蜡样杆菌(Bacillus cereus)M22 Mn-SOD cDNA片断的克隆

Cloning and Analysis of cDNA Fragment of Mn-SOD (Manganese Superoxide Dismutase) from Bacillus cereus M22

分析不同种细菌Mn SOD氨基酸序列的保守区域 ,设计一对简并引物进行RT PCR扩增 ,产物经T A载体连接转化大肠杆菌JM 1 0 9,筛选阳性克隆、测序并进行同源性分析 ,得到 4 36bpMn SODcDNA片段。根据此片段设计 5′端特异性引物 ,然后进行 5′RACE扩增 ,获得Mn SOD 5′端 387bpcDNA片断。将 2段序列进行拼接获得 5 94bpcDNA片断 ,编码 1 72个氨基酸。对推定氨基酸序列进行BLAST分析 ,结果表明其与炭疽芽孢杆菌 (Bacillusanthracis)同源性为 95 % ,且Gly77,Aly78,Phe86,Gln1 50 和Asp1 51 在已报道的Mn SOD中均存在 ,构成Mn SOD的活性中心。表明该序列为蜡样芽孢杆菌Mn

Analyzing the conserved domain of Mn SOD from thirteen different bacteria a pair of degenerate primers was designed, and RT PCR amplification was performed.The PCR product was then inserted into T A vector and transformed into E.coli JM109.Ten positive cloned colonies were selected and sequenced to analyze their homology.A 436bp cDNA fragment was obtained.Based on this fragment,specific primers were designed for 5' end and then 5' RACE amplification was performed, and a 5' end 387bp cDNA fragment of Mn SOD was obtained.Join these two fragments together a 594bp cDNA fragment was obtained encoding 172 amino acids.Comparison of the deduced amino acid sequence with BLAST revealed that its homology with Mn SOD of Bacillus anthracis was 95%.In addition,Gly\+\{77\},Aly\+\{78\},Phe\+\{86\},Gln\+\{150\},and Asp\+\{151\} all existed in the reported Mn SOD,which consisted of the active center of Mn SOD.All of these suggested that the sequence was the Mn SOD cDNA from B.cereus