CN102766617A - 噬菌体TSP4 dCTP脱氨酶和编码脱氨酶的多核苷酸 - Google Patents
噬菌体TSP4 dCTP脱氨酶和编码脱氨酶的多核苷酸 Download PDFInfo
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- CN102766617A CN102766617A CN2012102407704A CN201210240770A CN102766617A CN 102766617 A CN102766617 A CN 102766617A CN 2012102407704 A CN2012102407704 A CN 2012102407704A CN 201210240770 A CN201210240770 A CN 201210240770A CN 102766617 A CN102766617 A CN 102766617A
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- dctp
- tsp4
- deaminase
- desaminase
- bacteriophage
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Abstract
本发明公开了高温噬菌体TSP4的dCTP脱氨酶和编码脱氨酶的多核苷酸,该脱氨酶具有SEQIDNO.1所示的氨基酸序,多核苷酸具有SEQIDNO.2所示核苷酸序列及其互补序列;该dCTP脱氨酶来源于高温栖热菌的噬菌体TSP4,能催化dCTP进行脱氨作用,产生dUTP和NH3,其在60℃具有最高催化活性,可以应用于dUTP,dUMP及dTTP的工业合成过程,其核苷酸序列可以用于构建能催化形成以上产物的基因工程菌株。
Description
技术领域
本发明涉及噬菌体TSP4 dCTP脱氨酶和编码解旋酶的多核苷酸,dCTP脱氨酶为生物体中与核酸代谢相关的酶类,属于生物技术领域。
背景技术
dCTP脱氨酶是指催化dCTP进行脱氨作用,生产dUTP和NH3的一类酶类,属于dUTPase家族,其催化的反应方程式为: dCTP + H2O= dUTP + NH3。
dCTP脱氨酶广泛存在于细菌、真核和古菌中,能催化水解dCTP生成dUTP,dUTP在dUTPase的催化下生成dUMP和焦磷酸,dUMP是胸苷酸合成酶途径中dTTP合成的前体这一系列过程降低了细胞内dUTP/dTTP的比例,阻止了dUTP进入DNA,从而减少DNA的突变几率,dCTP脱氨酶是这些过程的重要催化酶类,其可以应用于dUTP,dUMP及dTTP的合成过程,其核苷酸序列可以用于构建能催化形成以上产物的基因工程菌株。
高温菌噬菌体TSP4为感染高温菌菌株Thermus TC16的一种高温病毒,李秋鹏(昆明理工大学硕士论文,2009)对该噬菌体基因组进行了初步的解析,本申请通过序列检索分析获得TSP4噬菌体dCTP脱氨酶的序列,该序列与来自于高温菌的P74-26的dCTP脱氨酶的核苷酸序列相似性为66%,该病毒分离自俄罗斯堪察加半岛的热泉,与已发表的其他dCTP脱氨酶核苷酸序列的相似性均在37%以下,为新的dCTP脱氨酶。来自于高温菌的P74-26的dCTP脱氨酶的核苷酸序列仅通过生物信息学预测其功能,未通过实验证明其具有生物学功能特征。
发明内容
本发明旨在提供一种TSP4噬菌体dCTP脱氨酶,其具有SEQ ID NO.1所示的氨基酸序。
本发明另一目的是提供一种编码噬菌体TSP4 dCTP脱氨酶的多核苷酸,其具有SEQ ID NO.2所示核苷酸序列及其互补序列。
本发明的另一个目的是提供含有编码dCTP脱氨酶的多核苷酸的重组载体,其是由前文所述的多核苷酸与质粒、病毒或运载体表达载体构建而成的重组载体。
本发明的有益效果:
本发明获得的dCTP脱氨酶,可特异的催化dCTP产生不可逆的脱氨反应,在核酸代谢中起着重要的作用,其可以应用于dUTP,dUMP及dTTP的工业合成过程,因其来源于高温噬菌体,使其具有较为特殊的高温催化能力及较高的热稳定性;其核苷酸序列可以用于构建能催化形成以上产物的基因工程菌株。
附图说明
图1是本发明dCTP脱氨酶基因的克隆片段电泳示意图;
