结核分枝杆菌感染诱导的人巨噬细胞细胞因子及其调控基因的差异表达谱

研究人巨噬细胞细胞因子在抗结核分枝杆菌感染免疫中的作用。利用表达谱芯片技术研究细菌感染后,宿主巨噬细胞基因表达情况,在全局表达谱分析基础上,重点分析细胞因子及相关基因的表达,并比较无毒株和临床分离有毒株在诱导细胞因子及其调控基因表达方面的差异。结果显示细菌感染影响较多细胞因子及其调控基因的表达,如IFN、TNF、TGF、IL系列及其受体、NFkappa B和TLR受体等;首次报道IL19参与了抗结核分枝杆菌感染免疫。被临床株感染影响的细胞因子较无毒株广泛和丰富。细胞因子及相关基因参与了宿主细胞对感染细菌的免疫应答,有关细胞因子及相关基因在抗结核免疫中的作用有待进一步研究。     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.     

狼洞穴空间格局及生境选择的分析

内蒙古东部草原地区狼洞穴空间分布格局及其生境选择的聚类分析结果表明,影响狼洞穴分布的主要因子为人类活动干扰和水源距离,其次为隐蔽条件、坡位和坡度,狼选择洞穴的最适生境为陡坡,洞口北向,隐蔽条件中等以上,距人为干扰大于１０００ｍ和距水源小于１０００ｍ的地方     

生态袋护坡技术在三峡水库消落带植被恢复中应用的可行性研究

通过对三峡水库重庆市巫山县双龙镇和巫峡镇段消落带开展生态袋护坡复绿试验7年后，生态袋上（内）、生态袋堆叠处上方和左侧消落带狗牙根（Cynodon dactylon）的种群密度、表型生长性状、地上和地下生物质量，以及土壤理化性质的测定，探讨以狗牙根为生态袋上的种植植物，将生态袋护坡技术用于三峡水库消落带植被恢复的可行性。结果表明：（1）各试验地生态袋上与其堆叠处上方和左侧消落带的狗牙根种群密度和地上生物质量差异不显著。（2）狗牙根的表型生长性状和根系生物质量因地和在生态袋堆叠处的方位不同而异。在双龙镇试验地，生态袋上比其堆叠处上方消落带上狗牙根的植株长度和节间长度低23.9%和22.6%（P<0.05），除此之外的各项指标差异均不显著；生态袋内0-5 cm土层的根系生物质量比其堆叠处上方消落带增加了75.7%（P<0.05），比其堆叠处左侧消落带降低了11.8%，在5-15 cm各土层降低了91.6%-96.9%（P<0.05），15-20 cm土层的差异不显著。在巫峡镇试验地，生态袋上与其堆叠处上方和左侧消落带的各表型生长性状的差异均不显著；生态袋内各土层的根系生物质量均比其堆叠处上方和左侧消落带增加了20.0%-138.7%。（3）各试验地生态袋内与其堆叠处上方和左侧消落带土壤容重的差异不显著，土壤化学性质因地和在生态袋堆叠处的方位不同而异。在双龙镇试验地，生态袋内的土壤全氮和速效氮含量比其堆叠处上方消落带分别降低了13.6%和40.9%（P<0.05），比其堆叠处左侧消落带分别降低了11.9%和33.0%（P<0.05）；速效钾含量比其堆叠处上方和左侧消落带分别增加了18.3%和34.1%（P<0.05）；除此之外各指标的差异均不显著。在巫峡镇试验地，生态袋内的土壤pH值和全氮含量比其堆叠处上方消落带分别降低了1.4%和27.9%（P<0.05），全钾含量增加了6.1%（P<0.05）；土壤全钾和速效钾含量比生态袋堆叠处左侧消落带分别降低了8.1%和24.9%（P<0.05）；除此之外各指标的差异也不显著。（4）狗牙根种群密度、大多数生长指标和生物质量与土壤理化指标相关不紧密。总体上，生态袋上（内）与其堆叠处上方和左侧消落带的大多数表型生长指标，地上和地下生物质量，以及土壤理化指标的差异不显著。狗牙根耐淹、抗旱、耐贫瘠，根系发达，且穿透力强，能够在生态袋上正常生长；生态袋透水不透土，且具有一定的保肥能力。因此，以狗牙根为生态袋上的种植植物，将生态袋护坡技术用于三峡水库消落带植被恢复具有一定的可行性。     

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.     

