CN107298698B - 一种非同源末端体系、制备方法及应用 - Google Patents
一种非同源末端体系、制备方法及应用 Download PDFInfo
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- CN107298698B CN107298698B CN201710414508.XA CN201710414508A CN107298698B CN 107298698 B CN107298698 B CN 107298698B CN 201710414508 A CN201710414508 A CN 201710414508A CN 107298698 B CN107298698 B CN 107298698B
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Abstract
本发明提供了一种非同源末端体系、制备方法及应用。所述非同源末端体系包括Ku K19R蛋白和LigD蛋白。所述的具有Ku K19R蛋白的非同源末端体系在连接双链DNA片段时，比传统的非同源末端体系具有更高的产物数量，即具有更佳的连接效果。
Description
技术领域
本发明涉及分子工程技术领域，特别涉及一种非同源末端体系、制备方法及应用。
背景技术
双链DNA片段的连接是分子生物学实验中的一个关键技术，该技术是基因重组和基因改造的重要中间环节。一般利用DNA连接酶催化两个相邻双链DNA片段的5’端磷酸与3’端羟基之间形成磷酸二酯键。T4噬菌体DNA连接酶是科研的首选连接酶，例如Promega公司出品的M1801、NEB公司出品的M0202等。T4噬菌体DNA连接酶的作用分三步：(1)连接酶与辅助因子ATP形成酶-AMP复合物；(2)酶-AMP复合物结合到具有5’-磷酸基切口的DNA末端，形成DNA-AMP复合物；(3)DNA-AMP复合物的3’-羟基和5’-磷酸基产生新的磷酸二酯键，释放AMP。连接酶作用的两个双链DNA片段的末端必须具有5’-磷酸基、3’-羟基和末端碱基互补的特性，一般通过限制性内切酶产生。
同源重组也在分子生物学实验中被用来进行目的与载体DNA片段之间的双链DNA片段连接，也被称为无缝克隆技术，是利用重组酶将末端具有15-20个同源碱基的载体与目的DNA片段连在一起。与连接酶相比，利用重组酶连接DNA的步骤更简略，不需要限制性内切酶产生互补的DNA末端，但是重组酶发挥作用的前提是两个DNA末端之间有同源碱基。
同源重组和非同源末端(non-homologous endjoining，NHEJ)体系是生物体内的两套断裂DNA修复体系。同源重组发生在基因组复制时期，需要同时存在两个拷贝的基因组才能发挥作用，而非同源末端连接可以在任何时期发挥作用。与同源重组相比，非同源末端体系的作用底物更广泛。非同源末端体系存在于真核细胞和少数原核细胞中，能够连接任意两个双链DNA，对DNA末端碱基无特殊要求。最小NHEJ体系由Ku和LigD蛋白组成。Ku蛋白是DNA末端结合蛋白，能够结合具有不同结构特征的DNA末端，包括平末端、5’或者3’凸出和DNA发夹结构。Ku蛋白以同源二聚体的形式结合双链DNA末端，随后特异性招募LigD蛋白，并促进LigD蛋白进行双链DNA末端之间的连接反应。
发明内容
本发明的目的在于提供一种非同源末端体系，其通过改变Ku蛋白的氨基酸序列，从而达到更佳的连接效果。
本发明是通过以下技术方案实现的：
一种非同源末端体系，包括Ku K19R蛋白和LigD蛋白，所述KuK19R蛋白是如下a)或b)蛋白：
a)序列表中的SEQ ID №.1所示的氨基酸序列组成的蛋白质；
b)将序列表中的SEQ ID №.1的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID №.1所示蛋白具有相同功能的由a)衍生的蛋白质；
所述LigD是如下c)或d)蛋白：
c)序列表中的SEQ ID №.2所示的氨基酸序列组成的蛋白质；
d)将序列表中的SEQ ID №.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID №.2所示蛋白具有相同功能的由c)衍生的蛋白质。
本发明还提供了制备非同源末端体系的方法，包括构建Ku K19R蛋白表达菌株和纯化Ku K19R蛋白。
所述构建Ku K19R蛋白表达菌株包括以下步骤：
1-1)将Ku基因与载体连接；
1-2)利用定点突变引物A和B将Ku基因的第19位赖氨酸突变为精氨酸，获得KuK19R表达质粒；
1-3)将完成定点突变的Ku K19R表达质粒转入受体细胞。
所述纯化Ku K19R蛋白包括以下步骤：
2-1)将Ku K19R蛋白表达菌株划平板培养，挑取单克隆菌落于LB培养基中，在37℃、200rpm/min的条件下振荡培养过夜，次日按1％接种量转接于LB培养基中；
2-2)将转接的LB培养基在200rpm/min的条件下振荡培养至OD600值在0.4-0.6之间，添加IPTG诱导剂至终浓度0.4mM，30℃继续诱导培养4小时后，在4℃、8000rpm/min的条件下离心收集菌株沉淀，以缓冲液清洗菌体一次，然后重悬于缓冲液；
2-3)将重悬于缓冲液的菌株超声破碎后，以12000rpm/min离心30min，上清液上样于经缓冲液平衡的Ni2+-NTA亲和层析柱，然后用不同浓度imidazole依次洗脱。
本发明还提供了利用非同源末端体系连接双链DNA片段的方法：将待连接的双链DNA片段与上述非同源末端体系混合，在37℃条件下孵育1小时。
与现有技术相比，本发明所述的具有Ku K19R蛋白的非同源末端体系在连接双链DNA片段时，比传统的非同源末端体系具有更高的产物数量，即具有更佳的连接效果。
附图说明
图1是实施例3的PCR检测连接产物的结果图；
图2是实施例3的线性双链DNA连接两端的产物数量统计结果图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：利用带有Ku K19R蛋白的非同源末端体系连接双链DNA片段
