CN103045574B - 冬虫夏草中国被毛孢尿苷酸合成酶、编码基因及其应用 - Google Patents
冬虫夏草中国被毛孢尿苷酸合成酶、编码基因及其应用 Download PDFInfo
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- CN103045574B CN103045574B CN201210535286.4A CN201210535286A CN103045574B CN 103045574 B CN103045574 B CN 103045574B CN 201210535286 A CN201210535286 A CN 201210535286A CN 103045574 B CN103045574 B CN 103045574B
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Abstract
本发明涉及一个来自“百令”生产菌冬虫夏草中国被毛孢参与乳清酸核苷酸出发合成代谢尿苷酸的酶,编码这个酶的基因及其应用;所述酶即为尿苷酸合成酶,氨基酸序列具有SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示氨基酸序列90%以上同源性,其编码基因对应具有SEQ ID No.2、SEQ ID No.4或SEQ ID No.6所示的核苷酸序列90%以上同源性;本发明从原理上对乳清酸核苷酸合成尿苷酸的代谢途径进行了详细研究,包含本发明所提供的核苷酸序列的克隆DNA可以用来通过转导、转化、结合转移的方法转入工程菌中,通过调节尿苷酸生物合成基因的表达,赋予宿主尿苷酸的高表达性,为扩大尿苷酸的产量提供了有效途径,具有重大应用前景。
Description
(一)技术领域
本发明涉及一个来自“百令”生产菌冬虫夏草中国被毛孢的参与乳清酸核苷酸成代谢尿苷酸的尿苷酸合成酶(uridine monophosphatesynthetase),编码这个酶的基因及其应用。
(二)背景技术
冬虫夏草(Cordyceps sinensis (Berk.) Sacc.)是冬虫夏草菌寄生在鳞翅目(Lepidoptera)蝙蝠蛾科昆虫(Hepialus armoricanusOberthur)幼虫上的子座及幼虫尸体上的复合体(包括子座和虫体)。冬虫夏草是一类珍惜的传统真菌药材资源,具有代谢产物和生物活性多样的特点,在生物医药领域展现出巨大的应用和发展前景。冬虫夏草以其多种药用功效广泛、明显而备受关注,在世界范围内备受推崇。中医认为,冬虫夏草入肺肾二经,既能补肺阴,又能补肾阳,主治肾虚,阳痿遗精,腰膝酸痛,病后虚弱,久咳虚弱,劳咳痰血,自汗盗汗等,是唯一的一种能同时平衡、调节阴阳的中药。现代药理学已证实,冬虫夏草具有免疫调节、抗菌、抗肿瘤、抗氧化、抗衰老、降血糖血脂、性激素样作用等广泛的生物活性。
冬虫夏草菌是一种子囊菌,在其生活史中具有分生孢子阶段(无性型)和子囊孢子阶段(有性型)。而在人工培养、液体发酵等实际生产中使用的是无性阶段的冬虫夏草菌,因而冬虫夏草无性型的鉴定非常重要。国内外学者在冬虫夏草资源调查、无性型确证、活性成分分离分析和作用机理、开发应用方面做了大量工作。冬虫夏草中国被毛孢已被证明是冬虫夏草的无性型存在形式,具有与天然冬虫夏草相同的活性成分和药效。
天然虫草具有严格的寄生性以及特殊的生态环境,故其产量很低,价格高昂。野生冬虫夏草由于受生长环境等因素制约,资源匮乏。由于近几年在人工栽培上进展不大,野生冬虫夏草代用品研究多集中在液体发酵上。利用液体深层发酵培养冬虫夏草菌丝体、提取物或发酵液,是解决冬虫夏草药源的一种有效途径。虫草发酵生产虫草替代品,既可有效保护虫草这一珍贵资源,又不受气候、地理环境和虫草寄生条件严格的限制,适合于工业化大规模生产。生产出的替代品如菌丝体其成分和药效也与天然虫草相似,因而国内外一直致力于虫草菌丝体的发酵培养。人工发酵培养冬虫夏草中国被毛孢得到的菌丝,经毒理、药理、植化研究,证明与天然虫草化学组成、药理作用基本一致,可代替天然虫草生产虫草制品,以弥补自然资源的短缺,通过对发酵条件的优化,菌丝体生物量和代谢产物的量均有明显提高。
近年来,随着天然产物化学和现代色谱技术的飞速发展,在对虫草产品研发中已逐步由虫草原料或粗提物的直接利用转向更深层次的功能性代谢产物研究。国内外已对虫草代谢产物做了大量的研究,代谢产物主要包括核苷、多糖、多肽、甾醇等几大类化合物,其中嘌呤类核苷、虫草多糖、甘露醇等具有代表性的功能性代谢产物在生物合成、药理作用等方面的研究初见成效。