图2是本发明dCTP脱氨酶基因保守结构域分析示意图;
图3是本发明重组质粒回收电泳图和质粒酶切图谱,其中:泳道1为Marker;泳道2为pET-32a(+)质粒,其大小为5900bp;泳道3为dCTP脱氨酶胶回收产物,其大小为564bp;泳道4为dCTP-pET-32a重组质粒;泳道5为dCTP-pET-32a重组质粒双酶切;
图4是本发明dCTP脱氨酶基因表达蛋白的SDS-PAGE分析检测电泳图,其分子量约为42000道尔顿,其中:泳道1为Protein Marker;泳道2为Rosetta-pET-32a诱导前;泳道3为Rosetta-pET-32a诱导后;泳道4为Rosetta-dCTP-pET-32a诱导前;泳道5为Rosetta-dCTP-pET-32a诱导后;
图5是本发明dCTP脱氨酶基因表达蛋白的可溶性分析SDS-PAGE检测电泳图,其中:泳道1为Protein Marker;泳道2为Rosetta-pET-32a诱导前;泳道3为Rosetta-pET-32a诱导后;泳道4为Rosetta-dCTP-pET-32a诱导后上清;泳道5为Rosetta-dCTP-pET-32a诱导后沉淀;
图6是本发明dCTP脱氨酶蛋白纯化SDS-PAGE分析检测电泳图,其中泳道1为Protein Marker;泳道2为Rosetta-dCTP-pET-32a诱导表达上清液;泳道3为上样后流穿液;泳道4为80mM咪唑洗脱液;泳道5为150mM咪唑洗脱液;泳道6为200mM咪唑洗脱液;泳道7和8为300mM咪唑洗脱液;泳道9和10为500mM咪唑洗脱液。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中使用的试剂和方法,如无特殊说明,均采用常规试剂和使用常规方法。
实施例1:噬菌体TSP4 dCTP脱氨酶的克隆和表达
1、TSP4噬菌体dCTP脱氨酶基因的扩增(以噬菌体TSP4 DNA为模板)
(1) TSP4噬菌体dCTP脱氨酶基因扩增所用引物序列如下:
正向引物:5’- CGCGGATCCATGCTGCTACCGAACTG -3’
反向引物:5’- CCGCTCGAGACCGTTCGTTCCGTGTAC -3’
(2)扩增体系如下:(表1)
表1:扩增反应体系组分
模板 | 10 ng |
Premix ExTaq | 0.5 μl |
10x Premix ExTaq Buffer | 2.5μl |
引物 | 各0.5μl |
dNTP | 2μl |
ddH2O | Up to 25μl |
(3)扩增条件如下:
将反应体系混匀,在94℃预变性4min,然后在94℃变性30 s,60℃退火30 s,72℃延伸30 s,30个循环后,72℃延伸10min。反应完后取产物2μl,在10g/L琼脂糖凝胶中进行电泳分析(见图1),其片段大小为564bp。
2、PCR产物的胶回收纯化
(1)在电泳仪中灌制1.0%琼脂糖凝胶;
(2)将待分离纯化的PCR产物点样电泳,于适当位置停止电泳;
(3)在紫外灯下切下含该目的片断的凝胶,转移到1.5ml的Ep管中;
(4)用索莱生物公司胶回收试剂盒进行目的片段的回收,回收方法按说明书进行操作。
将基因序列提交NCBI数据库Conserved Domain Database蛋白质保守序列分析软件进行分析,dCTP脱氨酶分析结果见图2,分析显示其具有dUTPase 超家族的保守结构域。
3、重组表达载体的构建
(1)带有粘性末端线性载体pET-32a(+)和目的片段的制备
为了将目的基因片断连接到表达载体pET-32a(+)上,就需要使目的片断带有粘性末端的片断,即带有酶切位点;同样,为了使目的片断能插入载体中,也需要使载体带有粘性末端,并且使它们的酶切位点相同。
A、pET-32a(+)质粒抽提:用质粒提取试剂盒(索莱宝),操作步骤如下:
②增菌并收集菌体:取氨苄青霉素5μl(终浓度100μg/ml)加入5ml LB培养基中;用接种环挑取阳性克隆,接种于Amp+-LB培养基中;然后放入37℃摇床150rmp培养过夜,并将培养的菌液室温12000 rpm离心1 min,使菌体沉淀,弃上清液;
⑦然后加750 μl漂洗液,12000rpm,室温离心1min,弃废液(重复一次);再室温离心一次,13000 rpm,2 min;
