用CODEHOP设计简并引物克隆反刍月形单胞菌K6乙酸激酶基因片段

利用CODEHOP设计细菌乙酸激酶的简并引物,选用1对引物ACKSe以高效丙酸生成菌反刍月形单胞菌K6基因组DNA进行PCR,得到749 bp PCR产物,产物经pMD18-T载体克隆转化至DH5α大肠杆菌中,测序后经Blastx比对,此DNA产物与其他菌属来源的乙酸激酶蛋白序列具有相似性,所克隆的序列即为K6的乙酸激酶基因片段。用CODEHOP程序化设计的简并引物可信性强,阳性率高。该基因的成功克隆为丙酸生成菌K6乙酸代谢工程研究提供了依据。

The cloning of K6 acetate kinase gene fragrnent of Selenomonas ruminantium de-generate primer design with CODEHOP

Degenerate PCR for acetate of Selenomonas ruminantium K6 was earried out using CODEHOP to design the degenerate primers,choseing a pair of degenerate primers named ACKSe and utilizing the propionic acid produ-cing bacteria genome DNA as template. 749 bp PCR product was obtained, transformed into the E. coli DH5_α through being linked with pMD18-T vector and sequenced after filtration. Similarity alignment showed that the prod-ucts of the cloned DNA were similar to those of acetate kinase gene from the other strains. The cloned sequence was putatively acetate kinase gene DNA fragment from K6 strain. The results indicated that the degenerate primers de-signed by the CODEHOP software could be used to obtain specific gene fragment. Cloning of gene fragment would give a scientific warrant for the metabolic engineering research of propionic acid producing bacteria,Selenomonas ru-minantium K6.