摘要
利用PCR技术从Staphylococcus aureus ATCC6538基因组中扩增出大小为1053 bp的镍钴转运酶基因NiCoT gene,将其连接到pET-3c载体上构建重组质粒,并转化至E.coli BL21.筛选阳性菌并经酶切分析和PCR扩增双重鉴定.核苷酸序列测定及分析结果与GenBank中报道的同类基因相似性高达97%以上,表明其具有正确的NiCoT基因核苷酸序列.重组菌的SDS-PAGE结果图谱中,在相对分子量为39000附近有特异性蛋白条带,大小符合预测值,表明NiCoT基因在E.coli BL21中成功表达.基因工程菌在IPTG用量为1.00 mmol.L-1,诱导时间为4 h的条件下培养对镍离子的富集能力最高.在不同镍离子浓度时,基因工程菌对溶液中Ni2+的平衡富集量为11.33 mg.g-1,与原始宿主菌相比提高了3倍.对基因工程菌吸附镍和钴的实验表明,Staphylococcus aureus ATCC6538的NiCoT对镍具有较高的特异性和富集容量,属于第Ⅲ类镍钴转运酶.
1 053 bp of the nickel/cobalt transferase gene, NiCoT gene, from Staphylococcus aureus ATCC6538 was amplified by PCR and ligated into vector pET-3c. The recombined plasmid was constructed and transferred into E. coli BL21 at appropriate temperature. The recombined strain was isolated and identified by restriction enzyme digestion and PCR amplification. Nucleotide sequence analysis showed that the identity was more than 97% between the nickel/cobalt transferase gene from S. aureus ATCC6538 and the reported gene sequence of other species or subspecies of S. aureus at GenBank. There was a characteristic protein strip near the relatively molecular weight of 39 000 in the SDS-PAGE picture, which was identical to the expected value. The result demonstrated that the NiCoT gene of S. aureus had been successfully expressed in E. coli BL21. The E. coli BI21 containiog the NiCoT gene had the highest bioaccumulation quantity when induced with 1.00 mmol. L^-1 IPTG for 4 h. The quantity of equilibrium accumulation of the genetically engineered E. coli BL21 was 11.33 mg·g^-1 , which was 3 times more than that of the original E. coli BL21 at different nickel concentrations. The NiCoT of S. aureus ATCC6538 was a highly selective and accumulative nickel transporter and belonged to the class Ⅲ of the nlckel/cobalt transferase.
出处
《环境科学》
EI
CAS
CSCD
北大核心
2007年第4期918-923,共6页
Environmental Science
基金
国家自然科学基金项目(50278040)
