CN114591993A - 快速鉴定Tn5转座酶活性的方法 - Google Patents
快速鉴定Tn5转座酶活性的方法 Download PDFInfo
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- CN114591993A CN114591993A CN202210500786.8A CN202210500786A CN114591993A CN 114591993 A CN114591993 A CN 114591993A CN 202210500786 A CN202210500786 A CN 202210500786A CN 114591993 A CN114591993 A CN 114591993A
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- transposase
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Abstract
本发明公开了一种快速鉴定Tn5转座酶活性的方法，其步骤为：（1）构建报告基因表达质粒以及Tn5转座酶表达质粒，所述报告基因表达质粒为荧光蛋白表达质粒，报告基因表达质粒复制子为与pBR322复制子兼容的大肠杆菌复制子，报告基因表达质粒的启动子为可诱导型启动子，在启动子两侧加装有被Tn5转座酶特异性识别的序列;（2）报告基因表达质粒和Tn5转座酶表达质粒共转化到表达细胞中；（3）诱导Tn5转座酶表达；（4）诱导报告基因表达；（5）检测报告基因表达水平，确定转座酶的相对活性。本发明通过检测荧光强度可迅速、灵敏地测试Tn5转座酶活性。整个活性测定过程仅需30‑60min，极大地缩短了酶活测定时间并大幅提高了测试通量。
Description
技术领域
本发明涉及一种快速鉴定Tn5转座酶活性的方法，属于生物工程技术领域。
背景技术
Tn5转座酶现已作为一种重要的工具应用于高通量测序技术，但转座酶的活性测定方法限制了酶的高通量优化筛选和稳定生产。目前转座酶的活性测定主要包括三种：第一种是基于转座酶介导报告基因（抗性基因或毒蛋白）插入基因组的原理，最终通过平板单克隆计数来间接确定转座酶的相对活性，实验组与对照组克隆数差异越大则转座酶活性越强。这种方法虽然全面考量了转座时“剪切与粘贴”两个过程，但最终单克隆数量即酶活的确定将受转化率与细胞状态的影响，测定结果存在较大误差。另外，由于工作量大、步骤繁琐，此种方法很难实现通量筛选测试。
第二种方法依赖于转座酶介导的猝灭基团与荧光基团的解离，通过荧光信号相对强弱判定酶活强弱。相比于第一种方法，其可大幅度减少测定误差获得重复性较好的酶活结果，但测活过程需要使用特定试剂及仪器。且由于在体外测活，需要将表达后的转座酶纯化，不适用于大规模突变体库的筛选和酶活测试。
第三种方法直接比对不同转座酶建库的质量，以文库的覆盖度指示酶活的强弱；或以接头引物的PCR产物浓度为标准，评估新的转座酶突变体的活性。此法测定的活性可直接与下游应用关联，但酶活测定所耗费时间及物料成本极高，尤其在开展大量筛选工作时性价比较低。而且，建库反应需要较高纯度的酶，对纯化工艺的要求更高，同样不适用于高通量的筛选。
因此，开发一种快速、准确的酶活相对定量方法迫在眉睫。
发明内容
本发明提供了一种快速鉴定Tn5转座酶活性的方法，可用于快速判断Tn5转座酶突变体或重组体的活性。
本发明采用的技术方案为：一种快速鉴定Tn5转座酶活性的方法，其步骤为：
（1）构建报告基因表达质粒以及Tn5转座酶表达质粒，所述报告基因表达质粒为荧光蛋白表达质粒，报告基因表达质粒复制子为与pBR322复制子兼容的大肠杆菌复制子，报告基因表达质粒的启动子为可诱导型启动子，在启动子两侧加装有被Tn5转座酶特异性识别的序列;
（2）报告基因表达质粒和Tn5转座酶表达质粒共转染到表达细胞中，构建双质粒报告系统；
（3）诱导Tn5转座酶表达；
（4）诱导报告基因表达；
（5）检测报告基因表达水平，确定转座酶的相对活性。
优选的，所述报告基因表达质粒中的荧光蛋白为红色、绿色或蓝色荧光蛋白；报告基因表达质粒复制子为p15A复制子；报告基因表达质粒启动子为P lac 、P BAD 或P tet，Tn5转座酶特异性识别的序列为IE、OE或ME。
优选的，所述Tn5转座酶表达质粒为表达Tn5转座酶的pET系列质粒。
优选的，所述Tn5转座酶表达质粒为pET21b(+)质粒。
优选的，所述表达细胞为大肠杆菌细胞。
优选的，所述表达细胞为BL21(DE3)、Rosetta(DE3)或其他含T7 RNA聚合酶的大肠杆菌细胞。
优选的，诱导Tn5转座酶表达的条件为0.5~5mM IPTG，37℃、250rpm培养4-5h，添加Mg2+作为激活剂。
优选的，报告基因表达的诱导条件为Tn5转座酶诱导1-1.5h后，按照启动子类型添加相应诱导剂，37℃、250rpm培养3-4h。
优选的，检测报告基因表达水平的方法为测试细胞内或细胞超声破碎后溶液内荧光蛋白的荧光强度，测定荧光强度信号后与对照的野生型Tn5转座酶组的荧光强度对比，得到突变型Tn5转座酶的相对荧光强弱。
优选的，检测报告基因表达水平的方法具体步骤为：
1）测定细胞培养液在600nm处的吸光值，OD值测定是为了确保每次实验取的细胞数量是一致的，其最终酶活才有可比性；
2）取含3-5OD的细胞培养液，离心；
3）用500μL PBS重悬细胞，离心；
4）重复洗涤菌体；
5）用PBS重悬细胞；
6）取重悬细胞样品于酶标板孔内，设定波长测试荧光强度。
