiosum ATCC 31565突变株HGF052体内合成C-3位为氨甲酰基的衍生物,并展现出良好的抗肿瘤活性(Organic Letters,2019,21(15):5823-5826.)。为进一步提高这类化合物的产量,本发明提供一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用。本发明的目标在于通过生物合成高效制备可用于合成ADC的氨甲酰美登醇,将推动美登木素抗体偶联药物的研发和临床应用,对于发现高特异性且具有自主知识产权的新型抗肿瘤药物具有重要意义。
本发明的技术方案如下:
一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇,其化学结构如下所示:
上述氨甲酰美登醇的合成方法,包括以下步骤:
(1)将能够表达氨甲酰基转移酶基因asc21b和正调控因子基因ascR4的基因工程菌HGF052+pSET5035n-ascR4-asc21b在ISP4固体培养基上划线活化,于30℃下培养3~5天,得到活化好的菌种;
(2)挑取活化好的菌种接种到YMG固体培养基上,于28~30℃下发酵培养9~12天,得到固体培养物;
(3)将固体培养物取出,切成1cm3的小方块,在20~30℃下用体积比为80:15:5的乙酸乙酯/甲醇/甲酸浸泡提取18~24小时,合并提取液,减压及35~40℃的温度下浓缩至干,得粗提物;再重复2~3次,合并粗提物;
(4)将粗提物溶于400~500mL纯净水中,使用等体积乙酸乙酯萃取2~4次,得到乙酸乙酯相;再将乙酸乙酯相减压及35~40℃的温度下浓缩至干,得EA提取物;
(5)将EA提取物溶于200~300mL甲醇中,再经等体积石油醚多次萃取,得到甲醇相;甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(6)将甲醇提取物依次经反相硅胶柱层析分离,凝胶柱层析分离,薄层层析和高效液相色谱分离,然后将组分相同的洗脱液进行合并,得到氨甲酰美登醇。
根据本发明优选的,步骤(1)中,所述基因工程菌HGF052+pSET5035n-ascR4-asc21b的构建方法,包括步骤如下:
(a)以asc基因簇DNA为模板进行PCR扩增,扩增得到正调控因子基因ascR4序列,PCR引物序列如下:
ascR4-F:5′-AAAGGAGGCGGACATATGCTGGACATCATCGGCCTGAGTG-3′,
ascR4-R:5′-CAATTGCCTCTAGATCACGGATGTGCCGAAGGCGAACCCG-3′;
(b)将步骤(a)中所述正调控因子基因ascR4序列插入到整合型表达载体pSET5035n上的Ned I和Xba I限制性酶切位点之间,使正调控因子基因ascR4位于kasop启动子的下游并受其调控,得到质粒载体pSET5035n-ascR4;
(c)以pSBT11-asc21b载体为模板进行PCR扩增,扩增得到带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列(ermE*p-asc21b),PCR引物序列如下:
asc21b-F:5′-GGCAATTGGGAATTCGTGCACGCGGTCGATCTTGACGGCT-3′,
asc21b-R:5′-TACAAAGATCGGAATTCAGGCCGGAGTGAGGGTGAAGAAG-3′;
(d)将步骤(c)中所述ermE*p-asc21b序列插入到质粒载体pSET5035n-ascR4的EcoRI限制性酶切位点,得到质粒载体pSET5035n-ascR4-asc21b;
(e)将质粒载体pSET5035n-ascR4-asc21b转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b;
(f)将供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b与珍贵橙色束丝放线菌突变株HGF052的菌丝体进行接合转移,得到基因工程菌HGF052+pSET5035n-ascR4-asc21b。
进一步优选的,步骤(a)中,所述正调控因子基因ascR4基因库登记号为KF813023.1,所述正调控因子基因ascR4的开放阅读框序列如SEQ ID NO.1所示;所述整合型表达载体pSET5035n的序列如SEQ ID NO.3所示。
