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Recombinant Streptococcus pneumoniae HMGR characterization and inhibitor screening

Author: CuiGuZhen
Tutor: LiuDeLi
School: Central China Normal University
Course: Biochemistry and Molecular Biology
Keywords: Streptococcus pneumoniae HMGR Characterization Homology modeling Competitive inhibitor
CLC: R378
Type: Master's thesis
Year: 2009
Downloads: 10
Quote: 2
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Abstract


Streptococcus pneumoniae (Streptoccus pneumoniae) is caused by meningitis, bacteremia, pneumonia and other diseases of the main pathogens in recent years due to environmental pollution and indiscriminate use of antibiotics caused by multi-drug resistant strains of Streptococcus pneumoniae increased year by year, to antibiotics resistance increased year by year. HMG-CoA reductase (HMG-CoA reductase, HMGR) is mevalonate (mevalonate) pathway limiting enzyme, catalytic reduction of HMG-CoA mevalonate. In this study, genomic DNA from Streptococcus pneumoniae was amplified by PCR mvaA gene is 1275bp complete coding sequence was cloned into pMD18-T vector, and transformed into E. coli DH5α. Recombinant plasmid was digested with Hind Ⅲ and BamHI analysis and sequencing confirmed the positive recombinants were subcloned into pET-28a () expression vector and transformed into E. coli BL21 (DE3). At 30 ℃ induced with 1mM IPTG, Ni-NTA column after purification by SDS-PAGE analysis showed that at about 47kD band at a specific expression. Streptococcus pneumoniae HMGR with NADPH as a coenzyme, kinetic analysis using UV spectrophotometry NADPH absorbance at 340nm changes. Test results show that the optimum pH of Streptococcus pneumoniae HMGR about 6.5, the optimum temperature is 37 ℃. Crude enzyme extract specific activity of 11.22U/mg, after purification specific activity of 31.98U/mg, the specific activity increased 2.8-fold. At 37 ℃, pH6.5 the Vmax and Km were 62.1U/mg and 260μM. Rosuvastatin is a competitive inhibitor of HMGR, against Streptococcus pneumoniae HMGR inhibition constant Ki is 362μM. Purified recombinant expression HMGR immune rabbits, extraction antisera, ELISA titer was detected 1:320000, Western hybridization with specific hybridization proved HMGR bands. Sequence analysis showed that, HMGR mainly there are two types, Ⅰ class is primarily found in eukaryotes and some archaea, Ⅱ type mainly found in prokaryotes and some archaea. Statins are a good class Ⅰ HMGR competitive inhibitor constant Ki in nM range, but statin class Ⅱ HMGR inhibitory effect is not obvious, the Ki values ​​in the μM range. To find more specific and effective for Class Ⅱ HMGR competitive inhibitor, this article Pseudomonas HMGR crystal structure as the template, SYBYL7.0 software, using homology modeling method to analyze the three-dimensional Streptococcus pneumoniae HMGR structure, based on their three-dimensional structure and construction of homology modeling HMGR competitive inhibitor screening. A recombinant S. pneumoniae HMGR, detection screening effect of suppressing the inhibitor. Preliminary results show that 30 compounds to be screened in the screening than the traditional one kinds competitive inhibitor rosuvastatin has better inhibitory effect of the compound, the inhibition constant Ki of approximately 76μM, such compounds can be used as further preselection of new antibacterial drugs drugs. Currently, computer analysis of three-dimensional structure as a means of using homology modeling approach to find structural analogues to study the development of new drugs is an effective way to further this experiment seeking specific effects inhibitors foundation.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria
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