以thyA基因为选择压力非抗性质粒载体的构建

CONSTRUCTION OF SHUTTLE VECTOR BY USING THYA GENE AS A SELECTIVE PRESSURE

以干酷乳杆菌Ｌ．ｃａｓｅｉ３４１０３染色体ＤＮＡ为模板，利用ＰＣＲ技术扩增胸苷酸合成酶（Ｔｈｙｍｉｄｙ－ｌａｔｅｓｙｎｔｈａｓｅ，ｔｈｙＡ）基因，回收纯化。选择以红霉素抗性为选择压力的可以在大肠杆菌和乳酸菌中穿梭表达的质粒ｐＷ４２５ｅ为基本质粒，以ｔｈｙＡ基因取代红霉素基因，获得重组载体并鉴定。此重组载体可以对ｔｈｙＡ基因缺陷的大肠杆菌Ｅ．ｃｏｌｉ Ｘ５１和嗜酸乳杆菌ＤＯＭＬａＳ １０７进行功能弥补。进而构建了以ｔｈｙＡ基因为选择压力的非抗生素抗性穿梭表达载体，其大小为３７１６ｂｐ，并命名为ｐＷ４２５ｔ。

Using chromosome DNA of Lactobacillus easei 34103 as template, thy A(Thymidylate synthase) gene was amplified by PCR with pfu DNA polymerase. Choosing pW425e vector, which can express shuttlely between E. coli and Lactobacillus, containing erythromycin resistance gene, as the basic plasmid. The PCR production of thyA gene was used to replace the erythromycin resistance gene of pW425e. The new plasmid vector consits of 3716bp as expected. Which can remedy the thyA mutant E. coli X51 and Lactobacillus DOMLaS107 successfully, and named as pW425t.