Expression, purification and properties of shikimate dehydrogenase from Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for alpha-helix, beta-sheet, beta-turn, and random coil were 29.2 %, 9.3 %, 32.7 %, and 28.8 %, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature 63(o)C. The apparent Michaelis constant for shikimic acid and NADP(+) were calculated to be about 29.5 microM and 63 microM. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of NADP(+) with an enzyme turnover number of 399 s(-1). Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.     

抗结核活性化合物HY-152E的作用靶点分析

抗结核活性化合物HY-152E是本实验室前期获得的具有良好抗结核活性并拥有授权专利（ZL201210088290.0）的小分子化合物（最低抑菌浓度≤0.09 μg/mL）。为深入探索HY-152E的抗结核机制,本研究利用药物亲和反应靶标稳定性（drug affinity responsive target stability,DARTS）技术并结合蛋白质谱技术,分析可能与HY-152E相互作用的结核分枝杆菌潜在靶标蛋白。将结核分枝杆菌H37Ra的菌体蛋白裂解液与HY-152E共同孵育互作,用不同浓度的链霉菌蛋白酶消化30、45、60 min后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）分离并比较与HY-152E互作前后菌体蛋白耐受蛋白酶消化的差异条带,分别在相对分子质量70 000和45 000~55 000处观察到差异蛋白条带。利用蛋白质谱技术分析差异条带的蛋白信息,共获得86个蛋白信息。结合结核分枝杆菌数据库及蛋白功能信息,最终筛选到9个蛋白可能是HY-152E的抗结核作用潜在靶标。这些潜在靶点的确定,为后续研究HY-152E的抗结核分子机制奠定了基础。     

Cell Surface Display of MerR on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for Biosorption of Mercury

The metalloregulatory protein MerR which plays important roles in mer operon system exhibits high affinity and selectivity toward mercury (II) (Hg²⁺). In order to improve the adsorption ability of Saccharomyces cerevisiae for Hg²⁺, MerR was displayed on the surface of S. cerevisiae for the first time with an α-agglutinin-based display system in this study. The merR gene was synthesized after being optimized and added restriction endonuclease sites EcoR I and Mlu I. The display of MerR was indirectly confirmed by the enhanced adsorption ability of S. cerevisiae for Hg²⁺ and colony PCR. The hydride generation atomic absorption spectrometry was applied to measure the Hg²⁺ content in water. The engineered yeast strain not only showed higher tolerance to Hg, but also their adsorption ability was much higher than that of origin and control strains. The engineered yeast could adsorb Hg²⁺ under a wide range of pH levels, and it could also adsorb Hg²⁺ effectively with Cd²⁺ and Cu²⁺ coexistence. Furthermore, the engineered yeast strain could adsorb ultra-trace Hg²⁺ effectively. The results above showed that the surface-engineered yeast strain could adsorb Hg²⁺ under complex environmental conditions and could be used for the biosorption and bioremediation of environmental Hg contaminants.     

Cloning and characterization of Rv0621 gene related to surfactant stress tolerance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

To understand how Mycobacterium tuberculosis (M. tuberculosis) could survive in human lung, Genomic expression library of M. tuberculosis in Escherichia coli (E. coli) had been prepared. Taking advantage of the genetic simplicity of E. coli and the functional conservation of some prokaryote proteins, a surfactant stress resistant gene Rv0621 was identified, which encodes a 37 kDa putative membrane protein. The E. coli colony with the partial Rv0621 gene insert, named S1, was able to grow in medium containing 0.4% sodium dodecyl sulfate, while the strain carried empty vector was unable to grow. The full length of the Rv0621 gene was then cloned into plasmid pET32a (+) expressed in E. coli BL21 (DE3). Using gas chromatographic–mass spectrometric (GC–MS), the fatty acid composition of the E. coli BL21 (DE3) carrying Rv0621–pET32a (+) and the E. coli BL21 (DE3) carrying empty vector pET32a (+) were compared. E. coli BL21 (DE3) carrying Rv0621–pET32a (+) contained more oleic acid. This suggests the gene may be involved in regulation of fatty acid synthesis and M. tuberculosis resistance to the surfactant defense of its host.