优选的，检测报告基因表达水平的方法具体步骤为：
1）测定细胞培养液在600nm处的吸光值；
2）取含3-5OD的细胞培养液，离心；
3）用500μL PBS重悬细胞，离心；
4）重复洗涤菌体；
5）用PBS重悬细胞；
6）超声破碎后离心，收集上清；
7）取上清样品于酶标板孔内，设定波长测试荧光强度。
优选的，还包括步骤（6）：测试报告基因表达质粒的丰度，复核转座酶活性。
优选的，测试报告基因表达质粒的丰度为测试细胞内不含启动子区域的报告基因表达质粒数量，采用同一对引物（如图2所示）在扩增含与不含启动子的两种报告质粒时形成大小两种PCR产物，根据小片段与大片段的比例确定转座酶活性，比例越大说明转座酶活性越高。
本发明所述的快速鉴定Tn5转座酶活性的方法，首先构建报告基因表达质粒，当其与Tn5转座酶的表达质粒共同转化至大肠杆菌细胞时，表达的Tn5转座酶识别报告基因前部启动子两侧的特异性序列，转座反应将启动子从质粒中剥离。随后诱导报告基因表达，通过测试报告基因表达水平，并与对照水平相比，最终确定Tn5转座酶活性。转座酶活性越高，则报告质粒基因表达水平越低。本发明通过检测荧光强度可迅速、灵敏地判断Tn5转座酶突变体或重组体相对于的野生型Tn5转座酶的活性差异。整个活性测定过程仅需30-60min，极大地缩短了酶活测定时间并大幅提高了测试通量。
附图说明
图1为Tn5转座酶快速活性测定原理示意图。pEGFP为由pET28a(+)改造而来的报告质粒；P BAD 为阿拉伯糖启动子，araC为阻遏蛋白表达基因，EGFP为增强型绿色荧光蛋白表达基因，KanR为卡那霉素抗性筛选基因；p15A ori为复制子。
图2为PCR酶活测定法的引物示意图。ME为转座酶特异性识别序列；引物 F和引物R为ME外侧的一对PCR引物；启动子为可诱导型启动子。
图3为Tn5转座酶相对定量的荧光强度测试结果。对照组为转座酶表达质粒中缺失转座酶序列的大肠杆菌胞内荧光蛋白的荧光强度；Tn5组为转座酶表达质粒表达野生型Tn5时的荧光强度；Mut组为转座酶表达质粒表达突变型Tn5的荧光强度。
图4为Tn5转座酶相对定量的PCR电泳结果。对照组为转座酶表达质粒中缺失转座酶序列时报告质粒的PCR结果；Tn5组为转座酶表达质粒表达野生型Tn5时的PCR结果；Mut组为转座酶表达质粒表达突变型Tn5的PCR结果。
具体实施方式
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或厂商提供的条件进行。下述实施例中所用的材料、试剂等，如无特别说明，均可从商业途径得到。
实施例1
1、EGFP报告质粒构建
如图1所示，以pET28a质粒为骨架，将pBR322复制子更换为p15A复制子；将乳糖操纵子更换为阿拉伯糖操纵子；然后在阿拉伯糖启动子下游接入绿色荧光蛋白基因（SEQ IDNO.1），并在启动子两侧各串联3组ME序列（SEQ ID NO.2）。
2、表达菌株构建
将Tn5转座酶表达质粒与报告质粒共转化至大肠杆菌BL21(DE3)细胞，构建双质粒报告系统。含双质粒的大肠杆菌可在卡那霉素和羧苄青霉素双抗生素环境下生长。Tn5转座酶表达质粒构建步骤如下：
1）合成如SEQ ID NO.3所示野生型Tn5基因序列（以下简称Tn5）及SEQ ID NO.4所示突变型Tn5基因序列（即E54K和L372P突变型Tn5，以下简称Mut），用NdeI/XhoI双酶切后连接至pET21b(+)载体，转化至DH5α感受态细胞，涂布羧苄抗性平板，37℃倒置培养过夜；
2）挑取单克隆菌落接种至5mL含羧苄青霉素的LB培养基中，37℃，250rpm培养过夜；
3）用上海翌圣生物公司的MolPure® Plasmid Mini Kit 质粒小量提取试剂盒提取质粒并测序，序列正确即表明完成转座酶表达质粒构建。
3、诱导转座酶表达
挑取大肠杆菌BL21(DE3)单克隆，接种至含卡那霉素、羧苄青霉素两种抗生素的5mL LB液体培养基中，37℃、250rpm培养3h后添加0.5mM的IPTG及终浓度为20mM的MgCl2诱导并激活Tn5转座酶。
4、诱导绿色荧光蛋白表达
转座酶表达1h后分别向培养基中加入0%、0.005%、0.02%、0.08%或0.32%的L-阿拉伯糖，37℃、250rpm培养4h。
5、收集菌体并超声破壁
分别测定各实验组菌液在600nm波长下的吸光值，取出5OD菌体，用PBS洗涤2次后离心弃尽上清。再用500μL PBS重悬菌体，在540Hz，4s/4s，超声3min后获得Tn5转座酶溶液。
6、荧光值测定
取100μL菌液加入96孔板，各实验组点3个复孔，设定激发光波长为485nm，发射光波长为538nm，在酶标仪下测定荧光信号。结果如图3所示，在各L-阿拉伯糖浓度下，Tn5组荧光均弱于对照，Mut组弱于Tn5组，即突变后的转座酶（Mut）活性显著优于野生型转座酶Tn5。表明此方法可直接用于确定突变后的转座酶与野生型转座酶的活性差异。
7、测定细胞内两种质粒占比
取步骤6剩余溶液各3μL为模板，用引物 F/引物 R引物扩增报告基因启动子区域，如图4电泳结果中可显见Mut组PCR产物的大小两个条带，而野生型转座酶组小片段不明显。灰度扫描结果表明Mut组小片段亮度约提升了25%，即Mut的相对酶活提升约25%。
所用引物序列：
引物 F: GAACCCCCCATGGTTAATTCCT
引物 R: CATTGCCGTCACTGCGTCTTTT。
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580 585 590
