超广谱β-内酰胺酶CTX-M-14中药抑制剂的筛选及芸香苷抑酶作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1105

摘要/Abstract

摘要：

β-内酰胺酶的产生是细菌产生耐药的常见机制,其主要代表为超广谱β-内酰胺酶,其中CTX-M-14在全世界呈现流行趋势,为寻找一种CTX-M-14抑制剂,使细菌恢复对β-内酰胺类抗生素的敏感性,本试验首先构建了CTX-M-14的原核表达载体,并以CTX-M-14为靶点借助虚拟筛选工具Autodock Vina进行筛选,得到结合作用较好的中药单体抑制剂芸香苷,通过DS Visualizer分析其相关作用力;通过联合抑菌试验验证芸香苷与抗生素的联合抑菌作用;通过酶动力学试验比较抑酶剂芸香苷与克拉维酸的抑酶作用与方式。结果表明,芸香苷与超广谱β-内酰胺酶CTX-M-14结合部位形成多个氢键和范德华力,其结合作用力为9.9 kcal/mol;芸香苷与头孢噻肟钠对大肠杆菌E320呈协同作用（FICI=0.236）;0.312 5 mg/mL的芸香苷与头孢噻肟钠对CTX-M-14蛋白阳性重组菌呈协同作用（FICI≤0.375）;1.25 mg/mL的芸香苷与头孢噻肟钠对导入空质粒pET-28a（+）的BL-21呈无关作用（FICI=1.5）;抑酶剂芸香苷与克拉维酸均为竞争性抑制剂,但克拉维酸抑酶作用优于芸香苷。本试验证明,芸香苷可以竞争性抑制CTX-M-14活性,具有β-内酰胺酶抑制剂潜质。

关键词: 虚拟筛选, CTX-M-14, 芸香苷, 联合抑菌, 酶动力学

Abstract:

The production of β-lactamases is a common mechanism for bacteria to develop drug resistance,and its main representatives are extended-spectrum β-lactamases,of which CTX-M-14 is showing an epidemic trend worldwide. In order to find an inhibitor to CTX-M-14 for restoring the susceptibility of bacteria to β-lactam antibiotics,in this experiment firstly a prokaryotic expression vector of CTX-M-14 was constructed,and CTX-M-14 was used as the target site for screening with the help of virtual screening tool Autodock Vina,and finally a monomeric inhibitor of Chinese medicine,rutin,with good binding effect was obtained. Its relevant potency by DS Visualizer was analyzed,the combined inhibitory effect of rutin and antibiotics was verified by co-inhibition test,and the enzyme inhibiting effect of rutin and clavulanic acid were compared by enzyme kinetic test. The results showed that rutin formed multiple hydrogen bonds and van der Waals forces at the binding site of CTX-M-14,and its binding force was 9.9 kcal/mol. Rutin and cefotaxime sodium showed synergistic effect on Escherichia coli E320（FICI=0.236）;0.312 5 mg/mL of rutin and cefotaxime sodium showed synergistic effect CTX-M-14 protein（FICI≤0.375）;1.25 mg/mL of rutin and cefotaxime sodium had no effect on BL-21 introduced into empty plasmid pET-28a（+）（FICI=1.5）;both rutin and clavulanic acid were competitive inhibitors,but clavulanic acid had better enzyme inhibitory effect than rutin. This experiment demonstrates that rutin may competitively inhibit the CTX-M-14 activity and has a potential as β-lactamase inhibitor.

Key words: virtual screening, CTX-M-14, rutin, combined antibacterial, enzyme kinetics

赵子玉, 王春光, 吕建存, 李继开, 张铁. 超广谱β-内酰胺酶CTX-M-14中药抑制剂的筛选及芸香苷抑酶作用研究[J]. 生物技术通报, 2022, 38(6): 235-244.

ZHAO Zi-yu, WANG Chun-guang, LV Jian-cun, LI Ji-kai, ZHANG Tie. Screening of β-lactamase CTX-M-14 Chinese Medicine Inhibitor and Enzyme Inhibition of Rutin[J]. Biotechnology Bulletin, 2022, 38(6): 235-244.

图/表 12

图1 PCR扩增产物凝胶电泳图 M：Marker;1：大肠杆菌E320;2：大肠杆菌25922

Fig. 1 Gel electrophoresis of PCR amplified product M：Marker;1：Escherichia coli E320;2：E. coli 25922

图2 转化后质粒经双酶切 M：Marker;1：双酶切后;2：E320

Fig. 2 Double enzyme digestion of transformed plasmid M：Marker;1：after double enzyme digestion;2：E320

图3 CTX-M-14测序结果比对图质粒：测序结果;ctx：预测基因序列

Fig. 3 Alignment of CTX-M-14 sequencing results Plasmid：sequencing result;ctx：predicted gene sequence

图4 重组蛋白的表达检测 A：CTX-M-14重组蛋白的诱导表达（M：蛋白marker;1：诱导前;2：诱导后）;B：重组蛋白的可溶性检测（M：蛋白marker;1：上清;2：沉淀）

Fig. 4 Expression detection of recombinant protein A：CTX-M-14 recombinant protein（M：Protein marker;1：before induction;2：after induction）. B：Soluble detection of recombinant protein（M：Protein marker;1：supernatant;2：precipitation）