核苷类是虫草最主要的活性物质之一,其中嘧啶类核苷包括胞嘧啶核苷和尿嘧啶核苷。尿嘧啶核苷(uracil riboside)又称尿苷(uIidine),别称由雪核糖苷、二氢嘧啶核苷、1-β-D-喃核糖基尿嘧啶。胞嘧啶核苷(cytosine riboside)又名胞苷(cytidine)、胞啶、1-β-D-呋喃核苷胞嘧啶。嘧啶核苷是多用途的核苷,可作为保健品和食品的添加剂。它作为美容和抗紫外辐射化妆品也逐渐成为人们生活的必需品,作为药物,已临床应用于中枢神经、泌尿、代谢和心血管等多方面的疾病,在美国,核酸类药品近年来在抗病毒、抗肿瘤方面显示了不可替代的作用。
研究表明胞苷主要用于生产抗肿瘤、抗病毒药物的中间体,随着抗病毒、抗肿瘤、治疗艾滋病药物研制的深入,对天然嘧啶核苷的需求量加大。胞苷作为嘧啶核苷,主要用于生产抗肿瘤、抗病毒药物的中间体,是制造阿糖胞苷(Ara—CR)、环胞苷(Cyclo C)、三磷酸胞苷(CTP)、胞二磷胆碱(CDP—Choline)等药物的主要原料。
尿苷是一种天然的核苷,是所有其他嘧啶核苷酸的前体,尿苷酸是构成细胞核糖核酸的4种主要单核苷酸之一,具有嘧啶环结构,它参与糖原合成,有助于提高细胞的耐缺氧能力。它也是构成动物细胞核酸的成分,能提高机体抗体水平,尿苷及其衍生物还可以用来阻断各种癌细胞和病毒的基因合成,治疗癌症和由病毒引起的疾病。
由于嘧啶核苷及其类似物的重要生理作用,国内外有关微生物生产嘧啶类核苷已有很多研究。然而,作为重要合成代谢嘧啶类核苷的冬虫夏草菌,还只停留在代谢产物成分分析和功效的研究上,对冬虫夏草菌嘧啶类核苷合成代谢途径中相关基因和蛋白的研究还很少见。
(三)发明内容
本发明目的在于针对以上存在的不足和需要解决的技术问题,对“百令”生产菌冬虫夏草中国被毛孢合成代谢尿苷酸的酶及其编码基因进行深入研究,提供了“百令”生产菌冬虫夏草中国被毛孢参与乳清酸核苷酸出发合成代谢尿苷酸的酶(即尿苷酸合成酶)、编码基因及其应用。
本发明采用的技术方案是:
本发明提供一种来自“百令”生产菌冬虫夏草中国被毛孢参与乳清酸核苷酸出发合成代谢尿苷酸的冬虫夏草中国被毛孢尿苷酸合成酶:所述酶具有SEQ ID No.1所示氨基酸序列、SEQ ID No.3所示氨基酸序列或SEQ ID No.5所示氨基酸序列90%以上同源性,优选所述酶氨基酸序列为SEQ ID No.1的pynC1蛋白、序列为SEQ ID No.3的pynC2蛋白或序列为SEQ ID No.5的pynC3蛋白;该酶可催化乳清酸核苷酸制备尿苷酸。由于氨基酸序列的特殊性,任何含有SEQ ID No.1、SEQ ID No.3或SEQ ID No.5所示氨基酸序列的肽蛋白的片段或其变体,如其保守性变体、生物活性片段或衍生物,只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在90%以上,均属于本发明保护范围之列。具体的所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换;其中,对于变体的保守性改变,所替换的氨基酸具有与原氨基酸相似的结构或化学性质,如用亮氨酸替换异亮氨酸,变体也可具有非保守性改变,如用色氨酸替换甘氨酸。
本发明还涉及所述尿苷酸合成酶在生物催化乳清酸核苷酸制备尿苷酸中的应用。
进一步,所述的应用为:以含冬虫夏草中国被毛孢尿苷酸合成酶菌体发酵培养获得的湿菌体经细胞破碎后的破碎混合液为催化剂,以乳清酸核苷酸为底物,于pH为6.5~8.5的缓冲溶液构成的转化体系中,在30℃、150rpm条件下转化反应2~3h,反应结束后,将反应液过滤,取滤液即为含尿苷酸的粗品,所述粗品分离纯化获得尿苷酸。
更进一步,所述底物的初始浓度为10g/L,所述催化剂的体积用量为100mL/g底物,所述催化剂的干菌体浓度为6.7mg/mL。
由乳清酸核苷酸合成代谢获得尿苷酸的路径如下式所示:
所述催化剂的制备方法为:将冬虫夏草中国被毛孢尿苷酸合成酶重组菌接种于5mL含有Kan抗性(50mg/L)的LB液体培养基中,37℃、250r/min培养过夜。取1mL培养物,将其转接于50mL新鲜的含有Kan抗性的LB液体培养基中37℃、250r/min培养至菌体浓度OD600约为0.6~0.8左右,向培养物中加入一定浓度(240mg/ml)的IPTG诱导培养8h,取诱导培养液过滤,收集湿菌体,称取收集的湿菌体0.5g用磷酸盐缓冲液(50mM、pH8.0)15mL悬浮,超声破碎(功率350W、破碎2s、间隔2s、共超声破碎5min)后获得的菌体混合液作催化用酶。
