重组肺炎链球菌HMGR特性分析及抑制剂筛选
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摘要
肺炎链球菌(Streptoccus pneumoniae)是引起脑膜炎、菌血症、肺炎等疾病的主要致病菌,近年来由于环境污染和抗生素的乱用而造成多药抗性的肺炎链球菌菌株逐年增加,对抗生素的抗性逐年增强。HMG-CoA还原酶(HMG-CoA reductase,HMGR)是甲羟戊酸(mevalonate)途径的限速酶,催化HMG-CoA还原为甲羟戊酸。本研究从肺炎链球菌基因组DNA中PCR扩增出mvaA基因全长1275bp的完整编码序列,将其克隆到pMD18-T载体中,转化大肠杆菌DH5α。重组质粒经HindⅢ和BamHI双酶切分析并测序证实的阳性重组子亚克隆到pET-28a(+)表达载体中,转化大肠杆菌BL21(DE3)。在30℃以1mM IPTG诱导表达,Ni-NTA层析柱分离纯化后经SDS-PAGE分析表明在约47kD处有特异性表达带。肺炎链球菌HMGR以NADPH作为辅酶,动力学分析采用紫外分光光度法检测NADPH在340nm处吸收值的变化。检测结果表明肺炎链球菌HMGR的最适pH约为6.5,最适温度37℃。粗酶提取物的比活为11.22U/mg,分离纯化后比活为31.98U/mg,比活提高2.8倍。在37℃,pH6.5时的Vmax和Km分别为62.1U/mg和260μM。罗伐他汀是HMGR的竞争性抑制剂,对肺炎链球菌HMGR的抑制常数Ki为362μM。表达纯化后的重组HMGR免疫新西兰大白兔,提取抗血清,ELISA检测其效价为1:320000,Western杂交证明HMGR具有特异性杂交条带。
序列分析表明,HMGR主要存在两类,Ⅰ类主要存在于真核生物和部分古细菌中,Ⅱ类主要存在于原核生物和部分古细菌中。他汀类药物是Ⅰ类HMGR的良好竞争性抑制剂,其Ki常数在nM范围内,但是他汀类药物对Ⅱ类HMGR的抑制效果并不明显,其Ki值在μM范围内。为了寻找更专一有效的针对Ⅱ类HMGR的竞争性抑制剂,本文以假单孢杆菌HMGR晶体结构为模板,通过SYBYL7.0软件,用同源模建的方法分析了肺炎链球菌HMGR的三维结构,根据其同源模建和三维结构筛选HMGR的竞争性抑制剂。用重组表达的肺炎链球菌HMGR,检测筛选到的抑制剂的抑制效果。初步分析结果表明,在待筛选的30种化合物中,筛选到一种比传统的竞争性抑制剂罗伐他汀具有更佳抑制效果的化合物,其抑制常数Ki约为76μM,此种化合物可作为进一步研究新型抗菌药物的预选药物。目前,以计算机分析三维结构为手段,用同源模建的方法寻找结构类似物来研究新药的开发是一种有效的途径,本实验为进一步寻找专一性的特效抑制剂奠定了基础。
Streptococcus pneumoniae is the major causative organisms of meningitis, bacteremia,and pneumonia.Resently,for the Environmental pollution the excess use of antibiotics,the drug resistance of S.pneumoniae to antibiotics showed ascending tendency.Hydroxymethylglutaryl-coenzyme A reductase(HMGR) is the rate-limiting enzyme in the classical mevalonate pathway,which converts HMG-CoA to mevalonate, the rate-limiting step in isopentenyl diphosphate(IPP) biosynthisis.The present study cloned the full-length HMG-CoA reductase(HMGR) gene of S.pneumoniae,and expressed in E.coli BL21(DE3).The recombinant protein was purified by Ni-NTA affinity chromatography and obtained the high activity HMGR.Activity was optimal at pH6.5 and approximately 37℃The specific activity of the crude extract and the Ni~(2+) -NTA were 11.22 U/mg and 31.48 U/mg respectively.The enzyme catalyzes the normal anabolic reaction using NADPH not NADU as a reductant,the V_(max)and K_m of the reductive deacylation of HMG-CoA to mevalonate were 62.1U/mg and 260μM respectively.Lovastatin inhibited competitively with HMG-CoA with the K_i362μM. New Zealand rabbits were immunized with the purify HMGR protein.The antiserum was detected by ELISA and the titer of these antibodies was up to 1:320,000.The specificity of these antibodies against HMGR was further confirmed by western blotting.
Comparison of the sequence of HMGR has revealed the existence of two classes of this enzyme,the classⅠenzymes found in eukaryotes and some archaea,and the classⅡfound in certain eubacteria and the archaea.Statins represent a kind of excellent competing inhibitor targeted for the classⅠHMGR,but were relatively poor to the classⅡenzyme of important bacterial pathogens.Potent HMGR inhibitors with Ki values in the nM range to the classⅠenzyme,but in theμM range to the classⅡenzyme,it is high 10~3-10~5 folders.In order to develop new inhibitors with more specific and stronger function targeted for the classⅡHMGR,the 3D model of S.pneumoniae HMGR was built based on structure template of HMGR of Pseudomonas mevalonii by homology modeling using composer module of SYBYL7.0 program.Acoding to the homology modeling of S.pneumoniae,we screen out one better inhibitor with the ki of 76μM from 30 kinds of inhibitors.Currently,using computer to analyze three-dimensional structure and using homology modeling to search for structural analogues and then develop new drugs is a new research area.