Ala Gly Gly Ala Thr Ala Ala Ala Cys Thr Gly Gly Cys Ala Cys Ala
595 600 605
Thr Ala Ala Thr Gly Ala Ala Cys Gly Thr Thr Thr Thr Gly Thr Gly
610 615 620
Gly Thr Thr Cys Gly Cys Thr Cys Thr Ala Ala Ala Cys Ala Thr Cys
625 630 635 640
Cys Thr Cys Gly Thr Ala Ala Ala Gly Ala Thr Gly Thr Gly Gly Ala
645 650 655
Ala Thr Cys Thr Gly Gly Thr Cys Thr Gly Thr Ala Thr Thr Thr Ala
660 665 670
Thr Ala Thr Gly Ala Thr Cys Ala Thr Thr Thr Ala Ala Ala Ala Ala
675 680 685
Ala Thr Cys Ala Gly Cys Cys Ala Gly Ala Ala Cys Thr Gly Gly Gly
690 695 700
Thr Gly Gly Cys Thr Ala Thr Cys Ala Gly Ala Thr Thr Thr Cys Ala
705 710 715 720
Ala Thr Thr Cys Cys Ala Cys Ala Gly Ala Ala Ala Gly Gly Cys Gly
725 730 735
Thr Thr Gly Thr Thr Gly Ala Thr Ala Ala Ala Cys Gly Cys Gly Gly
740 745 750
Thr Ala Ala Ala Cys Gly Thr Ala Ala Ala Ala Ala Thr Cys Gly Thr
755 760 765
Cys Cys Gly Gly Cys Thr Cys Gly Cys Ala Ala Ala Gly Cys Gly Ala
770 775 780
Gly Thr Cys Thr Gly Ala Gly Cys Thr Thr Ala Cys Gly Cys Thr Cys
785 790 795 800
Thr Gly Gly Thr Cys Gly Cys Ala Thr Thr Ala Cys Cys Cys Thr Gly
805 810 815
Ala Ala Ala Cys Ala Gly Gly Gly Thr Ala Ala Thr Ala Thr Cys Ala
820 825 830
Cys Cys Cys Thr Gly Ala Ala Thr Gly Cys Cys Gly Thr Gly Thr Thr
835 840 845
Ala Gly Cys Gly Gly Ala Ala Gly Ala Ala Ala Thr Thr Ala Ala Thr
850 855 860
Cys Cys Thr Cys Cys Thr Ala Ala Ala Gly Gly Cys Gly Ala Ala Ala
865 870 875 880
Cys Ala Cys Cys Ala Cys Thr Gly Ala Ala Ala Thr Gly Gly Cys Thr
885 890 895
Gly Cys Thr Gly Cys Thr Gly Ala Cys Ala Ala Gly Thr Gly Ala Ala
900 905 910
Cys Cys Ala Gly Thr Thr Gly Ala Ala Thr Cys Thr Thr Thr Ala Gly
915 920 925
Cys Ala Cys Ala Gly Gly Cys Ala Cys Thr Gly Cys Gly Cys Gly Thr
930 935 940
Gly Ala Thr Cys Gly Ala Thr Ala Thr Ala Thr Ala Thr Ala Cys Ala
945 950 955 960
Cys Ala Thr Cys Gly Thr Thr Gly Gly Cys Gly Thr Ala Thr Cys Gly
965 970 975
Ala Ala Gly Ala Ala Thr Thr Thr Cys Ala Thr Ala Ala Ala Gly Cys
980 985 990
Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Gly Gly Cys Gly Cys Gly
995 1000 1005
Gly Gly Cys Gly Cys Gly Gly Ala Ala Cys Gly Thr Cys Ala Gly Cys
1010 1015 1020
Gly Cys Ala Thr Gly Gly Ala Ala Gly Ala Ala Cys Cys Gly Gly Ala
1025 1030 1035 1040
Thr Ala Ala Thr Thr Thr Ala Gly Ala Ala Cys Gly Cys Ala Thr Gly
1045 1050 1055
Gly Thr Gly Ala Gly Thr Ala Thr Cys Cys Thr Gly Thr Cys Thr Thr
1060 1065 1070
Thr Thr Gly Thr Gly Gly Cys Cys Gly Thr Thr Cys Gly Cys Thr Thr
1075 1080 1085