本发明还涉及上述冬虫夏草中国被毛孢尿苷酸合成酶的编码基因,所述基因具有SEQ ID No.2所示核苷酸序列、SEQ ID No.4所示核苷酸序列或SEQ ID No.6所示核苷酸序列90%以上的同源性,优选所述基因序列为SEQ ID No.2的pynC1基因、序列为SEQ ID No.4的pynC2基因或序列为SEQ ID No.6的pynC3基因。由于核苷酸序列的特殊性,任何SEQ ID NO:2、SEQ ID No.4或SEQ ID No.6所示多核苷酸的变体,只要其与该多核苷酸具有90%以上同源性,均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的变位变异体或非生的变异体,包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个多核苷酸的取代、缺失或插入,但不会从实质上改变其编码的肽蛋白的功能。
本发明还涉及所述冬虫夏草中国被毛孢尿苷酸合成酶编码基因在构建能够生物催化乳清酸核苷酸制备尿苷酸的基因工程菌中的应用,以扩大尿苷酸或其衍生物的产量。
优选所述的应用为:构建含有所述冬虫夏草中国被毛孢尿苷酸合成酶基因的重组载体,将所述重组载体转化至大肠杆菌中,获得的重组基因工程菌进行诱导培养,培养液分离纯化获得含有冬虫夏草中国被毛孢尿苷酸合成酶的菌体细胞。
本发明所述冬虫夏草中国被毛孢尿苷酸合成酶的酶活定义为:在最适条件(30℃)下,在1分钟内能转化1微摩尔乳清酸核苷酸的酶量。
能够提供本发明所述冬虫夏草中国被毛孢尿苷酸合成酶及其编码基因的菌株为中国被毛孢(Hirsutella Sinensis)L0106,该菌种保藏在中国典型培养物保藏中心,保藏编号为CCTCC No:M 2011278,已在先前申请的专利CN102373190A中披露。
本发明的要点在于提供了SEQ ID NO:1、SEQ ID NO:3或SEQ ID NO:5所示的氨基酸序列和SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:6所示的核苷酸序列,在已知该氨基酸序列和核苷酸序列的情况下,该氨基酸序列和核苷酸序列的获得,以及相关载体、宿主细胞的获得,对于本领域技术人员来说均是显而易见的。
本发明的有益效果主要体现在:本发明从原理上对乳清酸核苷酸合成尿苷酸代谢途径进行了详细研究,包含本发明所提供的核苷酸序列的克隆DNA可以用来通过转导、转化、结合转移的方法转入工程菌中,通过调节尿苷酸生物合成基因的表达,赋予宿主尿苷酸的高表达性,为扩大尿苷酸或其衍生物的产量提供了有效途径,具有重大应用前景。
(四)附图说明
图1为“百令”生产菌冬虫夏草中国被毛孢总RNA的甲醛变性凝胶电泳图;
图2为嘧啶代谢途径注释图;
图3为尿苷酸合成酶基因PCR扩增产物凝胶电泳图;
图4为克隆载体pMD18-T Vector与表达载体pET-28a物理图谱;
图5为重组克隆质粒pMD18-T / pynC物理图谱;
图6为重组表达质粒pET-28a / pynC构建过程示意图;
图7为重组表达质粒pET-28a / pynC物理图谱;
图8为尿苷酸合成酶蛋白SDS-PAGE图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1: “百令”生产菌冬虫夏草中国被毛孢的培养
菌株来源:首先从青海采集天然冬虫夏草,并将其带回杭州进行分离筛选,得到了L0106菌株,并经菌种鉴定该菌株为中国被毛孢(Hirsutella Sinensis),该菌种保藏在中国典型培养物保藏中心,保藏编号为CCTCC No: M 2011278,已在先前申请的专利CN102373190A中披露。
将该菌种接种于斜面,培养基配方(此为固化之前的液体配方,按下述比例配制好之后再制成斜面)为葡萄糖2.0%(w/v,1%表示100mL培养基中含有1g,下同)、玉米粉1.0%、土豆汁0.5%、糊精0.5%、酵母粉0.5%、麸皮1.0%、蚕蛹粉2.0%、蛋白胨1.0%、硫酸镁0.05%、磷酸二氢钾0.05%、琼脂粉1.0%,余量为水,在12~16℃培养25天;然后将菌种接种于发酵培养基,培养基配方为葡萄糖1.0%、糖蜜1.0%、蚕蛹粉0.5%、黄豆饼粉1.0%、酵母膏0.5%、硫酸镁0.01%、磷酸二氢钾0.02%,余量为水,置于摇床上,温度12~16℃培养25天,培养结束后在无菌条件下,进行固液分离,并将固体(即湿菌体)置于无菌器具,备用。
