CN101962654B - 胸苷酸合成酶在大肠杆菌中的高效表达 - Google Patents
胸苷酸合成酶在大肠杆菌中的高效表达 Download PDFInfo
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Abstract
本发明为：胸苷酸合成酶在大肠杆菌中高效表达的新方法。TS是生物体内催化胸苷酸合成所必需的酶，在快速增殖的细胞中抑制TS活性可使DNA合成受阻，引起细胞死亡，多年来TS一直是肿瘤化学治疗的重要靶点之一，研究其酶学性质有助于开发新型抗癌药物。人类TS基因为真核基因，含有大量原核生物稀有密码子，传统的表达方法获得纯酶量少、操作量大。本发明采用分子生物学的方法、在不发生定点突变下，构建含有野生型人类胸苷酸合成酶基因的大肠杆菌表达质粒TS-pET-28b(+)，转入含有真核生物稀有密码子的菌株Rosetta(DE3)，通过发酵条件的优化，获得高效表达，因而本系统具有表达水平高、成本低等特点。
Description
技术领域：
本发明涉及野生型人类胸苷酸合成酶(thynidylate synthase，TS)基因在不发生定点突变情况下的克隆，含有该基因的原核表达载体TS-pET-28b(+)的构建，以及该基因在大肠杆菌中的高效表达。
技术背景：
世界经济的快速发展，人们越来越多接触包括烟草、酒精、病毒与感染、放射和辐射等一系列致癌物质，全球每年约有新发病例1100万。中国癌症发病率占全球的20.3％，每年新发病例超过220万，癌症已经成为全球的主要疾病负担，全球发病率也呈总体上升趋势。研发一种高效，低毒的抗癌药物已经成为各国政府，以及科研工作者孜孜以求的梦想，然而传统的研发手段低效性，限制了对抗癌药物的研究，为了获得一条快捷的研发路径，科学家逐渐倾向于从基因组学以及蛋白质组学的角度，开展研究工作。多年来TS一直是肿瘤化学治疗以及抑制微生物生长的重要靶点。
胸苷酸合成酶(thymidylate synthase，TS，EC 2.1.1.45)是由两个相同的亚单位构成二聚体蛋白，每个亚单位的分子量约36.0kDa，是生物体内催化胸苷酸合成所必需的酶，能催化生物体内的2′-脱氧尿苷-5′-磷酸(dUMP)甲基化形成2′-脱氧胸苷-5′-磷酸(dTMP)，dTMP进一步在细胞内代谢为脱氧胸苷三磷酸(dTTP)，dTTP为DNA合成所必需的前体物质。在快速增殖的细胞中抑制TS活性可使DNA合成受阻，引起细胞死亡，多年来TS一直是肿瘤化学治疗的重要靶点之一。基于TS在肿瘤研究中的重要地位，人们越来越多的去研究TS蛋白自身结构以及TS蛋白与抑制剂之间的关系，这就需要以大量的TS货白为基础。20世纪70年代，Lockshin，Dolnick等从人类肿瘤细胞中成功提取获得胸苷酸合成酶。随着基因异源重组以及原核表达系统，尤其是大肠杆菌表达系统的逐渐完善，通过原核表达来获得大量胸苷酸合成酶成为一条行之有效的方法。Daniel等首次实现人类胸苷酸合成酶(thymidylate synthase，TS)在大肠杆菌中表达，表达量占总蛋白的1.6％。Maley等对野生型TS cDNA中的编码序列进行定点突变，消除了稀有密码子，使TS的表达量达到总蛋白的25％-30％。但是基因序列分析发现人TS基因cDNA的编码序列中含有大量的原核生物稀有密码子，约占TS基因编码序列总数的10.09％(详见表1)，因此如果进行定点突变，将TS cDNA中编码序列中的真核生物密码子逐一突变成原核生物中常用的密码子，可以实现TS蛋白高效表达，但是实验操作量巨大，难度很高，经济适用差。因此急需要研制一种操作简单、高效表达、易于纯化、高效低价制备人TS货白的方法。
发明内容：
本发明的目的在于TS基因不发生定点突变的情况下将野生型TS编码序列与表达载体pET-28b(+)重组，转入含有真核生物稀有密码子的表达菌株Rosetta(DE3)中进行表达，并对发酵条件进行优化，实现了野生型TS基因的原核高效表达。该表达方法与现有的技术相比具有高效的表达率、产品接近天然TS蛋白活性，经济适用性好等特点，为研究TS的酶学性质、酶与抑制剂之间的相互作用提供充足的、廉价的纯酶来源。
本发明的另一目的在于提供一种6聚组氨酸-人类胸苷酸合成酶融合蛋白，减少后续重组TS蛋白纯化的难度。