进一步优选的,步骤(c)中,所述带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列如SEQ ID NO.2所示。
进一步优选的,步骤(f)中,所述珍贵橙色束丝放线菌突变株HGF052来自文献ApplMicrobiol Biotechnol 2016,100,2641-2649。
根据本发明优选的,步骤(1)中,所述种子培养条件为在30℃下培养5天,所述ISP4固体培养基成分为:可溶性淀粉10g,K2HPO4 1.0g,MgSO4.7H2O 1.0g,NaCl 1.0g,(NH4)2SO42.0g,CaCO3 2.0g,微量元素溶液1mL,琼脂粉15g,溶于1000mL蒸馏水中;
所述微量元素溶液为:将FeSO4·7H2O 0.1g,MnCl2·4H2O 0.1g,ZnSO4·7H2O 0.1g溶于100mL蒸馏水中。
根据本发明优选的,步骤(2)中,所述发酵培养条件为在30℃下培养10天,YMG固体培养基成分为:葡萄糖4g、麦芽提取物10g、酵母提取物4g、琼脂粉20g,溶于1000mL蒸馏水中。
根据本发明优选的,步骤(3)中,所述浸泡提取时间为20小时,重复次数为2次。
根据本发明优选的,步骤(5)中,所述提取采用的甲醇为95%甲醇。
根据本发明优选的,步骤(6)中,所述反相硅胶柱填料为C-18,凝胶柱型号为Sephadex LH-20,高效液相色谱的洗脱溶剂为37%乙腈。
根据本发明优选的,步骤(6)中,所述对甲醇提取物进行分离的具体步骤为:
甲醇提取物首先经反相硅胶柱层析分离,分别用30%、50%、70%、100%甲醇依次洗脱,每个组分洗脱1L,200~250mL/份接收,薄层层析检测,用CH2Cl2:MeOH=10:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并50%洗脱组分;继续用凝胶柱层析分离,甲醇洗脱,3~5mL/管,合并24~27管;继续高效液相色谱分离,洗脱溶剂为37%乙腈,接收同时合并15.6min洗脱峰,得到氨甲酰美登醇。
经药理试验研究表明,本发明提供的氨甲酰美登醇对人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性,IC50值分别为10.7和19.3nM,本发明还提供氨甲酰美登醇的制药用途,可用于制备抗肿瘤药物。
进一步优选的,所述的肿瘤为宫颈癌或乳腺癌。
一种抗肿瘤的药物组合物,包括本发明的氨甲酰美登醇和一种或多种药学上可接受载体或赋形剂。
本发明未详尽之处,均可采用现有技术。
本发明的有益效果在于:
1、经体外抗肿瘤活性试验表明,本发明提供的氨甲酰美登醇对人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性,IC50值分别为10.7和19.3nM,加之,其C-3位为氨甲酰基,更容易进行取代反应,因此可直接用于制备抗肿瘤药物,可与不同的抗体和连接子组成抗体偶联物。
2、本发明首次发现在细菌美登木素基因簇asc中AscR4调控蛋白属于TrmB家族调控因子,ascR4基因属于途径特异性的正调控因子,可以提高asc基因簇所产化合物的产量。然后本发明进一步通过异源表达正调控因子基因ascR4,提高了珍贵橙色束丝放线菌突变株中细菌美登木素类化合物的产量,并且分离得到一个新化合物—氨甲酰美登醇,其产量达到了8.3mg/L,比单独异源表达氨甲酰基转移酶asc21b基因的珍贵橙色束丝放线菌突变株中氨甲酰美登醇的产量提高了30.7倍,有效地克服了现有技术中存在的氨甲酰美登醇产量低的问题,可以降低了氨甲酰美登醇的生产成本。同时,正调控因子基因ascR4的发现和异源表达的成功,说明本发明可以应用于其他细菌美登木素生产菌株中,以实现各类美登木素衍生物产量的提高,可以用于并促进美登木素抗体偶联物药物的研究、生产和临床应用。
3、本发明通过生物合成方法得到的肿瘤抑制活性的氨甲酰美登醇,可以直接与连接子进行偶联,制备抗体药物偶联物,避免了传统的化学合成方法带来的原料浪费和难以去除的副产物。
附图说明
图1为HPLC检测实施例1共表达基因工程菌和对比例基因工程菌的培养物中氨甲酰美登醇产量。
