摘要
目的比较3种截短的泡球蚴Em18基因聚合酶链反应(PCR)扩增产物经TA克隆和直接酶切2种方法,在原核表达质粒构建中的运用。方法PCR扩增带有EcoRⅠ和XhoⅠ酶切位点的3种截短的泡球蚴Em18基因,经2种方法构建3种截短的Em18原核表达质粒：①PCR扩增产物与T载体连接构建T-Em18,重组质粒经EcoRⅠ/XhoⅠ酶切,电泳分离、纯化目的片段,再与经过EcoRⅠ/XhoⅠ酶切的原核表达载体pET41a(+)连接；②PCR扩增产物经EcoRⅠ和XhoⅠ酶切,电泳、纯化,直接与经过相同限制性内切酶酶切的pET41a(+)连接。结果先经TA克隆,再经酶切构建3种截短的泡球蚴Em18原核表达载体获得成功。而采用直接酶切PCR扩增产物的构建方法未获得成功。结论TA克隆可用于对直接酶切效果不好的PCR扩增片段与表达载体的连接构建,方法简便高效,可提高带有限制性酶切位点的PCR扩增片段克隆人原核表达载体的效率。
Objective To compare the efficiency of constructing three truncated Echinococcus multilocularis （Em）18 gene expression vector using two clone methods. Methods Using the following two methods, the pET41-Em 18 recombinants were constructed. The PCR fragments of three truncated Em 18 gene including EcoR I/Xho I site were ligated with T vector, and then, T-Em18 recombinant plasmids were digested by EcoR I/Xho I . The target fragments with EcoR I and Xho I site were purified with tagarose gel after electrophoresis and were connected with the pET41a （＋） expression vector that had been digested by the same restriction enzymes, The other method was that the PCR fragments were digested by EcoR I/Xho I and then were connected with pET41a（＋） which was digested using EcoR I/Xho I . Results The expression vectors of three truncated Eml8 gene were constructed successfully using TA clone method. In contrast, the pET41-Em18 was not obtained using the other method. Conclusions The TA clone is perhaps a more convenient and efficient method for ligating the amplified fragments from PCR production with expression vectors.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2006年第5期561-564,共4页
Chinese Jouranl of Endemiology
基金
国家自然科学基金(30360097)
新疆重点实验室开放课题基金(XJDX0202-2003-01)
关键词
克隆
分子
DNA
重组
质粒
Cloning, molecular
DNA, recombinate
Plasmids