本发明所述的能够制备人TS蛋白或者6聚组氨酸-人类胸苷酸合成酶融合蛋白的基因工程菌由重组质粒和宿主构成，其中宿主菌指的是Rosetta(DE3)，重组质粒指的是含有野生型人TS编码基因的质粒TS-pET-28b(+)，即重组菌TS-pET-28b(+)/Rosetta(DE3)(CGMCC No.3920)。
本发明提供的基因工程菌的进一步特征在于：所述的重组质粒是pET-28b(+)-PT7-6×His-TS，其中PT7是所述质粒pET-28b(+)的启动子；6×His是组氨酸纯化标签。
本发明所提够的一种高效表达重组人TS蛋白的基因工程菌的构建方法，包括设计PCR上游和下游引物，用PCR从人TS cDNA文库中扩增人TS基因编码片段，并通过表达载体转化到大肠杆菌，用PCR扩增的人TS编码片段经过TA克隆、测序、限制性内切酶酶切，克隆到表达质粒pET-28b(+)中，构建重组质粒TS-pET-28b(+)，转化大肠杆菌Rosetta(DE3)中，经诱导表达以后SDS-PAGE及Western-blotting鉴别后，证明获得高效表达重组人TS蛋白的基因工程菌。
具体来说，本发明的基因工程菌的构建方法，具体操作如下：
第一步引物的设计与合成
本发明中的野生型人类TS cDNA(GenBank ID：NM 001071)中编码序列全长942个碱基，含有人类TS的313个氨基酸残基的序列，其DNA编码序列及编码的氨基酸序列见序列表。PCR引物设计时，分别在5’端添加6聚组氨酸编码序列和内切酶Nde I识别序列，在3’端添加了内切酶Xho I的识别序列。
上游引物：5’-ATCAT ATGTA CCGCG CCATG CCTGT G-3’-(Nde I)
下游引物：5’-GCCTC GAGAA CCCTA AACAG CCATT TCCAT TT-3’-(Xho I)
第二步PCR扩增人TS编码片段及其T/A克隆
以野生型人类TS cDNA为模板，用上述的两条引物经PCR反应从cDNA中扩增出含有Nde I、Xho I酶切位点的、全长为971bp的野生型人类TS蛋白编码片段。扩增产物利用琼脂凝胶进行分离纯化，将胶回收得到的TS基因片段与克隆载体pMD-18T连接，并将连接产物转化感受态DH5α，经在LB/Amp/X-gal/IPTG平板上培养，挑取白斑菌落加入Amp LB培养基中过夜摇菌提取质粒，用PCR及NdeI、Xho I双酶切鉴定阳性克隆，阳性克隆命名为TS-pMD18-T。将通过鉴定的阳性克隆送上海博尚生物测序，证明基因工程菌含有完整的重组人TS基因编码片段。
第三步含有6聚His的人类胸苷酸合成酶大肠杆菌表达载体的构建
按照常规的分子克隆步骤，将TS-pMD18-T经过Nde I、Xho I双酶切切下目的基因(含有编码人类TS蛋白313个氨基酸残基的野生型基因)，再进一步克隆至大肠杆菌表达载体pET-28b(+)中，所得表达质粒命名为TS-pET-28b(+)。其构建过程详见附图1。
用含野生型人类胸苷酸合成酶编码基因的重组表达质粒TS-pET-28b(+)分别转化大肠杆菌BL21(DE3)、Rosetta(DE3)，并筛选表达TS-His的表达菌株，命名为TS-pET-28b(+)/BL21(DE3)、TS-pET-28b(+)/Rosetta(DE3)，详细过程见实施例。
第四步TS-pET-28b(+)/BL21(DE3)、TS-pET-28b(+)/Rosetta(DE3)的诱导表达、SDS-PAGE及Western-blotting鉴定
将TS-pET-28b(+)/BL21(DE3)单菌落接种于含卡那霉素(50μg/ml)的LB培养液中，37℃振荡过夜培养，次日以1∶100的比例转接到新鲜的含50μg/ml卡那霉素的较大体积的LB培养液中，37℃继续培养至OD600≈0.4-0.6，加诱导剂IPTG至终浓度1.5mM，30℃诱导8小时后，离心收集细胞。
将TS-pET-28b(+)/Rosetta(DE3)单菌落接种于含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的LB培养液中，37℃振荡过夜培养，次日以1∶100的比例转接到新鲜的含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的较大体积的LB培养液中，37℃继续培养至OD600≈0.4-0.6，加诱导剂IPTG至终浓度1.5mM，30℃诱导8小时后，离心收集细胞。