Ala Thr Thr Ala Cys Ala Gly Cys Thr Gly Cys Gly Cys Gly Ala Ala
1090 1095 1100
Thr Cys Thr Thr Thr Thr Ala Cys Cys Cys Thr Thr Cys Cys Ala Cys
1105 1110 1115 1120
Ala Gly Gly Cys Cys Thr Thr Ala Cys Gly Thr Gly Cys Thr Cys Ala
1125 1130 1135
Gly Gly Gly Thr Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Ala Ala
1140 1145 1150
Gly Cys Cys Gly Ala Ala Cys Ala Thr Gly Thr Thr Gly Ala Ala Thr
1155 1160 1165
Cys Thr Cys Ala Gly Ala Gly Cys Gly Cys Cys Gly Ala Ala Ala Cys
1170 1175 1180
Cys Gly Thr Thr Cys Thr Gly Ala Cys Ala Cys Cys Thr Gly Ala Thr
1185 1190 1195 1200
Gly Ala Ala Thr Gly Thr Cys Ala Gly Thr Thr Ala Thr Thr Ala Gly
1205 1210 1215
Gly Thr Thr Ala Thr Thr Thr Ala Gly Ala Thr Ala Ala Ala Gly Gly
1220 1225 1230
Cys Ala Ala Ala Cys Gly Cys Ala Ala Ala Cys Gly Cys Ala Ala Ala
1235 1240 1245
Gly Ala Ala Ala Ala Ala Gly Cys Cys Gly Gly Cys Thr Cys Ala Thr
1250 1255 1260
Thr Ala Cys Ala Gly Thr Gly Gly Gly Cys Cys Thr Ala Thr Ala Thr
1265 1270 1275 1280
Gly Gly Cys Gly Ala Thr Thr Gly Cys Ala Cys Gly Cys Thr Thr Ala
1285 1290 1295
Gly Gly Cys Gly Gly Thr Thr Thr Thr Ala Thr Gly Gly Ala Thr Thr
1300 1305 1310
Cys Thr Ala Ala Ala Cys Gly Ala Ala Cys Gly Gly Gly Cys Ala Thr
1315 1320 1325
Thr Gly Cys Cys Thr Cys Thr Thr Gly Gly Gly Gly Thr Gly Cys Cys
1330 1335 1340
Cys Thr Gly Thr Gly Gly Gly Ala Ala Gly Gly Thr Thr Gly Gly Gly
1345 1350 1355 1360
Ala Ala Gly Cys Ala Cys Thr Cys Cys Ala Gly Ala Gly Cys Ala Ala
1365 1370 1375
Ala Cys Thr Gly Gly Ala Thr Gly Gly Cys Thr Thr Thr Cys Thr Gly
1380 1385 1390
Gly Cys Cys Gly Cys Cys Ala Ala Ala Gly Ala Thr Thr Thr Ala Ala
1395 1400 1405
Thr Gly Gly Cys Cys Cys Ala Gly Gly Gly Thr Ala Thr Cys Ala Ala
1410 1415 1420
Ala Ala Thr Cys Thr Ala Ala
1425 1430
<210> 4
<211> 1431
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Ala Thr Gly Ala Thr Cys Ala Cys Gly Ala Gly Thr Gly Cys Cys Thr
1 5 10 15
Thr Ala Cys Ala Thr Cys Gly Cys Gly Cys Gly Gly Cys Cys Gly Ala
20 25 30
Thr Thr Gly Gly Gly Cys Cys Ala Ala Ala Ala Gly Thr Gly Thr Gly
35 40 45
Thr Thr Thr Thr Cys Ala Ala Gly Thr Gly Cys Thr Gly Cys Cys Thr
50 55 60
Thr Ala Gly Gly Cys Gly Ala Thr Cys Cys Ala Cys Gly Thr Cys Gly
65 70 75 80
Thr Ala Cys Cys Gly Cys Cys Cys Gly Cys Thr Thr Ala Gly Thr Thr
85 90 95
Ala Ala Thr Gly Thr Thr Gly Cys Cys Gly Cys Cys Cys Ala Gly Thr
100 105 110
Thr Ala Gly Cys Cys Ala Ala Ala Thr Ala Thr Ala Gly Cys Gly Gly