⑧将吸附柱移入新的1.5 ml的离心管中,在吸附膜中央加入70 μl 经65℃预热的洗脱液,室温放置2 min,然后,12000 rpm室温离心1 min,弃吸附柱,得到质粒;
⑨将所得的质粒取1μl进行电泳检测,并定量。
B、质粒pET-32a(+)目的片段酶切鉴定(见图3)
表2:反应体系组分
质粒 | 5 μl |
10×Buffer Tango | 2 μl |
BamHⅠ | 0.5 μl |
XhoⅠ | 0.5 μl |
ddH2O | 2μl |
总量 | 10 μl |
(2)将TSP4噬菌体dCTP脱氨酶基因片段连接到pET-32a(+)载体上,得到重组质粒。
表3:反应体系组分
PCR胶回收产物 | 5 μl |
Solution I | 4.5 μl |
pET-32a线性载体 | 0.5 μl |
总量 | 10 μl |
(3)重组质粒的转化
在100μl感受态细胞DH5a里,加入10μl连接产物,冰浴30min,然后42℃热击1~2min,再冰浴2min,之后加入400μl LB液体培养基,于37℃摇床,150rpm震荡培养1h。然后涂布含有氨苄青霉素(Amp+)的LB固体平板,于37℃倒置培养16h。
(4)通过菌落PCR检测含重组质粒的菌株,筛选出阳性菌落
② 裂解后用掌上离心机轻甩,裂解液用作菌落PCR模板。
③ 菌落PCR扩增反应体系如下:
表4:反应体系组分
模板 | 10 ng |
Premix ExTaq | 0.5 μl |
10×Premix ExTaq Buffer | 2.5μl |
引物 | 各0.5μl |
dNTP | 2μl |
ddH2O | Up to 25μl |
将反应体系混匀后,在94℃预变性4min;然后在94℃变性30 s,60℃退火30 s,72℃延伸30 s,30个循环;72℃延伸10min。反应完成后取产物5 μl,在1%琼脂糖凝胶中进行电泳分析。
(5)阳性克隆增菌
①取氨苄青霉素5μl(终浓度100μg/ml)加入5ml LB培养基中;
②用接种环挑取阳性克隆,接种于Amp+-LB培养基中;
③然后放入37℃摇床180rmp培养6-8h。
(6)重组质粒抽提 (采用质粒抽提试剂盒提供的方法)
①收集菌体:把步骤(5)中培养的菌液加入到5ml的离心管中,离心收集菌体沉淀,弃上清液;
②加溶液Ⅰ(含RNA酶)250μl,振荡至菌体彻底分散均匀,转入1.5ml的EP管中;
③加入溶液Ⅱ 250μl,轻轻混匀,使溶液Ⅰ的RNA酶消化细菌中RNA;
加入溶液Ⅲ 350μl,温和混匀,12000rpm离心10min;
⑤将上清液转入DNA吸附柱中,室温放置2min,混匀,12000rpm离心1 min,弃收集管中废液;
⑦室温13000rpm,2min再离心一次,吸附柱敞口放置数分钟,以去除残存的漂洗液;
⑧将吸附柱移入新的1.5ml的离心管中,在吸附膜中央加入70μl 在65℃预热的洗脱液,室温放置1min,然后12000rpm室温离心2min,再重复一次以提高回收率;
⑨将所得到的质粒取1 μl电泳分析(见图3)。
(6)重组质粒的酶切鉴定(见图3)
表5:反应体系组分
质粒 | 5 μl |
10×Buffer Tango | 2 μl |
BamHⅠ | 0.5 μl |
XhoⅠ | 0.5 μl |
ddH2O | 2μl |
总量 | 10 μl |
(7)重组质粒dCTP-pET-32a转化表达菌株Rosetta(DE3)
在100μl感受态细胞Rosetta(DE3)里,加入重组质粒3μl,冰浴30min,然后42℃热击1~2min,再冰浴2min,之后加入400μl LB液体培养基,于37℃,150rpm摇床培养1h。然后涂布含有氨苄青霉素(Amp+)的LB固体平板,于37℃倒置培养10h。
(8)TSP4噬菌体dCTP脱氨酶蛋白的诱导表达和纯化
① TSP4噬菌体dCTP脱氨酶蛋白在Rosetta(DE3)中的诱导表达
将构建好的重组载体dCTP-pET-32a转化至表达宿主菌Rosetta(DE3)中,含重组质粒的菌株经37℃培养过夜,菌液按1%比例接种到50ml Amp+-LB(氨苄青霉素终浓度100μg/ml)培养液中,放入37℃摇床培养至其OD600为0.6;取出1ml菌液用作对照实验;其余菌液加入100mM IPTG储液至终浓度1mM,80 rpm摇床培养诱导表达8小时。