将收集的三组重组表达菌超声破碎至澄清，Bradford测定蛋白浓度，15％SDS-聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis，SDS-PAGE)分析在不同菌株中的表达效果(见图4)，并通过Western-blotting鉴定重组蛋白(见图5)，详细过程见实施例。
第五步TS高效表达的条件
通过进一步优化发酵条件得出重组野生型人TS蛋白的最佳表达条件为，表达菌株：Rosetta(DE3)；表达温度：37℃；诱导剂IPTG浓度：1.5mM；诱导时间8h，表达量占菌体总蛋白量的43.5％以下为图表说明：
序列表：本发明克隆的野生型人类TS的DNA序列及由它所编码的氨基酸序列，以及本发明所生产的 重组人类TS蛋白TS-His融合蛋白的氨基酸序列。
表1：野生型人类胸苷酸合成酶编码基因cDNA中稀有密码子分析
图1：大肠杆菌表达质粒TS-pET-28b(+)构建示意图
图2：重组质粒TS-pET-28b(+)的PCR鉴定电泳图
其中M：DNA Marker E；1-5：阳性克隆子的PCR产物
图3：重组质粒TS-pET-28b(+)的酶切鉴定图
其中M1：1kb DNA ladder Marker；1：TS-pET-28b(+)的Nde I酶切产物；2：TS-pET-28b(+)的Xho I酶切产物：3：TS-pET-28b(+)双酶切产物；M2：DNA marker E
图4：本发明所获得的高效表达野生型人类TS基因的重组表达菌株---TS-pET-28b(+)/BL21(DE3)、TS-pET-28b(+)/Rosetta(DE3)，与传统方法表达菌株TS-pET-28b(+)/BL21(DE3)的SDS-PAGE电泳图
其中M：低分子量蛋白Marker；1：TS-pET-28b(+)/BL21(DE3)未诱导；2：TS-pET-28b(+)/B L21(DE3)已诱导；3：TS-pET-28b(+)/Rosetta(DE3)未诱导；4：TS-pET-28b(+)/Rosetta(DE3)已诱导
图5：重组人TS蛋白Western-blotting鉴定图
其中M：低分子量蛋白Marker；1：TS-pET-28b(+)/Rosetta(DE3)未诱导；2：TS-pET-28b(+)/Rosetta(DE3)已诱导；3：TS-pET-28b(+)/Rosetta(DE3)未诱导western-blotting；4：TS-pET-28b(+)/Rosetta(DE3)已诱导western-blotting
具体实施方式：
以下通过实例对本发明做进一步的说明。
实施实例：
野生型人类胸苷酸合成酶基因在大肠杆菌中的高效表达及鉴定
第一步引物设计与合成
根据已知的野生型人类TS cDNA中的蛋白编码序列(Genbank ID：NM_001071)，设计并合成两个引物，其序列如下：
上游引物：5’-ATCAT ATGTA CCGCG CCATG CCTGT G-3’-(Nde I)
下游引物：5’-GCCTC GAGAA CCCTA AACAG CCATT TCCAT TT-3’-(Xho I)
第二步PCR扩增人TS编码片段及其T/A克隆
以野生型人类TS cDNA为模板，用上述的两条引物经PCR反应从cDNA中扩增出含有Nde I、Xho I酶切位点的、全长为971bp的野生型人类TS蛋白编码片段。反应条件为95℃5min，94℃1min，64.6℃30s，72℃1.5min，30个循环，72℃10min，4℃pause。扩增产物利用琼脂凝胶进行分离纯化，将胶回收得到的TS基因片段与克隆载体pMD-18T连接，并将连接产物转化感受态DH5α，经在LB/Amp/X-gal/IPTG平板上培养，挑取白斑菌落加入Amp/LB培养基中过夜摇菌提取质粒，用PCR及Nde I、Xho I双酶切鉴定阳性克隆。将通过鉴定的阳性克隆送上海博尚生物测序。结果显示某一阳性亚克隆子的基因序列未发生突变，可以用于下一步实验，将测序正确的克隆命名为TS-pMD18-T。本发明所克隆得到的人类TSDNA序列编码的氨基酸序列见序列表。
第三步含有6聚His的人类胸苷酸合成酶大肠杆菌表达载体的构建