115 120 125
Cys Ala Ala Ala Ala Gly Thr Ala Thr Thr Ala Cys Ala Ala Thr Cys
130 135 140
Thr Cys Thr Ala Gly Thr Gly Ala Ala Gly Gly Thr Ala Gly Thr Ala
145 150 155 160
Ala Ala Gly Cys Thr Gly Cys Gly Cys Ala Gly Gly Ala Ala Gly Gly
165 170 175
Thr Gly Cys Cys Thr Ala Thr Cys Gly Cys Thr Thr Thr Ala Thr Cys
180 185 190
Cys Gly Cys Ala Ala Thr Cys Cys Thr Ala Ala Thr Gly Thr Gly Ala
195 200 205
Gly Thr Gly Cys Cys Gly Ala Ala Gly Cys Cys Ala Thr Thr Cys Gly
210 215 220
Cys Ala Ala Ala Gly Cys Gly Gly Gly Cys Gly Cys Cys Ala Thr Gly
225 230 235 240
Cys Ala Gly Ala Cys Cys Gly Thr Gly Ala Ala Ala Thr Thr Ala Gly
245 250 255
Cys Cys Cys Ala Gly Gly Ala Ala Thr Thr Thr Cys Cys Gly Gly Ala
260 265 270
Ala Cys Thr Gly Cys Thr Gly Gly Cys Thr Ala Thr Cys Gly Ala Ala
275 280 285
Gly Ala Thr Ala Cys Gly Ala Cys Gly Ala Gly Cys Thr Thr Ala Ala
290 295 300
Gly Thr Thr Ala Thr Cys Gly Thr Cys Ala Thr Cys Ala Gly Gly Thr
305 310 315 320
Thr Gly Cys Cys Gly Ala Ala Gly Ala Ala Cys Thr Gly Gly Gly Cys
325 330 335
Ala Ala Ala Thr Thr Ala Gly Gly Thr Ala Gly Thr Ala Thr Cys Cys
340 345 350
Ala Gly Gly Ala Thr Ala Ala Ala Thr Cys Thr Cys Gly Thr Gly Gly
355 360 365
Thr Thr Gly Gly Thr Gly Gly Gly Thr Thr Cys Ala Thr Ala Gly Thr
370 375 380
Gly Thr Thr Cys Thr Gly Thr Thr Ala Cys Thr Gly Gly Ala Ala Gly
385 390 395 400
Cys Thr Ala Cys Gly Ala Cys Cys Thr Thr Thr Cys Gly Cys Ala Cys
405 410 415
Thr Gly Thr Gly Gly Gly Thr Cys Thr Gly Thr Thr Ala Cys Ala Thr
420 425 430
Cys Ala Gly Gly Ala Ala Thr Gly Gly Thr Gly Gly Ala Thr Gly Cys
435 440 445
Gly Thr Cys Cys Gly Gly Ala Thr Gly Ala Thr Cys Cys Ala Gly Cys
450 455 460
Cys Gly Ala Thr Gly Cys Thr Gly Ala Thr Gly Ala Ala Ala Ala Ala
465 470 475 480
Gly Ala Ala Thr Cys Thr Gly Gly Cys Ala Ala Ala Thr Gly Gly Thr
485 490 495
Thr Ala Gly Cys Ala Gly Cys Ala Gly Cys Gly Gly Cys Thr Ala Cys
500 505 510
Cys Thr Cys Ala Cys Gly Cys Cys Thr Gly Cys Gly Thr Ala Thr Gly
515 520 525
Gly Gly Thr Ala Gly Thr Ala Thr Gly Ala Thr Gly Ala Gly Thr Ala
530 535 540
Ala Thr Gly Thr Gly Ala Thr Thr Gly Cys Cys Gly Thr Thr Thr Gly
545 550 555 560
Cys Gly Ala Thr Cys Gly Cys Gly Ala Ala Gly Cys Ala Gly Ala Thr
565 570 575
Ala Thr Thr Cys Ala Thr Gly Cys Ala Thr Ala Thr Thr Thr Ala Cys
580 585 590
Ala Gly Gly Ala Thr Ala Ala Ala Cys Thr Gly Gly Cys Ala Cys Ala
595 600 605
Thr Ala Ala Thr Gly Ala Ala Cys Gly Thr Thr Thr Thr Gly Thr Gly
610 615 620
