干酪乳杆菌胸苷酸合成酶基因的分子克隆与序列测定

本研究的目的是以干酪乳杆菌 Ｌ． ｃａｓｅｉ ３４１０３染色体 ＤＮＡ为模板，利用 ＰＣＲ技术扩增出胸苷酸合成酶（Ｔｈｙｍｉｄｙｌａｔｅｓｙｎｔｈａｓｅ，　ｔｈｙＡ）基因，利用玻璃奶回收纯化试剂盒回收纯化ｔｈｙＡ基因，并将其克隆入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，再转化Ｘ５１感受态细胞，筛选阳性克隆，提取质粒，进行酶切鉴定、ＰＣＲ扩增鉴定，并对ｔｈｙＡ基因片段进行测序，与已知序列进行同源性比较，结果表明成功克隆了 ｔｈｙＡ基因，全长约 １．１ｋｂ，与国外报道的 ｔｈｙＡ基因同源性达９９％。这为构建以ｔｈｙＡ基因为选择压力的非抗生素抗性穿梭表达载体奠定了基础。

Clning and Sequencing of the Lactobacillus Casei Thymidylate Synthase Gene

Using chromosome DNA of Lactobacillus casei 34103 as template,Thymidylate synthase(thyA)gene was amplified by PCR with pfu DNA polymerase, and purified by giassmilk purification kit. Choosing pW425e vector, which can express shuttlely between E. coli and Lactobacillus, containing erythromycin resistance gene, the PCR production of thyA gene was cloned into it. Then X51 (thyA-) competent cells were transformed with the ligation.The recombinant plasmid containing thyA gene were selected and to test the inserted thyA gene by cleaved with restriction endonuclease HincII and by PCR amplification and by sequenced. The result showed that: the whole thyA gene was cloned already (about 1. 1kb). Sequence analysis indicated that thyA gene displayed 99% nuclotide identities with the published sequences. The thyA gene proved a good foundation for the construction of shuttle vector by using thymidylate synthase(thyA) gene as a selective pressure.