如图1所示，将质粒TS-pMD18-T及表达载体pET-28b(+)，分别进行Nde I和Xho I双酶切，并进行凝胶回收。将回收后的TS基因片断与pET-28b(+)片段在T4 DNA连接酶的作用下，16℃过夜连接，连接 产物转化感受态E.coli DH5α细胞，经在含有卡那霉素的LB固体培养基上培养后，挑取数个单菌落加卡那霉素的LB液体培养基过夜培养，碱法提取质粒。PCR鉴定，电泳显示1、5、6泳道出现一条带，且与目的基因长度相符，约为1000bp(见附图2)。这表明菌落1、5、6是含有TS基因的阳性克隆子。选取1号克隆进一步酶切鉴定，酶切结果显示(见附图3)，Nde I、Xho I单酶切结果均出现大小为6kb的条带，大小与pET-28b(+)及TS之和相当；Nde I/Xho I双酶切电泳结果显示在5.3kb及1.0kb左右各出现了一条条带，大小与pET-28b(+)及TS编码片断大小一致。证明含有特异性酶切位点的重组野生型人TS原核表达载体TS-pET-28b(+)构建成功。
将TS-pET-28b(+)表达质粒转化大肠杆菌BL21(DE3)，筛选卡那霉素抗性的阳性转化体。
将TS-pET-28b(+)表达质粒转化大肠杆菌Rosetta(DE3)，筛选卡那霉素及氯霉素双抗性的阳性转化体。
第四步TS-pET-28b(+)/BL21(DE3)、TS-pET-28b(+)/Rosetta(DE3)的诱导表达、SDS-PAGE及Western-blotting鉴定
a、菌种的培养及IPTG诱导
将TS-pET-28b(+)/BL21(DE3)单菌落接种于含卡那霉素(50μg/ml)的LB培养液中，37℃振荡过夜培养，次日以1∶100的比例转接到新鲜的含50μg/ml卡那霉素的较大体积的LB培养液中，37℃继续培养至OD600≈0.4-0.6，加诱导剂IPTG至终浓度1.5mM，30℃诱导8小时后，离心收集细胞。
将TS-pET-28b(+)/Rosetta(DE3)单菌落接种于含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的LB培养液中，37℃振荡过夜培养，次日以1∶100的比例转接到新鲜的含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的较大体积的LB培养液中，37℃继续培养至OD600≈0.4-0.6，加诱导剂IPTG至终浓度1.5mM，30℃诱导8小时后，离心收集细胞。
将收集的三组重组表达菌超声破碎至澄清，Bradford测定货白浓度，15％SDS-聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis，SDS-PAGE)分析在不同菌株中的表达效果(见附图4)。变性条件下，TS单亚基的分子量大小为36.0kDa。通过以上实验表明，从电泳结果分析，重组BL21(DE3)和Rosetta(DE3)表达可见在36.0kDa处均有目的蛋白带出现，重组BL21(DE3)中目的蛋白诱导效果很差，通过蛋白凝胶分析，重组Rosetta(DE3)目的蛋白TS的表达量占细胞总蛋白含量的40％左右，初步证明野生型人TS货自己在Rosetta(DE3)后的高效表达。
b、重组野生型人TS蛋白的Western-blotting鉴定
将TS-pET-28b(+)/Rosetta(DE3)单菌落接种于含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的LB培养液中，37℃振荡过夜培养，次日以1∶100的比例转接到新鲜的含卡那霉素(50μg/ml)、氯霉素(34μg/ml)的较大体积的LB培养液中，37℃继续培养至OD600≈0.4-0.6，加诱导剂IPTG至终浓度1.5mM，30℃诱导8小时后，离心收集细胞。将收集的重组表达菌加PBS缓冲液悬浮，超声破碎至澄清。样品处理完毕后，进行PAGE电泳分析，经转膜、封闭之后，加第一抗体为鼠抗人TS-antibody孵化1h，洗涤之后加辣根酶标记山羊抗鼠gIG为第二抗体继续孵化1h，加入酶联碱性磷酸酶(Ab)生色底物显色，结果见附图5。从图中可以看出，在约36.0kDa处出现了明显的条带，而控制组却没有出现，证明在TS cDNA不进行特异性定点突变的情况下，通过在宿主中添加原核生物稀有密码子tRNA，可以实现TS的原核高效表达。
第五步
通过进一步优化发酵条件得出重组野生型人TS蛋白的最佳表达条件为，表达菌株：Rosetta(DE3)；表达温度：37℃；诱导剂IPTG浓度：1.5mM；诱导时间8h，表达量占菌体总蛋白量的43.5％。
表1：野生型人类胸苷酸合成酶编码基因cDNA中稀有密码子分析
注：大肠杆菌中密码子使用频率是根据GenBank Codon Usage Database中Escherichia coli B[gbbct]：11的使用频率计算得出。
