以淀粉酶基因为筛选标记的E.coli克隆载体的构建

以E．coli质粒pJRD184为基础，将多酶切点引入地衣芽胞杆菌（Bacilluslicheniformis）的a－淀粉酶基因信号肽编码区的PstI切点，构建了能表达并将a－淀粉酶（Amy）渗漏到胞外的E．coli克隆载体pLAM9712．该载体可根据在含有可溶性淀粉的LB平板上菌落周围有没有淀粉水解圈直接筛选重组子，从而避免了使用异丙基硫代－β－D－半乳糖苷（IPTG）、5－溴－4－氯－3－吲哚－β－D一半乳糖苷（X－gal），代之以廉价的碘、淀粉．实验进一步验证了pLAM9712在基因克隆中的可行性及质粒本身的稳定性．

CONSTRUCTION OF AN E. COLI CLONING VECTOR EMPLOYING THE AMYLASE GENE AS A SCREEN MARKER

Based on pJRD184, an E. coli cloning vector pLAM9172 has been constructed by inserting MCS into PstI restriction site of signal sequence coding region of a-amylase gene from Bacillus lichenlers. It prossesses the advantage over existing E. coli cloning vectors: Directly screening the recombinants on LBS plates by the loss of the hola around the colonies, inexpensive indicators, iodine/starch, instead of the very expensive ones, IPTG/X-gal; Furthermore, it has been proved that the cloning vector is not only stable in E. coli but also available in gene cloning.