②TSP4噬菌体dCTP脱氨酶蛋白进行SDS-PAGE检测
取出少量诱导后菌液测OD值,根据菌液OD值不同取不同的菌液量,使其菌体量相等,12000rpm,离心5min,弃上清;加入80 μl ddH2O,20 μl 5×上样缓冲液,振荡使菌体散开后;在98℃下热裂解10min,使菌体释放蛋白质;将样品上样于电泳仪中在100V,60mA,电泳2h后,加入100ml R250考马斯亮蓝染色液染色,摇床振荡,100 rpm,20min后;将染色液倒出,用清水漂洗,再加入适量脱色液100 rpm,10h,最后用扫描仪扫描凝胶,检测蛋白表达情况(见图4),结果得到大小为42kD左右的目的蛋白条带。
将2mL菌液进行超声破碎,12000rpm,离心5min,分别取上清和沉淀按照上述方法制备样品跑SDS-PAGE电泳进行蛋白表达情况的检测(见图5)。
③重组解旋酶蛋白的纯化
利用上述方法大量诱导含重组质粒dCTP-pET-32a的Rosetta菌株,菌液经离心收集菌体(4℃,5000x g,10 min)。菌体悬浮于适量(使菌悬液的OD600达到20)缓冲液A[20 mM磷酸钠,500 mM NaCl ,30 mM 咪唑,pH 7.4] 中,冰上超声破碎细胞,4℃,10000x g离心15 min,上清上样于已用缓冲液A平衡好的His Trap HP柱(1 ml,GE Healthcare)中,结合的蛋白用20 ml 含有咪唑梯度(0-500 mM)的缓冲液B[20 mM 磷酸钠, 500 mM NaCl , 30 mM 咪唑,pH 7.4]洗脱,分段收集洗脱液,经SDS-PAGE检测后,最后用缓冲液[50 mM Tris-HCl, 2 mM Mg2+,pH7.4] 透析过夜,纯化的重组蛋白组分进行SDS-PAGE分析(见图6),结果显示重组蛋白分子量约为42kD,与目标蛋白一致。
实施例2:重组dCTP脱氨酶蛋白的酶活的测定
实验原理如下:
dCTP脱氨酶水解dCTP反应产生氨和dUTP,再通过偶联谷氨酸脱氢酶作用,对产生的氨进行显色,从而得出dCTP脱氨酶的活性。
依据南京建成生物工程研究所提供的腺苷酸脱氨酶ADA试剂盒调整后进行测定,具体内容见下表:
酶活定义:在60℃下和底物作用60min产生1μg氨为一个酶活单位。
计算方法如下:
dCTP脱氨酶比活性=×标准应用液浓度(25μg/ml)÷蛋白含量(mg/ml)
经计算,获得的重组dCTP脱氨酶的比活性为4.12U,同时也证明其具有高温催化活性,具有一定的热稳定性。
SEQUENCE LISTING
<110> 昆明理工大学
<120> 噬菌体TSP4 dCTP脱氨酶和编码脱氨酶的多核苷酸
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 187
<212> PRT
<213> TSP4噬菌体
<400> 1
MET Leu Leu Pro Asn Trp Phe Trp Trp Leu Phe Pro Gly Thr Val Glu Pro Tyr Arg Pro
1 10 20
Glu Arg Val Asn Pro Thr Ser Tyr Asp Leu Thr Ile Asp Ser His Val Ile Leu Gln Val
21 30 40
Tyr His Gly Lys Pro Leu Asn Lys Arg Gly Glu Val Val Gly Gly Phe Ile Tyr Leu Asp
41 50 60
Asp Gly Thr Arg Val Pro Asn Leu Phe Leu Pro Gly Asp Ser Val Leu Ala Ser Thr Phe
61 70 80
Glu Val Leu Arg Val Pro Arg Phe Val Arg Leu Gln Gly MET Leu Lys Ser Ser Val Ala
81 90 100
Arg Glu Gly Leu Asp His Arg Thr Ala Leu Tyr Ile Asp Pro Gly Phe Arg Gly Pro Val