Gly Thr Thr Cys Gly Cys Thr Cys Thr Ala Ala Ala Cys Ala Thr Cys
625 630 635 640
Cys Thr Cys Gly Thr Ala Ala Ala Gly Ala Thr Gly Thr Gly Gly Ala
645 650 655
Ala Thr Cys Thr Gly Gly Thr Cys Thr Gly Thr Ala Thr Thr Thr Ala
660 665 670
Thr Ala Thr Gly Ala Thr Cys Ala Thr Thr Thr Ala Ala Ala Ala Ala
675 680 685
Ala Thr Cys Ala Gly Cys Cys Ala Gly Ala Ala Cys Thr Gly Gly Gly
690 695 700
Thr Gly Gly Cys Thr Ala Thr Cys Ala Gly Ala Thr Thr Thr Cys Ala
705 710 715 720
Ala Thr Thr Cys Cys Ala Cys Ala Gly Ala Ala Ala Gly Gly Cys Gly
725 730 735
Thr Thr Gly Thr Thr Gly Ala Thr Ala Ala Ala Cys Gly Cys Gly Gly
740 745 750
Thr Ala Ala Ala Cys Gly Thr Ala Ala Ala Ala Ala Thr Cys Gly Thr
755 760 765
Cys Cys Gly Gly Cys Thr Cys Gly Cys Ala Ala Ala Gly Cys Gly Ala
770 775 780
Gly Thr Cys Thr Gly Ala Gly Cys Thr Thr Ala Cys Gly Cys Thr Cys
785 790 795 800
Thr Gly Gly Thr Cys Gly Cys Ala Thr Thr Ala Cys Cys Cys Thr Gly
805 810 815
Ala Ala Ala Cys Ala Gly Gly Gly Thr Ala Ala Thr Ala Thr Cys Ala
820 825 830
Cys Cys Cys Thr Gly Ala Ala Thr Gly Cys Cys Gly Thr Gly Thr Thr
835 840 845
Ala Gly Cys Gly Gly Ala Ala Gly Ala Ala Ala Thr Thr Ala Ala Thr
850 855 860
Cys Cys Thr Cys Cys Thr Ala Ala Ala Gly Gly Cys Gly Ala Ala Ala
865 870 875 880
Cys Ala Cys Cys Ala Cys Thr Gly Ala Ala Ala Thr Gly Gly Cys Thr
885 890 895
Gly Cys Thr Gly Cys Thr Gly Ala Cys Ala Ala Gly Thr Gly Ala Ala
900 905 910
Cys Cys Ala Gly Thr Thr Gly Ala Ala Thr Cys Thr Thr Thr Ala Gly
915 920 925
Cys Ala Cys Ala Gly Gly Cys Ala Cys Thr Gly Cys Gly Cys Gly Thr
930 935 940
Gly Ala Thr Cys Gly Ala Thr Ala Thr Ala Thr Ala Thr Ala Cys Ala
945 950 955 960
Cys Ala Thr Cys Gly Thr Thr Gly Gly Cys Gly Thr Ala Thr Cys Gly
965 970 975
Ala Ala Gly Ala Ala Thr Thr Thr Cys Ala Thr Ala Ala Ala Gly Cys
980 985 990
Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Gly Gly Cys Gly Cys Gly
995 1000 1005
Gly Gly Cys Gly Cys Gly Gly Ala Ala Cys Gly Thr Cys Ala Gly Cys
1010 1015 1020
Gly Cys Ala Thr Gly Gly Ala Ala Gly Ala Ala Cys Cys Gly Gly Ala
1025 1030 1035 1040
Thr Ala Ala Thr Thr Thr Ala Gly Ala Ala Cys Gly Cys Ala Thr Gly
1045 1050 1055
Gly Thr Gly Ala Gly Thr Ala Thr Cys Cys Thr Gly Thr Cys Thr Thr
1060 1065 1070
Thr Thr Gly Thr Gly Gly Cys Cys Gly Thr Thr Cys Gly Cys Thr Thr
1075 1080 1085
Ala Thr Thr Ala Cys Ala Gly Cys Thr Gly Cys Gly Cys Gly Ala Ala
1090 1095 1100
Thr Cys Thr Thr Thr Thr Ala Cys Cys Cys Cys Thr Cys Cys Ala Cys