图中:A为共表达基因工程菌HGF052+pSET5035n-ascR4-asc21b;B为对比例菌株HGF052+pSET5035n-asc21b;1为氨甲酰美登醇。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
安丝菌素AP-3*,上海麦克林生化科技股份有限公司有售。
人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231),中科院上海细胞库有售。
pSBT11-asc21b载体来自文献Organic Letters 21(15):5823-5826。
珍贵橙色束丝放线菌突变株HGF052菌株来自文献Appl Microbiol Biotechnol2016,100,2641-2649。
在以下实施例中所述化合物1的化学结构式如下图所示(结构式中的阿拉伯数字是化学结构中碳原子的标位):
所述ISP4固体培养基成分为:可溶性淀粉10g,K2HPO4 1.0g,MgSO4.7H2O 1.0g,NaCl 1.0g,(NH4)2SO4 2.0g,CaCO3 2.0g,微量元素溶液1mL(微量元素溶液:FeSO4·7H2O0.1g,MnCl2·4H2O 0.1g,ZnSO4·7H2O 0.1g溶于100mL蒸馏水中),琼脂粉15g,溶于1000mL蒸馏水中。
所述YMG固体培养基成分为:葡萄糖4g、麦芽提取物10g、酵母提取物4g、琼脂粉20g,溶于1000mL蒸馏水中。
实施例1、构建基因工程菌
一种基因工程菌HGF052+pSET5035n-ascR4-asc21b的构建方法,包括步骤如下:
(a)以asc基因簇DNA为模板进行PCR扩增,扩增得到正调控因子基因ascR4序列,PCR引物序列如下:
ascR4-F:5′-AAAGGAGGCGGACATATGCTGGACATCATCGGCCTGAGTG-3′,
ascR4-R:5′-CAATTGCCTCTAGATCACGGATGTGCCGAAGGCGAACCCG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足蒸馏水至50μL;
PCR扩增程序:变性,98℃10sec;退火,60℃30sec;延伸,72℃30sec(30~35个循环);终止延伸,72℃10min;最后4℃保温;
所述正调控因子基因ascR4的基因库登记号为KB913032.1,所述基因ascR4的开放阅读框序列如SEQ ID NO.1所示;
(b)将步骤(a)中所述正调控因子基因ascR4序列插入到整合型表达载体pSET5035n上的Ned I和Xba I限制性酶切位点之间,使正调控因子基因ascR4位于kasop启动子的下游并受其调控,得到质粒载体pSET5035n-ascR4;
(c)以pSBT11-asc21b载体为模板进行PCR扩增,扩增得到带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列(ermE*p-asc21b),PCR引物序列如下:
asc21b-F:5′-GGCAATTGGGAATTCGTGCACGCGGTCGATCTTGACGGCT-3′,
asc21b-R:5′-TACAAAGATCGGAATTCAGGCCGGAGTGAGGGTGAAGAAG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足蒸馏水至50μL;
PCR扩增程序:变性,98℃10sec;退火,60℃30sec;延伸,72℃30sec(30~35个循环);终止延伸,72℃10min;最后4℃保温;
所述asc基因簇DNA的基因库登记号为KB913032.1,其中包含有氨甲酰基转移酶基因asc21b和正调控因子基因ascR4的基因序列;所述带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列如SEQ ID NO.2所示;
(d)将步骤(c)中所述ermE*p-asc21b序列插入到质粒载体pSET5035n-ascR4的EcoRI限制性酶切位点,得到质粒载体pSET5035n-ascR4-asc21b;
(e)将质粒载体pSET5035n-ascR4-asc21b转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b;
