摘要
目的对嗜麦芽窄食单胞菌所产L1型金属β内酰胺酶基因进行测序、原核表达。方法PCR扩增L1酶的编码基因,将其亚克隆入pUCmT载体后测定核苷酸序列,将L1酶基因克隆至表达载体pET41a(+)后在大肠埃希菌BL21(DE3)中表达,十二烷基硫酸钠聚丙烯酰胺凝胶电泳检测表达情况,等电聚集电泳测定表达蛋白的等电点。结果本实验中L1的编码基因与国外同类酶的氨基酸同源性为92.44%。此L1酶基因可在大肠埃希菌BL21(DE3)中大量表达,等电点为6.7。结论对L1型金属β内酰胺酶的克隆测序及成功表达为进一步研究该酶的分子生物学特性奠定了基础。
Objective This study was designed to conduct sequence analysis and prokaryotic expression of L1 metallo-β-lactamase gene from Stenotrophomonas maltophilia. Methods The encoding gene of L1 metallo-β-lactamase was amplified by PCR and sequenced after being subcloned into pUCm-T vector. The target gene was cloned into pET-41a (+) vector and expressed in Escherichia coli BL21 (DE3). The expression of L1 was confirmed by SDS-PAGE. The isoelectric point of recombinant protein was determined by isoelectric focusing electrophoresis. Results Sequence analysis of the target gene showed 92.44% amino acid identity to L1 found in other country and L1 can be profusely expressed in E. coli BL21 (DE3) with isoelectric point of ~6.7 . Conclusions The sequencing and successful prokaryotic expression of L1 laid a good foundation for further molecular biological study of L1.
出处
《中国抗感染化疗杂志》
CAS
2005年第3期156-159,共4页
Chinese Journal of Infection and Chemotherapy
基金
国家自然科学基金资助(编号:30472109)
广东省自然科学基金(编号:34616)
汕头大学研究与发展基金(编号:L00010)。
关键词
嗜麦芽窄食单胞菌
金属Β内酰胺酶
原核表达
Stenotrophomonas maltophilia
Metallo-β-lactamase
Prokaryotic expression
