结核分枝杆菌Rv1884基因真核表达质粒的构建及表达

目的:构建结核分枝杆菌Rv1884基因真核表达载体。方法:PCR扩增Rv1884基因,测序正确后克隆入真核表达载体pcDNA3.1(-);经酶切鉴定正确的重组质粒酶以阳离子聚合物转染P815细胞后,以RT-PCR方法检测mRNA的表达,以间接免疫荧光技术检测目的蛋白的表达。结果:构建了重组质粒pcDNA-Rv1884;RT-PCR结果证明Rv1884可在P815细胞中转录;用间接免疫荧光检测,表达有Rv1884蛋白的细胞着染。结论:构建了结核分枝杆菌Rv1884基因的真核表达载体pcDNA-Rv1884,Rv1884基因可以在P815细胞中表达。     

Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase

The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.     

Rv2131c gene product: an unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K(m) of 0.22+/-0.03mM for inositol-1-phosphate and K(m) of 0.45+/-0.05mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC(50) approximately 60mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV).     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

结核分枝杆菌H37Rv新基因Rv2742克隆表达及纯化

Rv2742是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacteriumtuberculosis H37Rv中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv漏注释蛋白Rv2742的可溶性诱导表达、纯化体系,为进一步探索Rv2742基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742及pMAL-c2X-Rv2742原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16℃及0.5mmol/LIPTG诱导条件下表达量最高。直链淀粉树脂(Amyloseresin)亲和层析柱纯化获得较纯的产物,经LC-MS/MS验证确认是Rv2742融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。     

新型抗结核分枝杆菌药物筛选模型的建立

为建立基于酶水平和细胞水平的新型抗结核分枝杆菌（Mycobacterium tuberculosis）药物的筛选模型,以M.tuberculosis H37Rv基因组DNA为模板,PCR特异性扩增异柠檬酸裂解酶（ICL）基因,构建表达载体,在E.coli BL21（DE3）中高效表达,使用N i2＋亲和层析柱纯化重组ICL,检测其活性。优化ICL酶反应条件,考察待筛选样品溶剂对酶活性的影响,建立ICL抑制剂酶水平筛选模型;考察与优化耻垢分枝杆菌（Mycobacterium smegma）在以乙酸盐为唯一碳源的培养基中的生长状况,建立基于M.sm egma的乙醛酸代谢途径抑制剂的细胞水平筛选模型;利用上述2种筛选模型对1 060种可能具有拮抗活性的微生物代谢样品进行初筛和复筛,两者筛选结果正相关性较好。     

结核分枝杆菌esat6-rpfD融合基因的原核表达及产物纯化

目的：构建结核分枝杆菌融合基因esat6-rpfD的原核表达载体,表达和纯化ESAT6-RpfD融合蛋白。方法：从结核分枝杆菌H37Rv株基因组中经PCR分别扩增esat6和慢周基因,克隆入pMD19-T载体,测序后克隆入原核表达载体pProExHTB,酶切重组质粒,转化大肠杆菌DH5α,IPTG诱导表达融合蛋白,亲和层析纯化融合蛋白。结果：PCR扩增的esat6、rpfD基因序列与GenBank报道一致;诱导表达后,经SDS-PAGE和Western blot分析,在相对分子质量约30000处有目的条带,融合蛋白以包涵体形式表达。结论：构建了esat6-rpfD融合基因原核表达载体,并在大肠杆菌中表达并纯化得到ESAT6-RpfD融合蛋白。     

结核分枝杆菌Rv3871抗原优势肽段的克隆表达及抗原性鉴定

目的:为了提高结核病诊断试剂的特异性和敏感性,克隆表达结核分枝杆菌H37Rv株RD1区Rv3871抗原优势肽段,并应用ELISA法对其抗原性进行初步鉴定。方法:利用Biosun生物信息学软件对Rv3871抗原进行表位分析,通过PCR从结核分枝杆菌H37Rv基因组中扩增Rv3871抗原优势肽段编码基因,在大肠杆菌HB101中进行表达,采用间接ELISA法初步评价其抗原性。结果:Rv3871-1肽段检测的敏感性为32.5%(13/40),特异性为97.2%(35/36);Rv3871-2敏感性为45%(18/40),特异性为100%;Rv3871-3敏感性为37.5%(15/40),特异性为91.6%。结论:结核分枝杆菌RD1区Rv3871抗原优势肽段Rv3871-2有较高的特异性和敏感性,有望作为候选抗原用于结核病患者的血清学检测。     

Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan-peptidoglycan linker, leads to irreversible loss of viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Expression and purification of a functionally active recombinant GDP-mannosyltransferase (PimA) from Mycobacterium tuberculosis H37Rv

Lipoarabinomannans (LAM), especially mannose-capped LAM, abundant in the cell wall of Mycobacterium tuberculosis (Mtb) exhibit a broad spectrum of immunomodulatory functions and emerge as key virulence factors that may be relevant drug targets. The pimA gene of mycobacteria encodes a alpha-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of LAM. PimA has been proposed to play an essential role in the growth of mycobacteria. In this study, the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the Mtb PimA in fusion with a histidine-rich peptide on the N-terminal. The Mtb PimA was purified from the supernatant of the lysed cells under native conditions by immobilized metal affinity chromatography. The purity and molecular weight of Mtb PimA were determined by high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Circular dichroism spectroscopy study on Mtb PimA showed that the protein was folded. The enzyme assays revealed that Mtb PimA showed a requirement for Mg(2+) for the activity and the K(m) and V(max) values of Mtb PimA were estimated at 18 +/- 2 microM and 0.1 +/- 0.05 nmol/min/microg, respectively. This is the first report describing cloning and expression of GDP-mannosyltransferase gene of M. tuberculosis in E. coli.     

