超广谱-内酰胺酶SHV-5的基因重组表达及酶活性鉴定

Cloning and expression of the extended-spectrum β-lactamase gene bla_(SHV-5) from Klebsiella pneumoniae

目的对blaSHV-5进行基因重组表达,为建立SHV类β-内酰胺酶抑制剂筛选模型研究奠定基础。方法以产blaSHV-5肺炎克雷伯菌基因组DNA为模板,自行设计一对寡核苷酸引物,通过PCR扩增获得blaSHV-5基因片断,定向克隆入质粒载体pBK-CM V构建重组质粒,对重组质粒进行酶切鉴定和DNA测序鉴定,重组质粒转化大肠埃希菌XL 1-B lue M RF′后,N itrocefin显色结合SDS-PAGE分析目的基因在大肠埃希菌中的表达及其酶活性。结果PCR扩增获得了大小约为900bp的DNA条带,定向克隆入质粒载体pBK-CM V后筛选到大小约为5.4kb的重组质粒,S acⅠ和E coRⅠ双酶切后可切下大小约900bp的插入片段,DNA测序发现插入片断的基因序列与G enB ank中肺炎克雷伯菌基因序列完全一致,重组质粒转化大肠埃希菌XL 1-B lue M RF′后可表达分子量约为31500的重组蛋白。重组菌裂解液对n itrocefin显示出较强水解活性。结论超广谱β-内酰胺酶SHV-5在大肠埃希菌XL 1-B lue M RF′中实现了基因重组表达。为进一步进行酶的纯化等相关后续研究奠定了基础。

Objective Cloning and expression of the extended-spectrum β-lactamase gene blaSHV-5 from Klebsiella pneurnoniae and analysing the enzyme activity of the recombinant protein. Methods A pair of oligonucleotide primers were designed according to the published blaSHV-5 gene sequence on Gen Bank. The genefragments were obtained by using polymerase chain reaction. After digestion by restriction enzyme Sac Ⅰ and EcoR Ⅰ , the gene fragments were inserted into plasmid vector pBK-CMV to construct the recombinant plasmid. Gene expression was analysed by using SDS-PAGE. Enzyme activity was detected by using nitrocefin.Results It was found that PCR products contained DNA fragments about 900 base pairs in 1% agarose gel electrophoresis. A 5.4kb recombinant plasmid was obtained. The recombined plasmid expressed a recombinant protein with relative molecular mass 31,500 in E. coli XL1-Blue MRF＇. The cell lysates of E. coli XL1-Blue MRF＇ containing the recombinant plasmid can hydrolyzed nitroeefin to show red colors. Conclusion The blaSHV-5 gene was successfully cloned and expressed in E. coli XL1-Blue MRF＇, which provided the basis for further screening of the SHV enzyme inhibitors.