101 110 120
Thr Leu Glu Leu Ser Phe His Val Pro Gly Arg Leu Val Pro Tyr Lys Pro Ile Ile Gln
121 130 140
Val Glu Ala His Phe Val Pro Thr Phe Ser Val Tyr Pro Gly Arg Tyr Asn Asp Gln Arg
141 150 160
Arg Pro Gln Pro Asn Leu Asn Pro Asp Ile Ala Phe Gln Ala Val Tyr Glu Thr Pro Ser
161 170 180
Leu Val His Gly Thr Asn Gly
181 187
<210> 2
<211> 561
<212> DNA
<213> TSP4噬菌体
<400> 2
atgctgctac cgaactggtt ctggtggttg tttcccggca ccgtagaacc 50
ttaccgtcca gaacgggtca atcccacttc ttatgacctg actattgaca 100
gccatgttat actacaggtg taccacggaa agccgctgaa caagcgggga 150
gaggttgtgg gcggctttat ctacctggat gacggcacgc gtgtgcccaa 200
cctgttcctg cctggcgaca gcgtcttggc cagtaccttt gaggtcttgc 250
gggttcccag gtttgtccgc ctgcagggga tgctcaagag ctctgtggcc 300
agggagggcc tggaccaccg cacggccctc tatattgacc caggctttcg 350
tggtcctgta accctggagc tctccttcca tgtccctggt aggctggtcc 400
cttataagcc catcatccag gtggaggccc actttgtccc caccttcagc 450
gtctatcctg gccgctacaa cgaccagagg aggcctcagc ctaacctgaa 500
cccggacatc gctttccagg ctgtttatga gaccccaagc cttgtacacg 550
gaacgaacgg t 561
Claims (2)
1.噬菌体TSP4 dCTP脱氨酶,其特征在于:具有SEQ ID NO.1所示的氨基酸序列。
2.编码权利要求1所述噬菌体TSP4 dCTP脱氨酶的多核苷酸,其特征在于:其具有SEQ ID NO.2所示核苷酸序列及其互补序列。
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CN110172451A (zh) * | 2019-05-05 | 2019-08-27 | 昆明理工大学 | 一种高通量分离噬菌体的方法 |
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《中国优秀硕士学位论文全文数据库》 20120510 李秋鹏 栖热菌噬菌体TSP4基因组解析及其解旋酶表达 全文 1-2 , * |
李秋鹏: "栖热菌噬菌体TSP4基因组解析及其解旋酶表达", 《中国优秀硕士学位论文全文数据库》, 10 May 2012 (2012-05-10) * |
薛寒等: "栖热菌噬菌体TSP4密码子偏嗜性及其对基因异源表达的影响", 《云南大学学报(自然科学版)》, vol. 34, no. 1, 10 January 2012 (2012-01-10), pages 107 - 112 * |
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CN110172451A (zh) * | 2019-05-05 | 2019-08-27 | 昆明理工大学 | 一种高通量分离噬菌体的方法 |
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