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

差显技术分析结核杆菌H37Rv与H37Ra差异表达的基因

采用差异显示技术比较了结核分支杆菌强毒株H37Rv和弱毒株H37Ra在体外培养条件下的基因表达差异。通过20种引物组合进行mRNA差异显示,克隆到了两菌株间的20余个差异表达基因,经序列分析及杂交鉴定发现其中2个基因仅在H37Rv中表达。其中Rv1894c基因编码的可能是H37Rv中的一个新蛋白。而在H37Ra的基因组中含有这2个基因的编码序列,但均未检测到基因的表达。     

The significance and effect of tandem repeats within the Mycobacterium tuberculosis leuA gene on α-isopropylmalate synthase

The 57-bp tandem repeats located in the Mycobacterium tuberculosis leuA gene code for the α-isopropylmalate synthase (α-IPMS). It is unique to this pathogen. It was previously demonstrated that the leuA -coding sequence Rv3710, containing the tandem repeats, can be translated to an active α-IPMS. The objective of the present study was to investigate the significance and effect of the two 57-bp tandem repeats upon gene expression and the general properties of α-IPMS. The putative M. tuberculosis H37Rv leuA gene with and without the tandem repeats was cloned by PCR and expressed in an Escherichia coli host. The enzyme product was studied for general properties, comparing that from a native leuA gene containing two repeats and that from the 57-bp tandem repeats deletion mutant. Upon deletion of the two 57-bp tandem repeats, the expression level of leuA from M. tuberculosis H37Rv was comparable with that of the native form. The general properties of the two types of enzymes were similar. They were both functional with the same range of optimal temperature and optimal pH for activity and with similar enzyme stability. Deletion of the repeats had no detectable effect on leuA expression level or the general properties of the enzyme product.     

利用表型芯片系统高通量分析H37Ra与H37Rv差异表型

H37Rv是结核分枝杆菌标准有毒株,H37Ra是从H37Rv获得的稳定减毒株,但目前H37Ra毒力减弱原因尚不完全清楚。本研究利用表型芯片系统,高通量分析H37Ra生长表型,并与H37Rv表型比较,筛选两菌株表型差异,分析与H37Ra毒力减弱可能的相关表型及分子机制。结果发现,与H37Rv相比,H37Ra耐酸及耐渗透压能力显著下降,且不能利用丁二酸单甲酯和吐温40作为碳源。结核分枝杆菌耐酸能力直接影响其在吞噬体中的生存和代谢,耐高渗能力影响其必需营养物质的跨膜运输,代谢途径的改变影响其在宿主内的能量摄取,三者改变均可能与H37Ra毒力减弱相关。     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.     

结核分枝杆菌持续感染期抗原Rv1733c 的表达、纯化和鉴定

目的:克隆结核分枝杆菌持续感染期抗原Rv1733c基因,构建其原核表达载体,并在大肠杆菌中进行表达和纯化。方法:采用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组中扩增出Rv1733c基因片段,克隆入pMD18-T载体,序列测定正确后将其亚克隆入原核表达载体pPro-EXHTb,并在大肠杆菌DH5α中进行表达,表达蛋白经SDS-PAGE及Western-blot分析后,以Ni-NTA亲和层析纯化蛋白。结果:成功克隆了Rv1733c基因片段并构建了其原核表达载体pPro-EXHTb-1733c,转化E.ColiDH5α后能表达大小约30 KD的蛋白,Western-blot分析表明表达产物正确。通过亲和层析获得纯化蛋白。结论:成功构建结核分枝杆菌持续感染期抗原Rv1733c原核表达载体pPro-EXHTb-1733c,并获得纯化蛋白,为研究新型结核疫苗的靶抗原奠定了基础。     

结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步检测

目的:克隆38kD、ESAT-6、CFP10和MPT64等4种结核分枝杆菌抗原基因,利用大肠杆菌表达系统分别表达重组蛋白,纯化并初步评价其抗原性。方法:通过PCR方法从结核分枝杆菌H37Rv株基因组中扩增38kD、ESAT-6、CFP10和MPT64抗原的基因,连接入pBVIL1表达载体,在大肠杆菌HB101株中进行表达,以间接ELISA方法初步评价其抗原性。结果:获得了结核分枝杆菌抗原38kD、ESAT-6、CFP10和MPT64的基因,并在大肠杆菌中进行了高效表达,初步验证所纯化获得的抗原具有良好的抗原性。结论:pBVIL1表达载体可以高效表达多种结核分枝杆菌抗原,38kD、ESAT-6和CFP10抗原均可作为结核病血清学诊断的候选抗原。