实施例2:“百令”生产菌冬虫夏草中国被毛孢总RNA的提取
用TRIzol试剂提取总RNA,步骤具体为:1)液氮研磨:取1g新鲜菌体放入研钵中,反复加入液氮充分研磨至粉末状,分装到预冷的1.5mL离心管中,加入1mL TRIzol试剂,混匀,冰上静置5min,使核酸蛋白复合物完全分离。2)RNA分离:加入0.2mL氯仿,用力震荡混匀15s,冰上静置2~3min,4℃、12000rpm离心15min,分层,取上层水相,约600μL。3)RNA沉淀:加入500μL异丙醇,在冰上静置10min,4℃、12000rpm离心10min,弃上清。4)RNA洗涤:加入1mL 75%(v/v)乙醇,将沉淀悬起,冰上静置10min,4℃、7500rpm离心15min;重复上面洗涤步骤,再洗一遍。5)溶解RNA:将离心管置于冰上敞开干燥5~10min,加适量DEPC水溶解,获得“百令”生产菌冬虫夏草中国被毛孢总RNA。
实施例3:“百令”生产菌冬虫夏草中国被毛孢RNA样品测序
提取样品总RNA后,用带有Oligo(dT)的磁珠富集mRNA。加入fragmentation buffer将mRNA打断成短片段(200~700bp),以mRNA为模板,用六碱基随机引物(random hexamers)合成第一条cDNA链,然后合成第二条cDNA链,再经过QiaQuick PCR试剂盒纯化并加EB缓冲液洗脱之后做末端修复、加polyA并连接测序接头,然后用琼脂糖凝胶电泳进行片段大小选择,最后进行PCR扩增,建好的测序文库用IlluminaGA IIx进行测序。测序得到的原始图像数据经base calling转化为序列数据,即raw data或raw reads。除去原始测序reads中只含adaptor序列的reads,备以后续分析。
实施例4:“百令”生产菌冬虫夏草中国被毛孢RNA短读序列组装
使用短reads组装软件SOAPdenovo(Li, Zhu et al. De novoassembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010, 20: 265-272.)做转录组从头组装。SOAPdenovo首先将具有一定长度overlap的reads连成更长的不含N的Contig片段。然后将reads比对回Contig,通过paired-end reads确定来自同一转录本的不同Contig以及这些Contig之间的距离,SOAPdenovo将这些Contig连在一起,中间未知序列用N表示,这样就得到Scaffold。进一步利用paired-end reads对Scaffold做补洞处理,最后得到含N最少,两端不能再延长的Unigene序列。最后,将Unigene序列与蛋白数据库nr、Swiss-Prot、KEGG和COG做blastx比对(evalue<0.00001),取比对结果最好的蛋白确定Unigene的序列方向。如果不同库之间的比对结果有矛盾,则按nr、Swiss-Prot、KEGG和COG的优先级确定Unigene的序列方向,跟以上四个库皆对比不上的Unigene用软件ESTScan(Iseli, Jongeneel et al.ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences[J]. In Proceedings of 9th InternationalConference onIntelligent Systems for Molecular Biology. AAAIPress,Menlo Park, CA, pp. 1999, 138-148.)预测其编码区并确定序列的方向。对于能确定序列方向的Unigene给出其从5'到3'方向的序列,对于无法确定序列方向的Unigene给出组装软件得到的序列。
实施例5:“百令”生产菌冬虫夏草中国被毛孢Unigene功能注释