1105 1110 1115 1120
Ala Gly Gly Cys Cys Thr Thr Ala Cys Gly Thr Gly Cys Thr Cys Ala
1125 1130 1135
Gly Gly Gly Thr Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Ala Ala
1140 1145 1150
Gly Cys Cys Gly Ala Ala Cys Ala Thr Gly Thr Thr Gly Ala Ala Thr
1155 1160 1165
Cys Thr Cys Ala Gly Ala Gly Cys Gly Cys Cys Gly Ala Ala Ala Cys
1170 1175 1180
Cys Gly Thr Thr Cys Thr Gly Ala Cys Ala Cys Cys Thr Gly Ala Thr
1185 1190 1195 1200
Gly Ala Ala Thr Gly Thr Cys Ala Gly Thr Thr Ala Thr Thr Ala Gly
1205 1210 1215
Gly Thr Thr Ala Thr Thr Thr Ala Gly Ala Thr Ala Ala Ala Gly Gly
1220 1225 1230
Cys Ala Ala Ala Cys Gly Cys Ala Ala Ala Cys Gly Cys Ala Ala Ala
1235 1240 1245
Gly Ala Ala Ala Ala Ala Gly Cys Cys Gly Gly Cys Thr Cys Ala Thr
1250 1255 1260
Thr Ala Cys Ala Gly Thr Gly Gly Gly Cys Cys Thr Ala Thr Ala Thr
1265 1270 1275 1280
Gly Gly Cys Gly Ala Thr Thr Gly Cys Ala Cys Gly Cys Thr Thr Ala
1285 1290 1295
Gly Gly Cys Gly Gly Thr Thr Thr Thr Ala Thr Gly Gly Ala Thr Thr
1300 1305 1310
Cys Thr Ala Ala Ala Cys Gly Ala Ala Cys Gly Gly Gly Cys Ala Thr
1315 1320 1325
Thr Gly Cys Cys Thr Cys Thr Thr Gly Gly Gly Gly Thr Gly Cys Cys
1330 1335 1340
Cys Thr Gly Thr Gly Gly Gly Ala Ala Gly Gly Thr Thr Gly Gly Gly
1345 1350 1355 1360
Ala Ala Gly Cys Ala Cys Thr Cys Cys Ala Gly Ala Gly Cys Ala Ala
1365 1370 1375
Ala Cys Thr Gly Gly Ala Thr Gly Gly Cys Thr Thr Thr Cys Thr Gly
1380 1385 1390
Gly Cys Cys Gly Cys Cys Ala Ala Ala Gly Ala Thr Thr Thr Ala Ala
1395 1400 1405
Thr Gly Gly Cys Cys Cys Ala Gly Gly Gly Thr Ala Thr Cys Ala Ala
1410 1415 1420
Ala Ala Thr Cys Thr Ala Ala
1425 1430
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaacccccca tggttaattc ct 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cattgccgtc actgcgtctt tt 22
Claims (14)
1.一种快速鉴定Tn5转座酶活性的方法，其特征在于其步骤为：
（1）构建报告基因表达质粒以及Tn5转座酶表达质粒，所述报告基因表达质粒为荧光蛋白表达质粒，报告基因表达质粒复制子为与pBR322复制子兼容的大肠杆菌复制子，报告基因表达质粒的启动子为可诱导型启动子，在启动子两侧加装有被Tn5转座酶特异性识别的序列；
（2）报告基因表达质粒和Tn5转座酶表达质粒共转化到表达细胞中，构建双质粒报告系统；
（3）诱导Tn5转座酶表达；
（4）诱导报告基因表达；
（5）检测报告基因表达水平，确定转座酶的相对活性。
2.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述报告基因表达质粒中的荧光蛋白为红色、绿色或蓝色荧光蛋白；报告基因表达质粒复制子为p15A复制子；报告基因表达质粒启动子为P lac 、P BAD 或P tet，Tn5转座酶特异性识别的序列为IE、OE或ME。
3.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述Tn5转座酶表达质粒为表达Tn5转座酶的pET系列质粒。
4.根据权利要求3所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述Tn5转座酶表达质粒为pET21b(+)质粒。
