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重组β-内酰胺酶基因的诱导表达及蛋白质的分离纯化研究摘要：目的：诱导表达三种临床危害严重的β-内酰胺酶基因，并分离、纯化表达的蛋白质，为新型β-内酰胺酶抑制剂的筛选研制提供药靶蛋白。方法：利用IPTG诱导表达三种临床危害严重的β-内酰胺酶重组基因，包括CTX-M-15、OXA-45和VIM-1；采取超声波破碎及离心等方法获取含β-内酰胺酶的上清液，利用亲和层析法分离、纯化β-内酰胺酶，利用BCA法进行蛋白含量检测，SDS-PAGE法进行检测β-内酰胺酶纯度。结果：获得了高纯度的CTX-M-15、OXA-45、VIM-1三种β-内酰胺酶蛋白质，为新型β-内酰胺酶抑制剂的高通量生化筛选提供了高纯度的药靶蛋白。32512
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毕业论文关键词：β-内酰胺酶；CTX-M-15；OXA-45；VIM-1；亲和层析；蛋白纯化
Study on expression of recombinant beta lactamase genes and separation and purification of protein
Abstract: Objective In this experiment, we try to induce the expression of three kinds of clinical serious harm of beta - amidase recombinant gene and purify the beta lactamase protein by physical and biochemical methods. Methods three clinical serious harmfully CTX-M-15、OXA-45、VIM-1 beta lactamase recombinant gene is induced by IPTG to express, then the supernatant containing enzymes is been acquired by taking the physical methods such as ultrasonication and centrifugation ,beta lactamase protein is obtained by biochemical methods such as affinity chromatography. Finally, to detect the purity of protein by electrophoresis and the content of protein by BCA method. Result CTX-M-15, OXA-45, VIM-1 three beta lactamase protein whose purity is high are obtained which is the first step of high-throughput biochemical screening been carried out and provides it with the high purity target protein.
Key words: Lactamase; CTX-M-15; OXA-45; VIM-1; affinity chromatography; purification of protein
目录
引言 1
1材料与方法 2
1.1 菌株与设备 2
1.1.1 菌株 2
1.1.2 仪器 2
1.2 试剂 2
抗生素 2
LB固、液体培养基（1L,pH 7.2） 2
IPTG (即异丙基硫代-β- D 半乳糖苷，0.1M) 3
His-tag亲和层析 需配试剂： 3
SDS-PAGE电泳 需配试剂： 4
测酶活 需配试剂： 5
1.3方法与步骤 5
1.3.1流程图 5
1.3.2 OXA-45重组蛋白的表达与纯化 6
1.3.2.1 工程菌的培养及诱导表达 6
1.3.2.2 测β-内酰胺酶酶活 7
1.3.2.3亲和层析 7
1.3.2.4 BCA（bicinchoninic acid）法检测蛋白浓度 8
1.3.2.5 浓缩 8
1.3.2.6 SDS-PAGE 电泳测蛋白纯度 8
1.3.3 CTX-M-15 、VIM-1重组蛋白的纯化 9
2 结果与分析 10
2.1 分离纯化重组OXA-45蛋白 10
2.1.1 OXA-45发酵液上清及洗脱液的β-内酰胺酶酶活性测定 10
2.1.2 OXA-45 洗脱液BCA法测蛋白含量 10
2.1.3 OXA-45 SDS-PAGE电泳 11
2.2分离纯化重组CTX-M-15蛋白 11
2.2.1 CTX-M-15发酵液上清及洗脱液的β-内酰胺酶酶活性测定 11
2.2.2 CTX-M-15 洗脱液BCA法测蛋白含量 11
2.2.3 CTX-M-15 SDS-PAGE电泳 11
2.3分离纯化重组VIM-1蛋白 12
2.3.1 VIM-1发酵液上清及洗脱液的β-内酰胺酶酶活性测定 12