[0062]
<110>青岛科技大学
<120>胸苷酸合成酶在大肠杆菌中的高效表达
<140>201010226489.6
<141>2010-07-12
<160>3
<210>1
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<212>DNA
<213>人类(Homo sapiens)
<220>
<223>野生型人TS cDNA基因编码序列
<400>1
atgcctgtgg ccggctcgga gctgccgcgc cggcccttgc cccccgccgc acaggagcgg 60
gacgccgagc cgcgtccgcc gcacggggag ctgcagtacc tggggcagat ccaacacatc 120
ctccgctgcg gcgtcaggaa ggacgaccgc acgggcaccg gcaccctgtc ggtattcggc 180
atgcaggcgc gctacagcct gagagatgaa ttccctctgc tgacaaccaa acgtgtgttc 240
tggaagggtg ttttggagga gttgctgtgg tttatcaagg gatccacaaa tgctaaagag 300
ctgtcttcca agggagtgaa aatctgggat gccaatggat cccgagactt tttggacagc 360
ctgggattct ccaccagaga agaaggggac ttgggcccag tttatggctt ccagtggagg 420
cattttgggg cagaatacag agatatggaa tcagattatt caggacaggg agttgaccaa 480
ctgcaaagag tgattgacac catcaaaacc aaccctgacg acagaagaat catcatgtgc 540
gcttggaatc caagagatct tcctctgatg gcgctgcctc catgccatgc cctctgccag 600
ttctatgtgg tgaacagtga gctgtcctgc cagctgtacc agagatcggg agacatgggc 660
ctcggtgtgc ctttcaacat cgccagctac gccctgctca cgtacatgat tgcgcacatc 720
acgggcctga agccaggtga ctttatacac actttgggag atgcacatat ttacctgaat 780
cacatcgagc cactgaaaat tcagcttcag cgagaaccca gacctttccc aaagctcagg 840
attcttcgaa aagttgagaa aattgatgac ttcaaagctg aagactttca gattgaaggg 900
tacaatccgc atccaactat taaaatggaa atggctgttt ag 942
<210>2
<211>313
<212>PRT
<213>人类(Homo sapiens)
<220>
<223>野生型人TS cDNA基因编码氨基酸序列
<400>2
Met Pro Val Ala Gly Ser Glu Leu Pro Arg Arg Pro Leu Pro Pro Ala
1 5 10 15
Ala Gln Glu Arg Asp Ala Glu Pro Arg Pro Pro His Gly Glu Leu Gln
20 25 30
Tyr Leu Gly Gln Ile Gln His Ile Leu Arg Cys Gly Val Arg Lys Asp
35 40 45
Asp Arg Thr Gly Thr Gly Thr Leu Ser Val Phe Gly Met Gln Ala Arg
50 55 60
Thy Ser Leu Arg Asp Glu Phe Pro Leu Leu Thr Thr Lys Arg Val Phe
65 70 75 80
Trp Lys Gly Val Leu Glu Glu Leu Leu Trp Phe Ile Lys Gly Ser Thr
85 90 95