序列分析表明,HMGR主要存在两类,Ⅰ类主要存在于真核生物和部分古细菌中,Ⅱ类主要存在于原核生物和部分古细菌中。他汀类药物是Ⅰ类HMGR的良好竞争性抑制剂,其Ki常数在nM范围内,但是他汀类药物对Ⅱ类HMGR的抑制效果并不明显,其Ki值在μM范围内。为了寻找更专一有效的针对Ⅱ类HMGR的竞争性抑制剂,本文以假单孢杆菌HMGR晶体结构为模板,通过SYBYL7.0软件,用同源模建的方法分析了肺炎链球菌HMGR的三维结构,根据其同源模建和三维结构筛选HMGR的竞争性抑制剂。用重组表达的肺炎链球菌HMGR,检测筛选到的抑制剂的抑制效果。初步分析结果表明,在待筛选的30种化合物中,筛选到一种比传统的竞争性抑制剂罗伐他汀具有更佳抑制效果的化合物,其抑制常数Ki约为76μM,此种化合物可作为进一步研究新型抗菌药物的预选药物。目前,以计算机分析三维结构为手段,用同源模建的方法寻找结构类似物来研究新药的开发是一种有效的途径,本实验为进一步寻找专一性的特效抑制剂奠定了基础。
Streptococcus pneumoniae is the major causative organisms of meningitis, bacteremia,and pneumonia.Resently,for the Environmental pollution the excess use of antibiotics,the drug resistance of S.pneumoniae to antibiotics showed ascending tendency.Hydroxymethylglutaryl-coenzyme A reductase(HMGR) is the rate-limiting enzyme in the classical mevalonate pathway,which converts HMG-CoA to mevalonate, the rate-limiting step in isopentenyl diphosphate(IPP) biosynthisis.The present study cloned the full-length HMG-CoA reductase(HMGR) gene of S.pneumoniae,and expressed in E.coli BL21(DE3).The recombinant protein was purified by Ni-NTA affinity chromatography and obtained the high activity HMGR.Activity was optimal at pH6.5 and approximately 37℃The specific activity of the crude extract and the Ni~(2+) -NTA were 11.22 U/mg and 31.48 U/mg respectively.The enzyme catalyzes the normal anabolic reaction using NADPH not NADU as a reductant,the V_(max)and K_m of the reductive deacylation of HMG-CoA to mevalonate were 62.1U/mg and 260μM respectively.Lovastatin inhibited competitively with HMG-CoA with the K_i362μM. New Zealand rabbits were immunized with the purify HMGR protein.The antiserum was detected by ELISA and the titer of these antibodies was up to 1:320,000.The specificity of these antibodies against HMGR was further confirmed by western blotting.
Comparison of the sequence of HMGR has revealed the existence of two classes of this enzyme,the classⅠenzymes found in eukaryotes and some archaea,and the classⅡfound in certain eubacteria and the archaea.Statins represent a kind of excellent competing inhibitor targeted for the classⅠHMGR,but were relatively poor to the classⅡenzyme of important bacterial pathogens.Potent HMGR inhibitors with Ki values in the nM range to the classⅠenzyme,but in theμM range to the classⅡenzyme,it is high 10~3-10~5 folders.In order to develop new inhibitors with more specific and stronger function targeted for the classⅡHMGR,the 3D model of S.pneumoniae HMGR was built based on structure template of HMGR of Pseudomonas mevalonii by homology modeling using composer module of SYBYL7.0 program.Acoding to the homology modeling of S.pneumoniae,we screen out one better inhibitor with the ki of 76μM from 30 kinds of inhibitors.Currently,using computer to analyze three-dimensional structure and using homology modeling to search for structural analogues and then develop new drugs is a new research area.
引文
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2.陈保德,尹一兵.肺炎链球菌致病的分子机理.国外医学临床生物化学与检验学分册.2002,23(6):362-364.
3.胡晓彦,桂炳东.肺炎链球菌分子流行病学研究进展.江西医学检验.2007,25(5):499-490.
4.刘晓红.肺炎链球菌感染及耐药机制研究新进展.医学检验与临床.2007,18(1):56-58.
5.刘晓红,张绪利.肿炎链球菌耐药性分析.世界健康杂志.2007,3(4):74-75.
6.刘亚昆.武汉地区肺炎链球菌的红霉素耐药性分析及血清分型研究.2006,8(3):4-9.
7.李南,王非,胥文春等.肺炎链球菌双组份系统中组氨酸激酶(YycG)的同源模建与分析.生物工程学报.2009,25(2):207-214.
8.李南,王非,牛司强等.组氨酸激酶(Yyc G)在筛选肺炎链球菌抑制剂中的应用.中国生物工程杂志.2009,29(3):25-29.
9.吴金英,李少君,徐新波等.肺炎链球菌对大环内酯类抗菌药物耐药机制的研究.中华医院感染学杂志.2008,18(12):1681-1683.
10.孙爱华,樊欢,夏肖萍等.肺炎链球菌ciaH/R基因重组表达及C iaH/R与β-内酰胺类耐药相关性.浙江大学学报(医学版).2008,37(6):605-611.
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