TEM-105编码基因的功能研究

目的　获得浙江地区临床分离的 4株肺炎克雷伯菌所产的TEM型β 内酰胺酶编码基因的序列,鉴定其基因型,并了解其相关特性。方法　PCR扩增TEM型β 内酰胺酶编码基因片段,克隆入pGEM Teasy载体,表达于感受态细胞大肠埃希菌DH5α中,双脱氧链终止法测定核苷酸序列,确定基因型;其基因与原核表达载体 (pET 28c)连接,并在感受态细胞大肠埃希菌DH5α中进行原核表达,提取质粒,用PCR进行表达鉴定,用等电聚焦电泳测定原核表达菌株中酶的等电点(pIs),用ESBLs表型确认试验鉴定表达菌株的表型,另外对这些临床菌株进行接合试验。结果　PCR扩增结果显示这 4株细菌产生的TEM型β 内酰胺酶编码基因片段含1 009个核苷酸,其表达酶的性质均为非ESBLs,pIs均为5 4,临床菌株的质粒接合试验均为阳性。这 4株临床菌株所产生的TEM型β 内酰胺酶的编码基因已被GenBank命名为TEM 105(GenBank注册号:AF516720)。结论　浙江地区这 4株细菌所产TEM型β 内酰胺酶均为TEM 105,在世界上我们首次从临床分离株中发现TEM 105。

Studies on the function of the encoding gene of TEM-105 type of β-lactamases

Aim To obtain the encoding gene sequences of TEM-type β-lactamases produced by 4-strain Klebsiella pneumoniae in Zhejian g Province, identify their genotypes and study some properties of these TEM-typ e β-lactamases.Methods The encoding genes of TEM-type β-la ctamases produced by 4 isolates were amplified by PCR. The purified PCR products were ligated with pGEM-T easy vectors, expressed in E. coli DH 5α, and sequenced by Sanger's dideoxy chain termin ation composition method. Compared with anino acid sequences in the GenBank,TEM -types of the β-lactamases was determined. The genes of TEM β-lactamases were ligated with pET-28 c vector to express recombinant proteins in E. coli DH 5α. Plasmids were extracted from the p ronucleus expression strains and PCR was performed to determine whether the pron ucleus expression was successful or not. Their phenotypes were determined by ESB Ls phenotype affirmative test. The isoelectric points (pIs) of the recombinant p roteins were determined by isoelectric focus. Conjugation test was performed to determine whether their genes existed in plasmid or chromosome. Results The encoding genes of β-lactamases were determined as TEM by PCR. It s PCR product had 1 009 nucleotides. The pI of the novel TEM β-lactamase was 5.4. The enzyme was determined as non-ESBLs by ESBLs phenotype affirmative tes t.Transconjugants were successfully selected from the paternal producers in conj ugation tests. The TEM-type β-lactamase produced by 4 strains was determined as TEM-105(AF516720) by GenBank. Conclusion The β-lactamase produced by 4-strain K. pneumoniae from 4 patients in Zhejiang Province was TEM-105. It was the first report of TEM-105 type β-lactamase produced by 4-st ain K. pneumoniae from China in the world.