功能注释信息给出Unigene的蛋白功能注释、Pathway注释、COG功能注释和Gene Ontology(GO)功能注释。首先,通过blastx将Unigene序列比对到蛋白数据库nr、Swiss-Prot、KEGG和COG(evalue<0.00001),得到跟给定Unigene具有最高序列相似性的蛋白,从而得到该Unigene的蛋白功能注释信息。根据KEGG注释信息能进一步得到Unigene的Pathway注释。将Unigene和COG数据库进行比对,预测Unigene可能的功能并对其做功能分类统计。根据nr注释信息,使用Blast2GO软件(Conesa, Gotz et al. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research[J]. Bioinformatics, 2005, 21(18): 3674-3676.)得到Unigene的GO注释信息。得到每个Unigene的GO注释后,用WEGO软件(Ye, Fang et al. WEGO: a web tool for plotting GO annotations[J]. Nucleic Acids Research, 2006,34: 293-297.)对所有Unigene做GO功能分类统计,从宏观上认识该物种的基因功能分布特征。
实施例6:“百令”生产菌冬虫夏草中国被毛孢尿苷酸代谢途径分析
图2是KEGG代谢途径注释中的嘧啶代谢(map00240),已注释的酶是已检测到的“百令”生产菌冬虫夏草中国被毛孢嘧啶代谢途径相关酶类,从图中可以看出,检测到了从乳清酸核苷酸出发合成尿苷酸的尿苷酸合成酶3个Unigene。通过NCBI中的ORF Finder软件在线检测,找出了这个基因的开放阅读框(SEQ ID No.2、SEQ ID No.4和SEQ ID No.6)并得到了相应的蛋白质序列(SEQ ID No.1、SEQ ID No.3和SEQ ID No.5)。
实施例7:“百令”生产菌冬虫夏草中国被毛孢尿苷酸合成酶基因引物设计
运用GENE RUNNER引物设计软件根据预测得到的基因开放阅读框DNA序列设计引物,用于克隆“百令”生产菌中国被毛孢合成代谢尿苷酸的尿苷酸合成酶基因,引物由上海生工生物工程有限公司合成,引物序列如下所列:
pynC1基因:正向引物5’ AGAGAATTCATGTTGGCTGACGCTAGGGTC3’
反向引物5’ ATAAAGCTTTCACCCTCTGCCGCACCAACTTG3’
pynC1基因长度为414bp
pynC2基因:正向引物5’ ATTGGATCCATGCAGTACACTGGCGGCTC3’
反向引物5’ ATTGAGCTCTCAGCGAACTCTCTCGAGGTATG3’
pynC2基因长度为768bp
pynC3基因:正向引物5’ ATAGAATTCATGGCGTCTCAGCTGCCCGCG3’
反向引物5’ AGCAAGCTTTCACTTAGTTGGCGGGTACTTG3’
pynC3基因长度为738bp
实施例8:“百令”生产菌冬虫夏草中国被毛孢cDNA第一链的制备
先按照实施例1提供的方法培养出中国被毛孢发酵菌丝体后,再按照实施例2所提供的方法对中国被毛孢进行总RNA的提取,得到总RNA后按下述进行“百令”生产菌冬虫夏草中国被毛孢cDNA第一链的合成,用于后续各基因克隆实验。
采用PrimeScript 1st Strand cDNA Synthesis Kit试剂盒(TaKaRa)从Total RNA中反转录合成cDNA第一链,实验步骤如下:
1)在Microtube管中配制下列混合液。
2)变性、退火操作有利于模板RNA的变性以及反转录引物和模板的特异性退火,可提高反转录反应效率,所以在PCR仪上进行变性、退火反应,条件设置如下:
65℃,5 min。
3)退火结束后离心数秒钟使模板RNA/引物等的混合液聚集于Microtube管底部。
4)在上述Microtube管中配制下列反转录反应液。
5)在PCR仪上按下列条件进行反转录反应。
42℃ 15~30 min
70℃ 15 min
一般情况,在真核生物mRNA 3’末端都有一个PolyA结构,A碱基的数量在十至几百个不等,利用这一结构可以利用Oligo(dT)引物,在反转录酶的作用下,以mRNA为模板合成cDNA第一链,本发明采用由TaKaRa独自开发的dT区域的序列(PrimeScript 1st Strand cDNA Synthesis Kit中提供)为引物,如果获得的mRNA完整性较好,那么通过逆转录过程可以得到物种中所有酶蛋白编码基因的cDNA第一链。
实施例9:“百令”生产菌冬虫夏草中国被毛孢合成代谢尿苷酸功能基因尿苷酸合成酶pynC基因的克隆、表达以及蛋白活力的检测
1、尿苷酸合成酶pynC1/C2/C3基因的PCR扩增