图5 蛋白表达条件的优化 A：诱导时间的优化（M：蛋白marker;1：诱导前;2：3 h;3：4 h;4：5 h;5：6 h）;B：诱导温度的优化（M：蛋白marker;1：诱导前;2：26℃;3：31℃;4：34℃;5：37℃）;C：IPTG浓度的优化（M：蛋白marker;1：诱导前;2：0.2 mmol/L;3：0.4 mmol/L;4：0.6 mmol/L;5：0.8 mmol/L;6：1.0 mmol/L;7：1.2 mmol/L）

Fig. 5 Optimization of protein expression conditions A：Optimization of induction time（M：Protein marker;1：before induction;2：3 h;3：4 h;4：5 h;5：6 h）. B：Optimization of induction temperature（M：Protein marker;1：before induction;2：26℃;3：31℃;4：34℃;5：37℃）. C：Optimization of IPTG concentration（M：Protein marker;1：before induction;2：0.2 mmol/L;3：0.4 mmol/L;4：0.6 mmol/L;5：0.8 mmol/L;6：1.0 mmol/L;7：1.2 mmol/L）

图6 重组蛋白纯化及活性鉴定 A：蛋白纯化鉴定（M：蛋白marker;1：纯化前;2：纯化后）;B：蛋白活性鉴定,上排：加入CTX-M-14重组蛋白组;下排：空白对照组

Fig. 6 Purification and activity identification of recomb-inant protein A：Protein purification and identification（M：Protein marker;1：before purification）;2：after purification. B：Identification of protein activity. Top row：CTX-M-14 recombinant protein group is added. Lower row：Blank control group

表1 Autodock Vina虚拟筛选的部分中药成分评分结果

Table 1 Scoring results of some Chinese herbal medicinal ingredients screened virtually by AutoDock Vina

配体Ligand	结合能Binding affinity/（kcal·mol^-1）	均方根偏差/最大值RMSD/UB	均方根偏差/最小值RMSD/LB
Rhein diglucoside	-11.5	0	0
Agastachin	-10.5	0	0
Glycyrrhizin	-10	0	0
Timosaponin A3	-10	0	0
Biepiasterolide	-10	0	0
Sennidin A	-9.9	0	0
Rutin	-9.9	0	0
Ciwujianoside	-9.9	0	0
Galloylpaeoniflorin	-9.8	0	0
Acaciin	-9.8	0	0

图7 芸香苷与超广谱β-内酰胺酶CTX-M-14作用力分析 A：芸香苷与CTX-M-14结合的2D结构示意图;B：芸香苷与CTX-M-14结合的3D结构示意图

Fig. 7 Interaction analysis of rutin with extended-spectrum β-lactamases CTX-M-14 A：2D structure diagram of the binding of rutin with CTX-M-14;B：3D schematic diagram of the binding structure of rutin with CTX-M-14

表2 大肠杆菌E320、重组蛋白阳性菌BL-21的MIC

Table 2 MIC of E. coli E320 and recombinant protein positive BL-21

药物Drug	E320	重组蛋白阳性菌BL-21 Recombinant protein-positive bacterium BL-21
芸香苷/（mg·mL^-1）	2.5	2.5
苯唑西林钠/（μg·mL^-1）	>256（R）	64（R）
氨苄西林钠/（μg·mL^-1）	>256（R）	64（R）
头孢唑啉钠/（μg·mL^-1）	>256（R）	16（R）
头孢他啶/（μg·mL^-1）	>256（R）	8（I）
头孢呋辛/（μg·mL^-1）	4（S）	16（I）
头孢曲松钠/（μg·mL^-1）	>256（R）	32（R）
头孢噻肟钠/（μg·mL^-1）	1024（R）	>1024（R）

表3 芸香苷和头孢噻肟钠联合抑菌试验

Table 3 Combined antibacterial test of rutin and cefotaxime sodium

菌株 Strain	药物 Drug	单药MIC MIC of single drug	联合用药MIC MIC of combination drugs	FICI	结果判定 Result determination
E320	芸香苷/（mg·mL^-1）	2.5	0.625	0.236	协同作用
E320	头孢噻肟钠/（μg·mL^-1）	1 024	16	0.236	协同作用
CTX-M-14蛋白阳性重组菌	芸香苷/（mg·mL^-1）	2.5	0.312 5	<0.375	协同作用
CTX-M-14蛋白阳性重组菌	头孢噻肟钠/（μg·mL^-1）	>1 024	256	<0.375	协同作用
导入空白质粒pET-28a（+）的BL-21	芸香苷/（mg·mL^-1）	2.5	1.25	1.5	无关作用
导入空白质粒pET-28a（+）的BL-21	头孢噻肟钠/（μg·mL^-1）	8	8	1.5	无关作用

表4 酶动力学参数

Table 4 Enzyme kinetic parameters

组别Group	K_m	V_max	K_i	K_m/V_max
空白对照组	7.13	21.33	-	0.33
溶剂对照组	7.11	20.97	-	0.34
芸香苷组	8.76	17.63	11.38	0.50
克拉维酸组	8.45	17.79	3.78	0.47

表5 抑酶保护率

Table 5 Inhibition rate of enzyme

组别 Group	初始OD值 Initial OD value	处理后OD值 OD value after treatment	OD值变化量 OD value change amount	抑酶保护率 Enzyme inhibition protection rate/%
空白对照组	1.15±0.035	0.33±0.015	0.82±0.026	-
溶剂对照组	1.14±0.020	0.32±0.025	0.81±0.015	-
克拉维酸组	1.19±0.025	0.87±0.020	0.32±0.020*	60.98
芸香苷组	1.50±0.030	1.14±0.010	0.35±0.020*	57.31

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