(一)Ku K19R和LigD蛋白的制备
1)构建Ku K19R和LigD蛋白表达菌株
从耻垢分枝杆菌(Mycobacterium smegmatis str.MC2 155)中，通过正向引物和反向引物，PCR扩增获得Ku基因编码片段和LigD基因编码片段，通过NEB公司货号分别为R3193、R3104和M0202酶将Ku基因编码片段连到pET28a载体(购自Novagen公司)，货号分别为R3136、R3104和M0202酶将LigD基因编码片段连到pET28a载体。利用定点突变引物A和B(SEQ ID №.3和SEQ ID №.4)将Ku基因的第19位赖氨酸突变为精氨酸。质粒pET28a-KuK19R和pET28a-LigD分别转到大肠杆菌BL21中。
2)纯化Ku K19R蛋白和LigD蛋白
E.coli BL21/pET28a-LigD蛋白表达菌株和E.coliBL21/pET28a-Ku K19R蛋白表达菌株分别划平板，培养时添加抗生素的终浓度为卡拉霉素30μg/mL。挑取单克隆于5mL LB试管培养基，37℃200rpm/min振荡培养过夜。次日按1％接种量分别转接于500mL LB培养基中，200rpm/min振荡培养至OD600在0.4-0.6之间，加IPTG至终浓度0.4mM，30℃继续诱导培养4小时后，4℃，8000rpm/min离心收集细胞，细胞沉淀以缓冲液A(10mmol/L imidazole，0.5mol/L NaCl，20mmol/L Tris-HCl，pH 7.9)清洗菌体一次，然后重悬于50mL缓冲液A。超声破碎后，以12000rpm/min 30min离心，上清上样于经缓冲液A平衡的Ni2+-NTA亲和层析柱，然后用不同浓度imidazole(40mmol/L,80mmol/L和200mmol/L)依次洗脱。根据紫外检测仪的读数收集对应的洗脱物，SDS-PAGE电泳检验纯度后，用2L透析缓冲液B(20mmol/LTris-HCl，pH 7.9，NaCl150mmol/L,5％甘油)透析。透析好的蛋白样品用离心超滤杯浓缩后分成小份保存于-80℃。
(二)准备待连接的DNA片段
pET-20b(购自Novagen公司，货号69739)质粒经EcoR V(购自NEB公司，货号R3195)酶切产生4kB双链片段，琼脂糖凝胶电泳分离，切胶回收准备待连接的DNA片段。
(三)片段连接
10μl反应体系包含LigD蛋白1μg，Ku K19R蛋白分别选取10ng、40ng、200ng，50μMdNTPs,50mM pH为7.5的Tris-HCl，10mM MgCl2，1mM ATP，10mM Dithiothreitol和80ng待连接的DNA片段。混匀后，37℃条件下孵育1h。
实施例2：传统非同源末端体系连接双链DNA片段
10μl反应体系包含LigD蛋白1μg，Ku蛋白分别选取10ng、40ng、200ng，50μM dNTPs,50mM pH为7.5的Tris-HCl，10mMMgCl2，1mM ATP，10mM Dithiothreitol和80ng待连接的DNA片段。混匀后，37℃条件下孵育1h。
实施例3：检测连接产物
1.PCR方法检测实施例1和2所得的连接产物
3μl反应体系作为模板，10μM引物对T7terminator(5’-TAATACGACTCACTATAGGG-3’)和T7promoter(5’-GCTAGTTATTGCTCAGCGG-3’)各1μl、5μl 2XTaq mixture(Genstar，货号A012)，混匀，进行PCR扩增。扩增反应程序94℃，5min；94℃，15s，55℃，15s，72℃，30s，30个循环；72℃，5min。琼脂糖凝胶电泳检测PCR反应产物，结果如图1所示，泳道5、7、8和9中在500bp处出现扩增条带，证明连接成功。
2.产物转化大肠杆菌方法检测连接产物
2μl反应体系中加入protease K，65℃，30min，转化E.coliDH5a。转化程序为冰上孵育30min，42℃热激90s，冰上静置5min，添加500μl LB培养基，37℃振荡培养1.5h；4000rpm/min离心，沉淀菌体全部涂布含氨苄青霉素100μg/ml LB固体培养基；37℃过夜培养后，菌落计数。结果如图2所示，单菌落产生，说明连接反应完成，并且反应体系中相同质量的Ku K19R蛋白(R19折线)比Ku蛋白(K19折线)产生的菌落数(CFU)多。
SEQUENCE LISTING
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Val Gly Glu Val Arg Tyr Ser Glu Arg Thr Ser Asp Gly Arg Leu Arg
725 730 735
Gln Pro Ser Trp Arg Gly Leu Arg Pro Asp Lys Thr Pro Asp Glu Val
740 745 750
Val Trp Glu
755
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
tgtagacgcg gaccggcacg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
cgtgccggtc cgcgtctaca 20
Claims (3)
1.一种非同源末端体系，其特征在于，包括Ku K19R蛋白和LigD蛋白，所述Ku K19R蛋白是如序列表中的SEQ ID№.1所示的氨基酸序列组成的蛋白质，所述LigD蛋白是如序列表中的SEQ ID№.2所示的氨基酸序列组成的蛋白质。
2.权利要求1所述的非同源末端体系在连接双链DNA片段的应用。
3.根据权利要求2所述的非同源末端体系在连接双链DNA片段的应用，包括步骤：将待连接的双链DNA片段与非同源末端体系混合，在37℃条件下孵育1小时。
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