以实施例8中得到的cDNA第一链为模板,用实施例7中合成的pyn C1基因引物:5’ AGA GAA TTC ATG TTG GCT GAC GCT AGG GTC 3’、5’ ATA AAG CTT TCA CCC TCT GCC GCA CCA ACT TG 3’、 pyn C2基因引物:5’ ATT GGA TCC ATG CAGTAC ACT GGC GGC TC 3’、5’ ATT GAG CTC TCA GCG AAC TCT CTC GAG GTA TG 3’、 pyn C3基因引物:5’ ATAGAA TTC ATG GCG TCT CAG CTG CCC GCG 3’、5’ AGCAAGCTTTCACTTAGTTGGCGGGTACTTG 3’进行Pfu DNA聚合酶PCR扩增反应,条件设置如下:
Pfu PCR扩增反应体系:
Pfu DNA Ploymerase PCR扩增条件:
2、尿苷酸合成酶pynC1、pynC2、pynC3基因PCR产物凝胶电泳检测
具体检测方法为:1)将配制好的0.9%的琼脂糖凝胶用微波炉加热使其溶解均匀;2)取15mL凝胶,待凝胶冷却至50℃左右时,加入1μL染色液Gold view,混合均匀后倒入电泳凝胶板上,除去气泡后插入点样梳;3)凝胶凝固后,小心取出点样梳,将胶板放入电泳槽中(点样孔一端靠近电泳槽的负极),在电泳槽中加入TAE电泳缓冲液;4)取5μL样品然后加入6×Loading Buffer 1.5μL和ddH2O 4μL混合后用移液枪上样,上样量为10 μL;5)连接电泳槽与电泳仪之间的电源线,正极为红色,负极为黑色;6)开启电源,开始电泳,最高电压不超过5 V/cm;7)当样品跑过胶板的2/3时可终止电泳;8)切断电源后,将凝胶取出放入凝胶成像仪中观察、拍照。
转录组测序预测尿苷酸合成酶pynC1、pynC2和pynC3基因的大小为414bp、768bp、738bp,琼脂糖凝胶电泳结果(图3)表明已成功扩增出了尿苷酸合成酶pynC1、pynC2和pynC3基因。
3、尿苷酸合成酶pynC1、pynC2和pynC3基因PCR产物的加碱基A处理以及纯化
由于Pfu DNA聚合酶PCR产物末端为平端,所以在胶回收后还需进行加碱基A处理、纯化后才可用于T载体连接。胶回收产物加碱基A体系如下:
PCR仪中72℃加A碱基20 min,最后用AxyPrep PCR 清洁试剂盒纯化。
4、尿苷酸合成酶pynC1、pynC2和pynC3基因与克隆载体的连接
克隆载体pMD18-T Vector购自TaKaRa公司(TaKaRa code D101A),其物理图谱见图4,将尿苷酸合成酶pynC1、pynC2和pynC3基因分别与克隆载体连接构建重组质粒pMD18-T/pynC1、pMD18-T/pynC2和pMD18-T/pynC3,物理图谱见图5,连接体系和连接条件如下。
连接体系:
连接条件:16℃,16h;灭活:65℃,15min。
5、尿苷酸合成酶重组质粒pMD18-T/pynC1、pMD18-T/pynC2、 pMD18-T/pynC3的转化
分别将重组质粒pMD18-T/pynC1、pMD18-T/pynC2、 pMD18-T/pynC3转入大肠杆菌E. coli JM109中构建携带尿苷酸合成酶pynC1、pynC2和pynC3基因的重组菌E.coliJM109/pMD18-T/pynC1、E.coliJM109/pMD18-T/pynC2、E.coli JM109/pMD18-T/pynC3。具体步骤为:1)将10μL反应体系转至感受态细胞E. coli JM109中,冰浴30min;2)热击:42℃,90s;3)冰浴:2-3min;4)加入800μL液体LB,37℃,250rpm,1h;5)涂布平板(含Amp抗性);6)37℃培养箱培养过夜。
6、尿苷酸合成酶E. coli JM109/pMD18-T/pynC1、E. coli JM109/pMD18-T/pynC2、E. coli JM109/pMD18-T/pynC3阳性重组菌的筛选
菌落PCR可不必提取基因组DNA,而直接以菌体热解后暴露的DNA为模板进行PCR扩增,该方法操作简便、快捷、可以快速鉴定菌落是否为含有目的质粒的阳性菌落。菌落首先,用牙签挑取单菌落加入含50μL无菌水的1.5mL离心管中,沸水浴30min,然后离心以上清作为模板,进行PCR扩增,PCR程序设定为Taq酶扩增一般程序。最后采用0.9%的琼脂糖凝胶电泳检测菌落PCR产物。