Asn Ala Lys Glu Leu Ser Ser Lys Gly Val Lys Ile Trp Asp Ala Asn
100 105 110
Gly Ser Arg Asp Phe Leu Asp Ser Leu Gly Phe Ser Thr Arg Glu Glu
115 120 125
Gly Asp Leu Gly Pro Val Tyr Gly Phe Gln Trp Arg His Phe Gly Ala
130 135 140
Glu Tyr Arg Asp Met Glu Ser Asp Tyr Ser Gly Gln Gly Val Asp Gln
145 150 155 160
Leu Gln Arg Val Ile Asp Thr Ile Lys Thr Asn Pro Asp Asp Arg Arg
165 170 175
Ile Ile Met Cys Ala Trp Asn Pro Arg Asp Leu Pro Leu Met Ala Leu
180 185 190
Pro Pro Cys His Ala Leu Cys Gln Phe Tyr Val Val Asn Ser Glu Leu
195 200 205
Ser Cys Gln Leu Tyr Gln Arg Ser Gly Asp Met Gly Leu Gly Val Pro
210 215 220
Phe Asn Ile Ala Ser Tyr Ala Leu Leu Thr Tyr Met Ile Ala His Ile
225 230 235 240
Thr Gly Leu Lys Pro Gly Asp Phe Ile His Thr Leu Gly Asp Ala His
245 250 255
Ile Tyr Leu Asn His Ile Glu Pro Leu Lys Ile Gln Leu Gln Arg Glu
260 265 270
Pro Arg Pro Phe Pro Lys Leu Arg Ile Leu Arg Lys Val Glu Lys Ile
275 280 285
Asp Asp Phe Lys Ala Glu Asp Phe Gln Ile Glu Gly Tyr Asn Pro His
290 295 300
Pro Thr Ile Lys Met Glu Met Ala Val
305 310
<210>3
<211>334
<212>PRT
<213>人工序列
<220>
<223>本实验本发明所生产的重组人类TS蛋白TS-His融合蛋白的氨基酸序列
<400>3
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
-20 -15 -10
Arg Gly Ser His Met Met Pro Val Ala Gly Ser Glu Leu Pro Arg Arg
-5 -1 1 5 10
Pro Leu Pro Pro Ala Ala Gln Glu Arg Asp Ala Glu Pro Arg Pro Pro
15 20 25
His Gly Glu Leu Gln Tyr Leu Gly Gln Ile Gln His Ile Leu Arg Cys
30 35 40
Gly Val Arg Lys Asp Asp Arg Thr Gly Thr Gly Thr Leu Ser Val Phe
45 50 55
Gly Met Gln Ala Arg Thy Ser Leu Arg Asp Glu Phe Pro Leu Leu Thr
60 65 70 75
Thr Lys Arg Val Phe Trp Lys Gly Val Leu Glu Glu Leu Leu Trp Phe
80 85 90
Ile Lys Gly Ser Thr Asn Ala Lys Glu Leu Ser Ser Lys Gly Val Lys
95 100 105
Ile Trp Asp Ala Asn Gly Ser Arg Asp Phe Leu Asp Ser Leu Gly Phe
110 115 120
Ser Thr Arg Glu Glu Gly Asp Leu Gly Pro Val Tyr Gly Phe Gln Trp
125 130 135
Arg His Phe Gly Ala Glu Tyr Arg Asp Met Glu Ser Asp Tyr Ser Gly
