铜绿假单胞菌产TEM-29型ESBL基因的克隆及原核表达

Cloning, sequencing and prokaryotic expression of TEM-29 extended-spectrum β-lactamase-encoding gene in Pseudomonas aeruginosa

目的研究临床分离铜绿假单胞菌Pa3-104产生的超广谱β-内酰胺酶的基因型,并对其进行原核表达,了解它的相关特性。方法对该临床菌株进行接合试验;采用PCR扩增TEM型β-内酰胺酶编码基因的片段,克隆入pUCm-T载体,在E.coliJM109中表达,双脱氧链终止法测定核苷酸序列,确定基因型;并将TEM型β-内酰胺酶全编码基因克隆入原核表达载体pBK-CMV中进行原核表达;用三维试验检测重组菌产β-内酰胺酶的表型,等电聚焦电泳测定其表达蛋白的等电点(pI)。结果该临床菌株质粒接合试验为阳性;PCR扩增测序结果显示为TEM型,其基因片段含861个核苷酸,该TEM型β-内酰胺酶的编码基因与GenBank中大肠埃希菌产生的TEM-29的编码基因(Y17584)一致,等电聚焦电泳结果显示该酶的pI为5.4,三维试验证实为ESBL。结论在国内首次从临床分离铜绿假单胞菌中发现TEM-29型ESBL。

Objective To investigate the properties of extended-spectrum β-lactamase in a Pseudomonas aeruginosa clinical isolate Pa3-104. Methods Conjugation test was performed to determine whether the gene existed in plasmid or chromosome. The encoding gene of TEM-type beta-lactamase produced by clinical isolate was amplified by PCR and sequenced after being subcloned into pUCm-T vector. Compared with amino acid sequences in the GenBank, TEM-type of the beta- lactamase was determined. The gene of TEM beta-lactamase was cloned into pBK-CMV vector and expressed in E. coli JM109. Recombinant plasmids were identified by restriction enzyme digestion. The phenotype was determined by three-dimensional test. The isoelectric point （pI） of the recombinant protein was determined by isoelectric focus electrophoresis. Results Transconjugants were successfully selected from the paternal producers in conjugation test. The PCR products were encoding genes of beta-lactamase, and had 861 nucleotides. The encoding gene sequence of TEM-type beta-lactamase produced by Pa3-104 was the same as that of TEM-29 produced by Escherichia coli （#17584 in GenBank）. The enzyme was characterized as ESBL by three-dimensional test with pI of 5.4. Conclusions It was firstly reported that TEM-29 type extended spectrum beta-lactamase produced by Pseudomonas aeruginosa was found in China.