Xiao Mao Buttercuos Lactone Mycobaterium Smegmatis Recombinant The Eis Gene Expression

ObjectiveTB course stretches and recurrence Retention has long known clinicalfacts, but so far held the nature of stay bacteria Mycobacterium tuberculosis,there is still no exact knowledge of the mechanisms and clinical treatmentand clearly defined, especially in the Mycobacterium tuberculosis clinicaldrug treatment Held stay bacteria, almost at a standstill, so far no effectivedrugs to stay bacteria Mycobacterium tuberculosis (Mycobacteriumtuberculosis, MTB) held, the study found that MTB can enter macrophages,and enter macrophages after the presence of a particular gene expression, inorder to adapt the macrophage intracellular environment to achieveRetention recent study found that the EIS (Enhance intracellular survival)gene-specific expression of pathogenic MTB complex group havingimproved MTB in the ability to survive in macrophages, it is possible toachieve long Retention the eis gene product is a soluble secreted protein, amolecular weight of approximately42kd, capable of acting on themacrophage intracellular and extracellular, research has shown that the42kd protein can affect the macrophage interleukin (interlukin IL)-10and tumornecrosis factor (tunlornecrosisfactor TNF)-a secretion, inhibition ofmacrophage activation in MTB’s left holding a certain relationship. Indecreased immunity, Retention MTB in the body will resurgence of TB,tuberculosis comeback, significantly increased morbidity and mortality, heldto stay state of Mycobacterium tuberculosis is the resurgence of tuberculosisendogenous and course stretches the root causes of the proliferation ofMycobacterium tuberculosis MTB invalid targeting anti-TB drugs in thenon-proliferation of Retention state, which is one of the main obstacles to theGlobal Fund to Fight TB. In recent years, Chinese herbal medicine in thetreatment of chronic infection of TB increasingly receive attention, the studyfound the active ingredient of Cat’s Claw grass hairs buttercup the lactone(Ternatolide Tern) can significantly enhance the cytotoxic T lymphocyte(Cytotoxic lymphocyte, CTL) bactericidal capacity, to kill off held staybacteria [3], our previous experimental studies have shown MTBeis(Enhance intracellular survival, eis) gene is specifically expressed in thepathogenic MTB composite group, improve the viability of MTB inmacrophages with MTB held to stay are closely related.Tern is the active ingredient from the the catlike grass (Ranunculusternatus Thunb), Medicine believes that their sexual Weixin Gan, andphlegm, detoxification swelling and is filed under Yin drugs, mainly used inclinical treatment of tuberculosis, lymph node tuberculosis, and Tern EIS gene is currently no research, the study was designed to explore the Terngrowth and proliferation of recombinant Mycobacterium smegmatis(Mycobacterium smegmatis, MS), EIS gene, protein expression. Furtherresearch catlike grass Retention Mycobacterium active ingredient,mechanism of action and intensity.Methods1Picked from the plate the5×105MS-eis-PMV261were inoculatedat Tern (200mg/L) Kanamycin neomycin resistance of LB broth medium asthe test group, MS-eis-PMV261cultured alone as a control group,Also at37°C,250r/min, shaking culture, in both groups on days1,2,3,4,5and6depicting200μL measurement broth drawn based on the measured value ofA in the A value at the wavelength of600nm,growth curves.2Cultured for6days from3ml centrifugal Tern intervention in theexperimental group and the control group collected broth extracted bacterialDNA dissolved in20ul double distilled water extract broth DNA Tern eisgene expression detected by RT-PCR method.1ul as template RT-PCRamplification eis gene. The the eis gene detection primer sequence asfollows:upstream:5’-ATGTTCCTACTGGCCGCGGC-3’;Product reparation:5’-TCAGAACTCGAACGCGGTCTGG.-3’. PCR conditions:94°C initialdenaturation3min;94°C denaturation30s,60oC annealing30s,72oCextension for30s,35cycles after the the72oC extension of10min. PCRproducts were analyzed by1%agarose gel electrophoresis, and analyzed with Quantity One software strip gray value quantization level of geneexpression.3Western blot SDS-PAGE and western blot detection Tern EISprotein the3ml broth were cultured for6days12000g,2min centrifugalreserved the bacterial and30ul broth and add the boiling water bath for10min5x protein sample buffer. Preparation of a12%separating gel and5%stacking gel, the collection of the bacterium protein after centrifugation thesupernatant to SDS-PAGE electrophoresis, after electrophoresis with R-250Coomassie blue staining30min bleaching solution bleaching were observedafter overnight between42ku at protein expression. Semi-electricallytransferred by the protein band transferred to PVDF membranes,5%skimmilk powder is closed, to a1:100dilution of the sputum positive TB patientsserum as the primary antibody, a1:5000dilution of peroxidase labeled goatanti-human IgG secondary antibody, DAB color observation and analysis ofprotein bands.4Statistically obtained data SPSS18.0software independent samplest test.Results1Tern intervention trial group and not the proliferation curve theMS-eis-PMV261intervene in the control group had no significantdifference.2In granting Tern the trial group eisDNA bands compared with control group was significantly weakened.3broth the treatment underwent SDS-PAGE electrophoresis andwestern blot, the results show that the the MS-eis-PMV261containing EISgene not give Tern drug intervention the independent training control groupcan be expressed42kd at a significant bands (P <0.05) weakened, Ternintervention in the experimental group clause with expression.Conclusions1Tern does not directly kill the MS-eis-PMV261.2broth DNA extraction, Tern eis gene expression was detectedbyRT-PCR method, there are differences between the test group and thecontrol group, proved the Tern able to inhibit the replication of eisDNAMS-eis-PMV261.3found after the SAD-PAGE electrophoresis and western blot detectionthe given200mg/L Tern intervention,,42ku protein secretion decreasedprove Tern can inhibit the the eis gene’s expression.