(f)将供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b与珍贵橙色束丝放线菌突变株HGF052的菌丝体进行接合转移,得到基因工程菌HGF052+pSET5035n-ascR4-asc21b。
实施例2、化合物1的制备
一种使用实施例1所述基因工程菌制备化合物1的方法,包括以下步骤:
(1)将实施例1所述的基因工程菌HGF052+pSET5035n-ascR4-asc21b在ISP4固体培养基上划线活化,于30℃下用10块ISP4固体培养基培养5天,得到活化好的菌种;
(2)挑取步骤(1)活化好的菌种划线接种于18L的YMG固体培养基上,于30℃下发酵培养10天,得到固体培养物;
(3)将固体培养物从YMG固体培养基取出,切成1cm3的小方块,收集于5L三角瓶中,在25℃下用体积比为80:15:5的乙酸乙酯/甲醇/甲酸浸泡提取24小时,合并提取液,减压及37℃的温度下浓缩至干,得粗提物;重复以上步骤2次,合并粗提物;
(4)将粗提物溶于400mL蒸馏水中,使用等体积乙酸乙酯多次萃取,得到乙酸乙酯相;乙酸乙酯相减压及37℃的温度下浓缩至干,得EA提取物;
(5)将EA提取物溶于200mL甲醇中,再经等体积石油醚萃取3次,得到甲醇相;甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(6)将甲醇提取物首先经反相硅胶柱层析(RP-18,200g柱)分离,分别用30%、50%、70%、100%甲醇依次洗脱,每个组分洗脱1L,220mL/份接收,薄层层析检测,用CH2Cl2:MeOH=10:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并50%洗脱组分;继续用凝胶柱层析分离,甲醇洗脱,3mL/管,合并24~27管;继续高效液相色谱分离,洗脱溶剂为37%乙腈,接收并合并15.6min洗脱峰,得到化合物1(149.5mg)。
实施例3、氨甲酰美登醇的鉴定
电喷雾质谱法(ESI-MS)测得实施例2所得化合物1的准分子离子峰为m/z 608.24[M+H]+。1H和13C NMR显示,化合物1共含有29个碳原子(表1),包括3个甲基,2个甲氧基,1个氮甲基,3个亚甲基,10个次甲基和10个季碳。根据HMQC和HMBC的信号,确定了该化合物为氨甲酰美登醇。根据C-3位质子与C-1’的远程相关关系,确定了氨甲酰基的取代位置是在C-3位的O上,对全部的NMR光谱数据进行指定,确定为一新化合物,为氨甲酰美登醇。
表1、化合物1的核磁共振数据
实施例4氨甲酰美登醇的体外抗肿瘤活性试验
采用CCK-8试剂盒测定细胞生长抑制率。
具体方法如下:
1)将人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231)分别培养至对数生长期,胰酶消化,用新鲜的DMEM培养基调整细胞密度至3~7万个/mL制备细胞悬液;
2)在96孔板中接种细胞悬液,每孔约100μL,置于37℃、5.0% CO2饱和湿度的培养箱中培养过夜;
3)弃除细胞培养基,用DMEM培养基将氨甲酰美登醇稀释至2倍检测浓度,取100μL稀释液加入96孔板中,继续培养72h;
4)弃除细胞培养基,用DMEM培养基配置反应液,培养基和CCK-8溶液的体积比为10:1,每孔加入100μL反应液,继续培养4h;
5)使用酶标仪在波长450nm下测定给药孔和空白孔OD值,结果如表2所示。
表2、氨甲酰美登醇对2株肿瘤细胞的细胞毒试验结果(IC50,nM)
*为阳性对照药
细胞生长抑制率=(1-用药组平均OD值/对照组孔OD值)×100%。
试验结果的评判与解释:细胞半数生长抑制时的药物浓度IC50按照量效数据进行换算。每个实验重复三次,吸收值差异小于5%,IC50差异小于30%,以IC50≤100nM为有效标准。
由表2可知,本发明提供的氨甲酰美登醇对人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231)均显示了明显的细胞毒活性,其IC50值分别为10.7和19.3nM。
试验结论:通过药理学试验,可以看出本发明提供的氨甲酰美登醇对人宫颈癌细胞(HeLa)和人乳腺癌细胞(MDA-MB-231)均显示了明显的细胞毒活性。因此,本发明提供的氨甲酰美登醇可用于制备抗肿瘤药物,可与其他药物制成抗肿瘤药物组合物,还可以与不同的抗体和连接子偶联制成抗体偶联物。
对比例