7、尿苷酸合成酶重组质粒pMD18-T/pynC1、pMD18-T/pynC2、pMD18-T/pynC3的测序
对菌落PCR检测出的阳性重组菌液体LB培养基培养过夜后,取4mL菌液提取质粒,方法按AxyPrep质粒DNA小量试剂盒提供的操作说明。测序由上海桑尼生物科技有限公司完成。经测序验证,序列SEQ ID No.2、SEQ ID No.4和SEQ ID No.6已经分别重组至重组质粒pMD18-T/pynC1、pMD18-T/pynC2和pMD18-T/pynC3中。
8、尿苷酸合成酶重组表达质粒pET-28a/pynC1、pET-28a/pynC2、pET-28a/pynC3的构建
实验根据外源基因在大肠杆菌中表达的原则,以及表达载体pET-28a和尿苷酸合成酶pynC1、pynC2、pynC3基因酶切位点比对情况,确定了pynC1和pynC3为EcoRⅠ和Hind Ⅲ双酶切位点,pynC2为BamHI/SacI酶切位点,并对重组大肠杆菌E. coli JM109/pMD18-T/pynC1、E. coli JM109/pMD18-T/pynC2和 E. coli JM109/pMD18-T/pynC3进行液体LB试管摇床培养、重组质粒提取。
尿苷酸合成酶pynC1和pynC3基因的重组质粒pMD18-T/pynC1、和pMD18-T/pynC3及表达载体pET-28a分别用BamHI/SacI Ⅲ限制性内切酶在37℃分别酶切处理6h,pynC2基因的重组质粒pMD18-T/pynC2及表达载体pET-28a用BamHI/SacI限制性内切酶在37℃处理6h,酶切体系如下所示:
BamH I/SacI双酶切体系:
酶切结束后65℃灭活15min,然后分别用Axygen DNA凝胶回收试剂盒进行回收、纯化。
尿苷酸合成酶pynC1、pynC2、pynC3基因及表达载体pET-28a经双酶切、纯化后再用T4连接酶16℃连接过夜,构建重组表达质粒pET-28a/pynC1、pET-28a/pynC2、pET-28a/pynC3,其构建过程见图6,构建得到的重组表达质粒pET-28a/pynC1、pET-28a/pynC2、pET-28a/pynC3图谱见图7。连接体系组成如下:
连接体系:
9、尿苷酸合成酶重组表达质粒pET-28a/pynC1、pET-28a/pynC2、pET-28a/pynC3的转化以及阳性单克隆的筛选
将构建好的表达质粒热激转化至E. coli BL21宿主菌中,然后涂布到含有(50mg/L)卡那霉素(Kan)抗性的LB琼脂平板上,37℃培养过夜。从平板上随机挑选单菌落,以各功能基因的引物进行PCR扩增,挑选阳性克隆。
10、尿苷酸合成酶重组菌E. coli BL21/pET-28a/pynC1、E. coliBL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3的诱导表达
将鉴定为阳性的单克隆接种于5mL含有Kan抗性(50mg/L)的LB液体培养基中,37℃、250r/min培养过夜。取1mL培养物,将其转接于50mL新鲜含有Kan抗性(50mg/L)的LB液体培养基中37℃、250r/min培养至菌体浓度OD600约为0.6~0.8左右。向培养物中分别加入一定浓度(240mg/mL)的IPTG诱导培养8h。收集湿菌体供电泳分析以及酶活检测。
11、尿苷酸合成酶重组菌E. coli BL21/pET-28a/pynC1、E. coliBL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3表达产物SDS-PAGE分析
以转入空载体的E. coli BL21菌及未加入诱导剂IPTG的重组菌作为对照。鉴定为阳性的重组菌经IPTG诱导培养8h后,取0.5mL诱导培养物,离心收集菌体,重悬于50μL蒸馏水中,加入50μL上样缓冲液,混匀后煮沸10min,进行SDS-PAGE电泳分析,图8中的“C1” 、“C2”、 “C3”泳道即分别为重组菌E. coli BL21/pET-28a/pynC1、E.coli BL21/pET-28a/pynC2、E. coli BL21/pET-28a/pynC3表达的尿苷酸合成酶蛋白pynC1(经测序验证其氨基酸序列为SEQ ID No.1所示)、pynC2(经测序验证其氨基酸序列为SEQ ID No.3所示)、pynC3(经测序验证其氨基酸序列为SEQ ID No.5所示)的SDS-PAGE图。
