结核分枝杆菌Rv3881c抗原鼠单克隆抗体的制备及初步鉴定

目的:制备抗结核分枝杆菌Rv3881c抗原鼠mAb。方法:采用杂交瘤技术,获得了11株针对结核分枝杆菌Rv3881c抗原鼠mAb杂交瘤细胞株,对其中的5株进行了小鼠腹水的制备及相关鉴定。结果:5株mAb的腹水效价达到1∶32 000～1∶512 000,将这5株mAb进行了纯化,纯化后纯度大于90%,抗体亚类(型)均为IgG1/κ型,ELISA结果显示制备的mAb与结核分枝杆菌Rv3881c抗原可发生特异反应。结论:制备了抗结核分枝杆菌Rv3881c抗原鼠mAb,为结核分枝杆菌Rv3881c生物学功能的研究奠定基础。     

Evaluation of Structurally Diverse Benzoazepines Clubbed with Coumarins as Mycobacterium tuberculosis Agents

Tuberculosis caused by Mycobacterium tuberculosis remains a leading cause of mortality worldwide into 21st century. In continuation with our anti‐tuberculosis research programme, in this work, we have prepared molecularly diverse coumarins clubbed with benzothiazepines as well as its aza‐analogues‐benzodiazepines by molecular hybridization. The resulting compounds were screened for their M. tuberculosis activity against H₃₇Rv strains using microplate alamar blue assay. Among the designed diversity, the compounds 5k, 5n and 5o were found significantly active in primary anti‐tuberculosis assay at minimum inhibitory concentration <6.25 μm . Moreover, the IC₅₀ values of 5k and 5o in level‐2 screening were observed as >10 μg/mL and 3.63 μg/mL, respectively. Design and synthesis of more focused library and its three‐dimensional quantitative structure activity relationship analysis are underway.     

Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression

Background:

In prostate adenocarcinoma, the dissection of the expression behaviour of the eukaryotic elongation factors (eEF1A1/2) has not yet fully elucidated.

Methods:

The EEF1A1/A2 expressions were investigated by real-time PCR, western blotting (cytoplasmic and cytoskeletal/nuclear-enriched fractions) and immunofluorescence in the androgen-responsive LNCaP and the non-responsive DU-145 and PC-3 cells, displaying a low, moderate and high aggressive phenotype, respectively. Targeted experiments were also conducted in the androgen-responsive 22Rv1, a cell line marking the progression towards androgen-refractory tumour. The non-tumourigenic prostate PZHPV-7 cell line was the control.

Results:

Compared with PZHPV-7, cancer cells showed no major variations in EEF1A1 mRNA; eEF1A1 protein increased only in cytoskeletal/nuclear fraction. On the contrary, a significant rise of EEF1A2 mRNA and protein were found, with the highest levels detected in LNCaP. Eukaryotic elongation factor 1A2 immunostaining confirmed the western blotting results. Pilot evaluation in archive prostate tissues showed the presence of EEF1A2 mRNA in near all neoplastic and perineoplastic but not in normal samples or in benign adenoma; in contrast, EEF1A1 mRNA was everywhere detectable.

Conclusion:

Eukaryotic elongation factor 1A2 switch-on, observed in cultured tumour prostate cells and in human prostate tumour samples, may represent a feature of prostate cancer; in contrast, a minor involvement is assigned to EEF1A1. These observations suggest to consider EEF1A2 as a marker for prostate cell transformation and/or possibly as a hallmark of cancer progression.     

Mcl-1信号通路阻断剂对H37Rv感染小鼠巨噬细胞凋亡的影响

目的:探讨Mcl-1信号通路阻断剂在结核分枝杆菌H37Rv感染小鼠模型中对Mcl-1表达、巨噬细胞凋亡情况及结核分枝杆菌的影响。方法:小鼠腹腔注射H37Rv菌悬液,建立感染小鼠模型,针对Mcl-1的信号通路选用JAK/STAT信号通路阻断剂AG490、MAPK信号通路阻断剂PD98059和PI3K信号通路阻断剂LY294002用腹腔注射方式作用于各组感染小鼠模型,分为H37Rv感染组、AG490处理组、PD98059处理组、LY294002处理组和对照组。通过细胞抗酸染色观察结核分枝杆菌H37Rv感染小鼠腹腔巨噬细胞的动物模型是否建立成功;通过免疫细胞化学检测结核分枝杆菌H37Rv感染巨噬细胞的Mcl-1表达情况,使用流式细胞技术检测各组巨噬细胞的凋亡率,采用结核分枝杆菌菌落计数来判断巨噬细胞凋亡对结核分枝杆菌的清除效果。结果:细胞抗酸染色结果可见感染的巨噬细胞内散在排列的红色短小抗酸结核分枝杆菌。免疫细胞化学结果显示H37Rv感染组、AG490处理组和LY294002处理组中的Mcl-1蛋白为强阳性表达,PD98059处理组中Mcl-1蛋白为弱阳性表达,对照组Mcl-1蛋白为阴性表达。流式细胞术检测发现H37Rv感染组巨噬细胞凋亡率较对照组高,PD98059处理组的凋亡率显著高于各组,差异显著(P0.05)。结核分枝杆菌菌落计数结果显示PD98059处理组对H37Rv菌株抑菌作用最明显。结论:Mcl-1信号通路阻断剂通过抑制JAK/STAT、MAPK和PI3K信号通路增加结核分枝杆菌H37Rv感染巨噬细胞的凋亡率,抑制结核分枝杆菌生长;其中,MAPK信号通路干扰Mcl-1的作用最明显,感染的巨噬细胞凋亡率最高,抑菌作用最强。     