140 145 150 155
Gln Gly Val Asp Gln Leu Gln Arg Val Ile Asp Thr Ile Lys Thr Asn
160 165 170
Pro Asp Asp Arg Arg Ile Ile Met Cys Ala Trp Asn Pro Arg Asp Leu
175 180 185
Pro Leu Met Ala Leu Pro Pro Cys His Ala Leu Cys Gln Phe Tyr Val
190 195 200
Val Asn Ser Glu Leu Ser Cys Gln Leu Tyr Gln Arg Ser Gly Asp Met
205 210 215
Gly Leu Gly Val Pro Phe Asn Ile Ala Ser Tyr Ala Leu Leu Thr Tyr
220 225 230 235
Met Ile Ala His Ile Thr Gly Leu Lys Pro Gly Asp Phe Ile His Thr
240 245 250
Leu Gly Asp Ala His Ile Tyr Leu Asn His Ile Glu Pro Leu Lys Ile
255 260 265
Gln Leu Gln Arg Glu Pro Arg Pro Phe Pro Lys Leu Arg Ile Leu Arg
270 275 280
Lys Val Glu Lys Ile Asp Asp Phe Lys Ala Glu Asp Phe Gln Ile Glu
285 290 295
Gly Tyr Asn Pro His Pro Thr Ile Lys Met Glu Met Ala Val
300 305 310
Claims (2)
1.一种保藏号为CGMCC No.3920的基因工程菌TS-pET-28b(+)/Rosetta(DE3)的制备方法，其特征在于包括以下步骤：
第一步引物的设计与合成
以野生型人类TS cDNA为模板设计PCR引物，其中TS cDNA序列如SEQ IDNO：1所示；PCR引物设计时，分别在5’端添加6聚组氨酸编码序列和内切酶Nde I识别序列，在3’端添加了内切酶Xho I的识别序列；
上游引物：5’ATCAT ATGTA CCGCG CCATG CCTGT G-3’-(Nde I)；
下游引物：5’-GCCTC GAGAA CCCTA AACAGCCATT TCCAT TT-3’-(XhoI)；
第二步PCR扩增人TS编码片段及其T/A克隆
以SEQ ID NO：1所示野生型人类TS cDNA为模板，用上述的两条引物经PCR反应从cDNA中扩增出含有Nde I、Xho I酶切位点的、全长为971bp的野生型人类TS蛋白编码片段；扩增产物利用琼脂凝胶进行分离纯化，将胶回收得到的TS基因片段与克隆载体pMD18-T连接，并将连接产物导入感受态DH5α，经在LB/Amp/X-gal/IPTG平板上培养，挑取白斑菌落加入LB/Ampicillin培养基中过夜摇菌提取质粒，用PCR及NdeI、Xho I双酶切鉴定阳性克隆，阳性克隆命名为TS-pMD18-T；
第三步含有6聚组氨酸编码序列的人类胸苷酸合成酶大肠杆菌表达载体的构建
按照常规的分子克隆步骤，将TS-pMD18-T经过限制性内切酶酶切下含有编码人类TS蛋白313个氨基酸残基的野生型目的基因，再进一步连接至大肠杆菌表达载体pET-28b(+)中，所得重组表达质粒命名为TS-pET-28b(+)；将含野生型人类胸苷酸合成酶编码基因的重组表达质粒TS-pET-28b(+)导入大肠杆菌Rosetta(DE3)中，并筛选表达TS-His的菌株，命名为TS-pET-28b(+)/Rosetta(DE3)。
2.一种保藏号为CGMCC No.3920的基因工程菌TS-pET-28b(+)/Rosetta(DE3)生产野生型人类TS的方法，步骤为：
(1)菌种培养：将单个新鲜的菌落接种于LB/Kanamycin培养基中，37℃震荡过夜培养；次日以1：100的比例转接于新鲜的LB/Kanamycin培养基中，继续培养至OD600＝0.4-0.6之间；
(2)IPTG诱导：加入IPTG至终浓度1.5mM，37℃，诱导8小时。
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