T4 DNA聚合酶在大肠杆菌中高效表达、纯化及部分生物学活性分析  

Efficient Expression, Purification and Selected Biological Activities of Recombinant T4 DNA Polymerase in Escherichia coli

作　　者：付大伟[1] 陈启蒙[1] 孙莹莹[1] 徐伟[1] FU Dawei;CHEN Qimeng;SUN Yingying;XU Wei(Key Laboratory for Food Science and Engineering,Harbin University of Commerce,Harbin 150076,China)

机构地区：[1]哈尔滨商业大学食品科学与工程重点实验室,黑龙江哈尔滨150076

出　　处：《食品科学》2018年第16期214-220,共7页Food Science

基　　金：哈尔滨商业大学博士科研启动基金项目(92508633)

摘　　要：T4 DNA聚合酶(T4 DNA polymerase,T4 DP)是基因工程等相关领域中不可缺少的工具酶。为获得大量高纯度并具有生物活性的可溶性T4 DP,通过从T4噬菌体基因组中克隆出T4 dp基因,将其克隆到pET-22b(+)质粒中构建重组表达载体pET-22b(+)-T4 dp,经DNA测序检测成功后,转化到大肠杆菌Transetta(DE3)中进行自诱导表达,通过磁珠法高效准确检测T4 DP的可溶性表达情况,并对自诱导的温度和时间进行优化,确定最佳诱导条件为30℃诱导培养10 h,分别利用超声波法、超声波-溶菌酶法及化学裂解法对自诱导表达的重组菌体进行破碎,确定最佳破碎方法为化学裂解法,分别利用Ni Sepharose^(TM) 6 Fast Flow亲和层析柱和MagNi磁珠纯化重组菌体裂解后上清液中的T4 DP,MagNi磁珠能高效地纯化出高纯度的T4 DP,其质量浓度为3 073.960 μg/mL,成功获得了高纯度高质量浓度的可溶性T4 DP,并使其成功应用于克隆载体的构建,证明其具有较好的末端平滑化活性。T4 DNA polymerase(T4 DP)is an indispensable tool in genetic engineering and other related fields.To obtain a large quantity of high purity and bioactive soluble T4 DP,the recombinant expression vector pET-22b(+)-T4 dp was constructed by cloning the T4 dp gene from the T4 bacteriophage genome into pET-22b(+)plasmid,which was then transformed into Escherichia coli Transetta(DE3)competent cells after the positive clones were confirmed by sequencing.The expression of soluble T4 DP was efficiently and accurately detected by magnetic beads.The temperature and time of auto-induction were optimized to be 30℃and 10 h,respectively.The auto-inducing recombinant bacteria were disrupted by ultrasonic treatment,ultrasonic treatment after preincubation with lysozyme and chemical cleavage,and chemical cleavage was determined as the optimal disruption method.The soluble T4 DP in the supernatant after disintegrating recombinant bacteria was purified by Ni SepharoseTM 6 Fast Flow affinity column or MagNi magnetic beads.High purity T4 DP was efficiently purified to a concentration of 3 073.960μg/mL by MagNi magnetic beads.The high purity soluble T4 DNA polymerase was successfully applied in the construction of low background cloning vector demonstrating its terminal blunting activity.

关 键 词：T4DNA聚合酶 自诱导 磁珠法 低背景克隆载体 末端平滑化 

分 类 号：Q786[生物学—分子生物学]