12、尿苷酸合成酶重组菌E. coli BL21/pET-28a/pynC1、E. coliBL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3的蛋白活性检测
酶液制备:称取方法10收集的重组菌E. coli BL21/pET-28a/pynC1、E. coli BL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3湿菌体各0.5g(干重0.1g),分别用磷酸盐缓冲液(50mM、pH8.0)15mL悬浮,超声破碎(功率350W、破碎2s、间隔2s、共超声破碎5min)后用作催化用酶。
尿苷酸合成酶pynC转化体系:分别在50mL转化瓶中加入E. coli BL21/pET-28a/pynC1、E. coli BL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3超声破碎菌体混合液10mL、0.1g 乳清酸核苷酸,30℃、150r/min转化反应2~3h,转化结束后,将转化液离心,分别取上清备以后续检测。
检测方法:高效液相色谱法定量检测尿苷酸的转化情况。前处理:转化液离心后,上清液过0.22μm微孔膜用于高效液相色谱检测。条件:色谱柱:安捷伦C18柱(4.6mm×250mm i.d.5μm);柱温:35℃;进样体积:20μL;流速1mL/min;检测波长260nm;流动相:A:超纯水;B:甲醇。梯度洗脱:0min,15%B,保持3min;3.0-3.5min,15-24%B;3.5min,24%保持5min;8.5-9.0min,24-35%B;35%B保持6min;15.0-16.0min,35-85%B;85%B保持6min;22.0-22.5min,85%-15%B;15%B保持5min。
标准曲线的制作:精确称取尿苷酸标准品,约1.00mg-2.00mg,超纯水定容。标准品配制6个梯度(1μg/mL, 30μg/mL,60μg/mL,90μg/mL,120μg/mL,150μg/mL)。然后依次上样,每个浓度5次重复。取一定体积最高浓度的标准液混合在一起后,定容,然后HPLC分析尿苷酸检测的最优条件。
经过上述的色谱条件检测和计算,我们得出以下结论:尿苷酸合成酶重组菌E. coli BL21/pET-28a/pynC1、E. coli BL21/pET-28a/pynC2和E. coli BL21/pET-28a/pynC3所表达的尿苷酸合成酶氧化酶的最大比酶活(Specific Activity)分别为65mol/min/mg、63mol/min/mg、59mol/min/mg,底物转化率分别为76%、72%和69%。
SEQUENCE LISTING
<110> 浙江工业大学、杭州中美华东制药有限公司
<120> 冬虫夏草中国被毛孢尿苷酸合成酶、编码基因及其应用
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Claims (8)
1.一种冬虫夏草中国被毛孢尿苷酸合成酶,其特征在于所述酶的氨基酸序列为SEQ ID No.1所示。
2.如权利要求1所述冬虫夏草中国被毛孢尿苷酸合成酶在生物催化乳清酸核苷酸制备尿苷酸中的应用。
3.如权利要求2所述的应用,其特征在于所述的应用为:以含冬虫夏草中国被毛孢尿苷酸合成酶菌体发酵培养获得的湿菌体经细胞破碎后的破碎混合液为催化剂,以乳清酸核苷酸为底物,于pH为6.5~8.5的缓冲溶液构成的转化体系中,在30℃、150rpm条件下转化反应2~3h,反应结束后,将反应液过滤,取滤液即为含尿苷酸的粗品,所述粗品分离纯化获得尿苷酸。
4.如权利要求3所述的应用,其特征在于所述底物的初始浓度为10g/L,所述催化剂的体积用量为100mL/g底物,所述催化剂的干菌体浓度为6.7mg/mL。
5.一种编码权利要求1所述酶的基因。
6.如权利要求5所述基因,其特征在于所述基因的核苷酸序列为SEQ ID No.2所示。
7.如权利要求5或6所述基因在构建能够生物催化乳清酸核苷酸制备尿苷酸的基因工程菌中的应用。
8.如权利要求7所述的应用,其特征在于所述的应用为:构建含有所述冬虫夏草中国被毛孢尿苷酸合成酶基因的重组载体,将所述重组载体转化至大肠杆菌中,获得的重组基因工程菌进行诱导培养,培养液分离纯化获得含有冬虫夏草中国被毛孢尿苷酸合成酶的菌体细胞。
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