淋巴结核丸对H37Rv、耐多药结核菌体外药敏实验研究

目的:淋巴结核丸对强毒人型结核菌(H37Rv)及耐多药结核菌(mutiple drugs resistant Bacillus tuberculosis,MDR-TB)在体外药敏实验。方法:用美国B-D公司生产试剂12B培养液和Bectec-460检测仪检测。结果:实验表明淋巴结核丸对H37Rv具有抑制作用,最低抑菌浓度为22.5mg/ml;对MDR-TB具有显著的抗菌作用P<0.01,最低抗菌浓度为22.5mg/ml,而对照药异烟肼、利福平、乙胺丁醇无抑菌作用。结论:用淋巴结核丸对H37Rv、MDR-TB均具有抑菌作用。     

结核分枝杆菌Rv0901基因原核表达质粒的构建及其表达

目的　为研究结核分枝杆菌基因Rv0 90 1的功能提供材料。方法　PCR扩增Rv0 90 1基因编码序列 ,定向克隆入融合蛋白原核表达载体pGEX 1λT获得重组表达质粒 ,转化大肠杆菌后用IPTG进行诱导表达 ,通过SDS PAGE、GST 纯化试剂盒鉴定并纯化表达产物。结果　从结核杆菌H37Rv株基因组DNA中扩增出Rv0 90 1基因 ;成功构建了融合表达质粒pGEX Rv0 90 1;重组质粒经IPTG诱导后能在大肠杆菌中稳定表达 4 5kDa的GST Rv0 90 1融合蛋白。结论　本实验成功表达并纯化GST Rv0 90 1融合蛋白 ,为Rv0 90 1基因功能的研究打下了基础。     

Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N(ε)-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N(α)-acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.     

碘伏对3株分枝杆菌标准菌株杀灭效果的研究

目的 了解碘伏消毒液对龟分枝杆菌、结核杆菌标准株H37Rv、结核杆菌标准株H37Ra 3种标准菌株的杀灭效果.方法 采用悬液定量杀菌试验.结果 含有效碘250 mg/L作用3min、有效碘500mg/L作用1 min,对龟分枝杆菌脓肿亚种杀灭对数值＞4.00;含有效碘250 mg/L作用3min、有效碘500 mg/L作用1min对结核杆菌标准菌株H37Rv杀灭对数值＞4.00.含有效碘500 mg/L作用1 min对结核杆菌标准菌株H37Ra杀灭对数值＞4.00.结论 碘伏消毒液对龟分枝杆菌、结核杆菌标准株H37Rv、结核杆菌标准株H37Ra 3种标准菌株均具有良好杀菌作用,H37Ra对碘伏消毒液抗性最强,H37Rv次之,龟分枝杆菌最弱.     

结核分枝杆菌Rv0073基因在大肠杆菌中的表达及纯化

目的 构建结核分枝杆菌(MTB) Rv0073基因原核表达载体并进行表达和纯化.方法 以MTB H37Rv基因组DNA为模板,采用聚合酶链反应(PCR)扩增目的基因片段,构建原核表达载体pET26b-Rv0073,经测序确定无误后转化至大肠杆菌(E.coli)感受态细胞BL21中.用聚丙烯酰氨凝胶电泳(SDS-PAGE)方法检测重组蛋白表达,检测异丙基-β-D-硫代半乳糖苷(IPTG)诱导不同时间、不同温度条件下重组蛋白表达量.采用His镍磁珠进行外源蛋白小量纯化.结果 成功构建重组表达质粒,重组蛋白经IPTG诱导后,2h开始明显表达且表达量无时间依赖性,在不同温度诱导下,重组蛋白的表达量随温度的增高而减少.重组蛋白以包涵体形式存在,经His镍磁珠纯化后获得重组蛋白.结论 成功构建并表达Rv0073蛋白,为后续Rv0073的大量纯化及其功能研究奠定了基础.