5.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述表达细胞为大肠杆菌细胞。
6.根据权利要求5所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述表达细胞为BL21(DE3)、Rosetta(DE3)或其他含T7 RNA聚合酶的大肠杆菌细胞。
7.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：诱导Tn5转座酶表达的条件为0.5~5mM IPTG，37℃、250rpm培养4-5h，添加Mg2+作为激活剂。
8.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：报告基因表达的诱导条件为Tn5转座酶诱导1-1.5h后，按照启动子类型添加相应诱导剂，37℃、250rpm培养3-4h。
9.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：检测报告基因表达水平的方法为测试细胞内或细胞超声破碎后溶液内荧光蛋白的荧光强度，测定荧光强度信号后与对照的野生型Tn5转座酶组的荧光强度对比，得到Tn5转座酶的相对荧光强弱。
10.根据权利要求9所述的快速鉴定Tn5转座酶活性的方法，其特征在于：检测报告基因表达水平的方法具体步骤为：
1）测定细胞培养液在600nm处的吸光值；
2）取含相同OD数的细胞培养液，离心；
3）用PBS重悬细胞，离心；
4）重复洗涤菌体；
5）用PBS重悬细胞；
6）取重悬细胞样品于酶标板孔内，设定波长测试荧光强度。
11.根据权利要求9所述的快速鉴定Tn5转座酶活性的方法，其特征在于：检测报告基因表达水平的方法具体步骤为：
1）测定细胞培养液在600nm处的吸光值；
2）取含相同OD数的细胞培养液，离心；
3）用PBS重悬细胞，离心；
4）重复洗涤菌体；
5）用PBS重悬细胞；
6）超声破碎后离心，收集上清；
7）取上清样品于酶标板孔内，设定波长测试荧光强度。
12.根据权利要求1所述的快速鉴定Tn5转座酶活性的方法，其特征在于：还包括步骤（6）：测试报告基因表达质粒的相对丰度，复核转座酶活性。
13.根据权利要求12所述的快速鉴定Tn5转座酶活性的方法，其特征在于：测试报告基因表达质粒的丰度为测试细胞内不含启动子区域的报告基因表达质粒数量，采用同一对引物在扩增含与不含启动子的两种报告质粒时形成大小两种PCR产物，根据小片段与大片段的比例确定转座酶活性。
14.根据权利要求13所述的快速鉴定Tn5转座酶活性的方法，其特征在于：所述一对引物的序列如SEQ ID No.5和SEQ ID No.6所示。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060294606A1 (en) * | 2004-05-18 | 2006-12-28 | Istefo Moisyadi | Tn5 transposase-mediated transgenesis |
| CN108265101A (zh) * | 2016-12-30 | 2018-07-10 | 天津强微特生物科技有限公司 | 一种快速稳定的Tn5转座酶酶活测定方法 |
| CN112899296A (zh) * | 2019-12-04 | 2021-06-04 | 上海细胞治疗研究院 | 一种转座酶的筛选报告载体及其制备方法和应用 |
| CN112899252A (zh) * | 2019-12-04 | 2021-06-04 | 上海细胞治疗研究院 | 一种高活性转座酶及其应用 |
| CN114250266A (zh) * | 2021-12-20 | 2022-03-29 | 南京诺唯赞生物科技股份有限公司 | 一种转座酶活性测定方法 |
| CN114350693A (zh) * | 2021-12-30 | 2022-04-15 | 苏州译酶生物科技有限公司 | 一种Tn5转座酶及其制备方法 |
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2022
- 2022-05-10 CN CN202210500786.8A patent/CN114591993B/zh active Active
- 2022-05-10 CN CN202210833604.9A patent/CN116286713A/zh not_active Withdrawn
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| US20060294606A1 (en) * | 2004-05-18 | 2006-12-28 | Istefo Moisyadi | Tn5 transposase-mediated transgenesis |
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| CN112899252A (zh) * | 2019-12-04 | 2021-06-04 | 上海细胞治疗研究院 | 一种高活性转座酶及其应用 |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN114591993B (zh) | 2022-08-12 |
| CN116286713A (zh) | 2023-06-23 |
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