一种氨甲酰基转移酶基因asc21b异源表达菌株的构建,包括步骤如下:
1)以pSBT11-asc21b载体为模板进行PCR扩增,扩增带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列(ermE*p-asc21b),PCR引物序列如下:
asc21b-F:5′-GGCAATTGGGAATTCGTGCACGCGGTCGATCTTGACGGCT-3′,
asc21b-R:5′-TACAAAGATCGGAATTCAGGCCGGAGTGAGGGTGAAGAAG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足蒸馏水至50μL;
PCR扩增程序:变性,98℃10sec;退火,60℃30sec;延伸,72℃30sec(30~35个循环);终止延伸,72℃10min;最后4℃保温;
2)将步骤(1)中所述ermE*p-asc21b序列插入到质粒载体pSET5035n的EcoRI限制性酶切位点之后,得到质粒载体pSET5035n-asc21b;
3)将质粒载体pSET5035n-asc21b转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSET5035n-asc21b;
4)将供体菌ET12567/pUZ8002/pSET5035n-asc21b与珍贵橙色束丝放线菌突变株HGF052的菌丝体进行接合转移,得到共表达菌株HGF052+pSET5035n-asc21b。
试验例
按照实施例2所述方法对实施例1共表达基因工程菌HGF052+pSET5035n-ascR4-asc21b和对比例1的基因工程菌HGF052+pSET5035n-asc21b分别进行固体培养,然后对固体培养物进行HPLC检测,结果如图1所示。
HPLC检测使用Thermo ScientificTMUltiMateTM3000系统,使用SuperLu C18(2)色谱柱(250×4.6mm,5μm)。检测波长为254nm,流速为1mL/min,流动相A为蒸馏水,流动相B为乙腈,洗脱条件见表3。
表3发酵产物HPLC检测方法
由图1可知,以实施例1构建的基因工程菌HGF052+pSET5035n-ascR4-asc21b为生产菌株制备氨甲酰美登醇的产量为149.5mg,以YMG固体培养基计,氨甲酰美登醇的产量为8.3mg/L。而以对比例构建的菌株HGF052+pSET5035n-asc21b为生产菌株,制备氨甲酰美登醇的产量为4.8mg,以YMG固体培养基计,氨甲酰美登醇的产量为0.27mg/L。即本发明通过异源表达asc基因簇的正调控因子基因ascR4有效的提高了氨甲酰美登醇的产量,是对比菌株的30.7倍,有效克服了现有技术中存在的产量低的问题,可以降低美登木素抗体偶联物药物的生产成本,并促进这类药物的研究、生产和临床应用。
Claims (10)
2.如权利要求1所述氨甲酰美登醇的合成方法,其特征在于,包括以下步骤:
(1)将能够表达氨甲酰基转移酶基因asc21b和正调控因子基因ascR4的基因工程菌HGF052+pSET5035n-ascR4-asc21b在ISP4固体培养基上划线活化,于30℃下培养3~5天,得到活化好的菌种;
(2)挑取活化好的菌种接种到YMG固体培养基上,于28~30℃下发酵培养9~12天,得到固体培养物;
(3)将固体培养物取出,切成1cm3的小方块,在20~30℃下用体积比为80:15:5的乙酸乙酯/甲醇/甲酸浸泡提取18~24小时,合并提取液,减压及35~40℃的温度下浓缩至干,得粗提物;再重复2~3次,合并粗提物;
(4)将粗提物溶于400~500mL纯净水中,使用等体积乙酸乙酯萃取2~4次,得到乙酸乙酯相;再将乙酸乙酯相减压及35~40℃的温度下浓缩至干,得EA提取物;
(5)将EA提取物溶于200~300mL甲醇中,再经等体积石油醚多次萃取,得到甲醇相;甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(6)将甲醇提取物依次经反相硅胶柱层析分离,凝胶柱层析分离,薄层层析和高效液相色谱分离,然后将组分相同的洗脱液进行合并,得到氨甲酰美登醇。
3.如权利要求2所述氨甲酰美登醇的合成方法,其特征在于,步骤(1)中,所述基因工程菌HGF052+pSET5035n-ascR4-asc21b的构建方法,包括步骤如下:
(a)以asc基因簇DNA为模板进行PCR扩增,扩增得到正调控因子基因ascR4序列,PCR引物序列如下:
ascR4-F:5′-AAAGGAGGCGGACATATGCTGGACATCATCGGCCTGAGTG-3′,
ascR4-R:5′-CAATTGCCTCTAGATCACGGATGTGCCGAAGGCGAACCCG-3′;
(b)将步骤(a)中所述正调控因子基因ascR4序列插入到整合型表达载体pSET5035n上的Ned I和Xba I限制性酶切位点之间,使正调控因子基因ascR4位于kasop启动子的下游并受其调控,得到质粒载体pSET5035n-ascR4;
(c)以pSBT11-asc21b载体为模板进行PCR扩增,扩增得到带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列(ermE*p-asc21b),PCR引物序列如下:
asc21b-F:5′-GGCAATTGGGAATTCGTGCACGCGGTCGATCTTGACGGCT-3′,
asc21b-R:5′-TACAAAGATCGGAATTCAGGCCGGAGTGAGGGTGAAGAAG-3′;
(d)将步骤(c)中所述ermE*p-asc21b序列插入到质粒载体pSET5035n-ascR4的EcoRI限制性酶切位点,得到质粒载体pSET5035n-ascR4-asc21b;
(e)将质粒载体pSET5035n-ascR4-asc21b转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b;
(f)将供体菌ET12567/pUZ8002/pSET5035n-ascR4-asc21b与珍贵橙色束丝放线菌突变株HGF052的菌丝体进行接合转移,得到基因工程菌HGF052+pSET5035n-ascR4-asc21b。
4.如权利要求3所述氨甲酰美登醇的合成方法,其特征在于,步骤(a)中,所述正调控因子基因ascR4基因库登记号为KF813023.1,所述基因ascR4的开放阅读框序列如SEQ IDNO.1所示;所述整合型表达载体pSET5035n的序列如SEQ ID NO.3所示。
5.如权利要求3所述氨甲酰美登醇的合成方法,其特征在于,步骤(c)中,所述带有ermE*p启动子的氨甲酰基转移酶基因asc21b序列如SEQ ID NO.2所示。
6.如权利要求2所述氨甲酰美登醇的合成方法,其特征在于,步骤(1)中,所述种子培养条件为在30℃下培养5天;所述ISP4固体培养基成分为:可溶性淀粉10g,K2HPO4 1.0g,MgSO4.7H2O 1.0g,NaCl 1.0g,(NH4)2SO4 2.0g,CaCO3 2.0g,微量元素溶液1mL,琼脂粉15g,溶于1000mL蒸馏水中;
所述微量元素溶液为:将FeSO4·7H2O 0.1g,MnCl2·4H2O 0.1g,ZnSO4·7H2O 0.1g溶于100mL蒸馏水中。
7.如权利要求2所述氨甲酰美登醇的合成方法,其特征在于,步骤(2)中,所述发酵培养条件为在30℃下培养10天;所述YMG固体培养基成分为:葡萄糖4g、麦芽提取物10g、酵母提取物4g、琼脂粉20g,溶于1000mL蒸馏水中。
8.如权利要求2所述氨甲酰美登醇的合成方法,其特征在于,步骤(6)中,所述反相硅胶柱填料为C-18,凝胶柱型号为Sephadex LH-20,高效液相色谱的洗脱溶剂为37%乙腈;所述对甲醇提取物进行分离的具体步骤为:甲醇提取物首先经反相硅胶柱层析分离,分别用30%、50%、70%、100%甲醇依次洗脱,每个组分洗脱1L,200~250mL/份接收,薄层层析检测,用CH2Cl2:MeOH=10:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并50%洗脱组分;继续用凝胶柱层析分离,甲醇洗脱,3~5mL/管,合并24~27管;继续高效液相色谱分离,洗脱溶剂为37%乙腈,接收并合并15.6min洗脱峰,得到氨甲酰美登醇。
9.权利要求1所述氨甲酰美登醇在制备抗肿瘤药物中的应用。
10.一种抗肿瘤的药物组合物,其特征在于,包括权利要求1所述氨甲酰美登醇和一种或多种药学上可接受载体或赋形剂。
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