PCR法快速筛选重组克隆方法的改进

第39卷　第3期

2003年9月

青岛大学医学院学报

ACT A ACADE MIAE ME DICINAE QING DAO UNIVERSIT ATIS

V ol.39,N o.3September 2003

[收稿日期]2003203216;　[修订日期]2003204220

[作者简介]毛伟征(19622),男,博士,副主任医师,硕士生导师。

PCR 法快速筛选重组克隆方法的改进

毛伟征1,孙道升2,司春祥2,隋爱华3,刘湘萍3,陈　栋1,吕振华3

(1　青岛大学医学院附属医院普外科,山东青岛　266003;　2　青岛海慈医院检验科;　3　青岛大学医学院附属医院分子生物

学中心实验室)

[摘要]　①目的　以PCR 法在菌液中快速筛选重组克隆。②方法　提取小鼠巨噬细胞总RNA ,反转录PCR (RT 2PCR )获得肿瘤坏死因子2α(T NF 2α)基因片段,T A 克隆连接质粒载体、转化大肠杆菌JM109。在大肠杆菌培养中

的不同时间取样测定大肠杆菌A 600值及计数。以不同数量的大肠杆菌为模板进行系列PCR 扩增,获得适宜PCR 扩增条件的大肠杆菌数。③结果　以菌液为模板进行PCR 能够快速筛选重组体,大肠杆菌模板数在125～250个范围内,PCR 对阳性克隆的扩增可获得良好结果;获得常规培养条件下转化大肠杆菌的生长曲线,A 600值与大肠杆菌计数间存在显著正相关(r =0.93,P <0.001)。④结论　通过测定大肠杆菌培养液A 600值推算大肠杆菌数量,以适宜的大肠杆菌模板行PCR 法筛选重组克隆,快速准确并方便下游操作。

[关键词]　重组体筛选;聚合酶链反应;克隆,分子;大肠杆菌

[中图分类号]　Q784 [文献标识码]　A [文章编号]　167224488(2003)0320228203

A MODIFICATION OF RAPI D PCR SCREENING FOR RECOMBINANT C LONE MAO Wei 2zheng ,SUN Dao 2sheng ,SI Chun 2xiang ,et al (Department of G eneral Surgery ,The A ffiliated H ospital of Qingdao University M edical C ollege ,Qingdao 266003,China )

[ABSTRACT]　Objective T o establish a rapid screening of recombinant clones by PCR.　Methods T otal RNA of m ouse microphages was extracted.T NF 2genes were acquired by RT 2PCR.JM109,E.coli trans formed by recombinant plasmid was inoculated.At a series of time spots ,diluted 1L of medium was plated onto LB agar plate.M eanwhile ,A 600of media was detected with spectrophotometer.C olonies on agar were counted.M edia with different am ount of E.coli were used as a tem plate ,a series of PCR carried out.　Results PCR screening showed that an optimized result could be obtained with tem plates of media with E.coli am ount ranged from 125-250.The growth curve of trans formed E.coli was set up.There was a significant linear relationship between colonies and A 600from 4.5to 7.0hours after inoculation.　Conclusion The am ount of E.coli can be calculated through detecting A 600of the media.It is a fast and efficient way to screen recombinant clones with PCR ,which takes a proper am ount of E.coli as its tem plate.

[KE Y WOR DS]　screening ,recombinants ;polymerase chain reaction ;cloning ,m olecular ;Escherichia coli

外源基因经反转录PCR (RT 2PCR )获得理想的PCR 产物后,用定向克隆2T A 克隆等方法插入载体的多克隆位点,转化大肠杆菌感受态细胞,提取重组质粒后进行限制性酶切分析和DNA 测序等重组体分析。该工作颇为繁琐,费时、费力。我们在制备荧光定量PCR 阳性模板测定小鼠巨噬细胞肿瘤坏死

因子2

α(T NF 2α)mRNA 的实验中[1],以重组体转化的大肠杆菌为模板进行了PCR 扩增,通过对实验中各个参数的测定和分析,获得了对重组体筛选的快速、简便、准确方法,现介绍如下。1　材料与方法1.1　主要试剂

TRI zol 试剂:G ibco BR L ,Life T echnologies 公司;

反转录试剂盒:Promaga 公司;T aq DNA 聚合酶:华美

生物工程公司;Rsa Ⅰ限制性内切酶:Promaga 公司;PCR 纯化试剂盒:R oche 公司;质粒:p GE M 2TE ASY VECT OR SY STE M Ⅱ,Promaga 公司;大肠杆菌:E.coli JM109,大连宝生物公司。1.2　引物

根据G enBank 中小鼠T NF 2α(NM -013693)序列资料自行设计引物,上、下游引物分别为:5′2C AC AA 2G ATGG TGGG AC AG TG AC 23′和5′2G AC A 2G AGGG AAG CTG ACC ACTC 23′。扩增片段长度149bp 。1.3　目的基因

小鼠T NF 2

α基因片段,从小鼠巨噬细胞提取总RNA ,反转录为cDNA ,然后经PCR 扩增、回收纯化获得。