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To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Subject(s)
Apoptosis , Genes, Reporter , Luciferases, Firefly , TransfectionABSTRACT
Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to in-vestigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro-EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. Coli DHSa and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. Luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. Tuberculosis H37Ra which were in non-culturable' condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 x 103 and 12 x 103, the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. Luteus Rpf and Rpf domain from the E. Coli showed activity of stimulating the resuscitation of M. Luteus and M. Tuberculosis H37Ra in non-cuhurable' condition which could be inhibited by monoclonal antibodies of M. Luteus Rpf domain remarkably. Conclusion It was dem-onstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. Luteus Rpf domain protein is fully active as M. Lateus full-length Rpf.
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Objective To investigate the immunological properties of Rv1009 domain. Methods BALB/c mice were immunized with Rv1009 domain three times at 2-week interval. ELISA was used to detect the antiRv1009 domain antibody titer in the sera of immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. Levels of secreted IFN-γ, IL-10 and IL-12 upon specific antigen stimulation were detected by ELISA. The BALB/c mice immunized with Rv1009 domain were intravenously infected with MTB H37Rv. Four weeks after the final injection, the number of CFU in spleens was determined. Results The titer of the specific antibody in sera of the immunized BALB/c mice was 1:12 800. The SI of Rv1009 domain immunized group (2. 40±0. 18) was significantly higher than that of saline immunized group (0.90±0.21). The IFN-γ,IL-10 and IL-12 levels in culture supematant of spleen lymphecytes from the fusion proteins immunized mice was (1 432±30) ng/L, (503±11) ng/L and (311±11) ng/L respectively, significant different from that of saline immunized group[(256±20) ng/L, (76±6) ng/L and(56±8) ng/L,P<0.01]. Four weeks after the final injection,compared with normal saline immunized mice (6.64±0.13), dramatic reduction in MTB replication was observed in the spleen (4.86±0.14) from BALB/c mice immunized with fusion proteins following a subsequent MTB H37Rv challenge, but the protection efficacy of mice immunized with Rv1009 domain was not as good as that of BCG vaccination group (3.81±0.16). Conclusion Rv1009 domain can be used as a candidate for the new TB vaccine.
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OBJECTIVE To give experimental basis for further investigation on Staphylococcus epidermidis biofilm resistance,we investigated the erythromycin repression of the planktonic,resuspended and biofilm cells.METHODS log Reduction of the planktonic,resuspended and biofilm cells of S.epidermidis strain 1457 and wild isolate S 68 was tested under 100 MIC erythromycin.The cells inside biofilm were observed by transmission electron microscope.RESULTS log Reduction of the planktonic and resuspended cells of S.epidermidis 1457 were 5.56?0.11 and 5.28?0.08,respectively,after 4h under 100 MIC erythromycin,which was higher than that of biofilm cells(P0.05).The tendency of the log reduction of isolate S 68 was the same as that of strain 1457.The cells of biofilm without erythromycin were spherical and of even distributed,but the swelling,liquefied cells and cell debris were observed in the biofilm with erythromycin after 12h.The arrangement of cells inside biofilm with erythromycin was looser than that without erythromycin.CONCLUSIONS The erythromycin resistance of biofilm cells is higher.Biofilm can give protection for the bacteria to avoid the killing of action by antibiotics.
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<p><b>OBJECTIVE</b>To purify recombinant nuclear protein of Hantaan virus.</p><p><b>METHODS</b>The recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein is a fusion protein with GST and existed in inclusion bodies. The inclusion bodies were processed through denaturation and renaturation and were purified with Glutathione Sepharose 4B affinity chromatography column. Then the purified fusion protein and nuclear protein were examined by sandwich ELISA and Western blot.</p><p><b>RESULTS</b>The expressed fusion protein was separated from the mixture proteins by the first affinity chromatography. GST was cut from the purified fusion protein with thrombin. The nuclear protein was separated from GST by the second affinity chromatography. The crossed peak represents nuclear protein and the elute peak represents GST. The purified fusion protein and nuclear protein were single band by SDS-PAGE. Both of them had available antigen activity.</p><p><b>CONCLUSIONS</b>Purification of nuclear protein of Hantaan virus with Glutathione Sepharose 4B affinity chromatography is an effective method.</p>
Subject(s)
Blotting, Western , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Hantaan virus , Nuclear Proteins , Plasmids , Recombinant Fusion Proteins , Viral ProteinsABSTRACT
Objective To review the current situation and developments of researches into important pathogenic microorganisms domestically and abroad,and to suggest the orientation of research work and development in pathogenic microbiology in PLA.Methods The achievements and advances of research work achieved domestically and abroad in the past five years regarding important viruses(such as hepatitis viruses,human immunodeficiency virus,influenza virus,encephalitis viruses and hantaanvirus)and bacteria (such as Mycobacterium tuberculosis,Streptococcus suis serotype 2,Yersinia pestis,Bacillus anthracis and Helicobacterp ylori)were retrieved and reviewed using intelligence research methods.Results Infectious diseases caused by pathogenic microorganisms were the most severe hazards to health and life of human beings.Especially in the past thirty years,newly emerging infectious diseases and recurrence of previonsly controlled infectious diseases had received wide attention.Infectious diseases control had been greatly improved owing to the increasing discoveries in the knowledge about pathogenic microorganisms.Conclusions During the period of "Twelfth Five-Years Plan" ,a big team of science and technology personnel with strong innovative ability in the domain of medical microbiology should be brought up in PLA;and a number of advanced and consummate research bases and technology platforms should be built up;to apply for and realize a batch of major research projects,strive to make a number of scientific achievements with innovation and important application prospects,improve the transformation efficiency of scientific and technological achievements and contribution of scientific and technological progress,and strive to achieve important progresses and breakthrough in mainstream research.
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Aim To express hepatitis C virus glycoprotein E (gE) deleting carboxy-terminal 31 amino acids, and detect anti-E antibody in HCV patients using expressed gE. Methods E gene derived from HCV H strain was inserted into baculovirus transfer vector containing a polyhedrin promotor. The recombinant plasmid was cotransfected into insect cell sf9 with a viral expression vector. The expression of gE was analyzed with Western blot, and the cells were used for dectecting antibodies against E1 and E2 in 35 hepatitis C patients by indirect immunofluorescence. Results A series of proteins with different relative molecular masses(Mr) could be detected by Western blot. Results from indirect immunofluorescence staining showed and only 4 patients were anti-E antibody positive gE was located in cytoplasm. Conclusion HCV gE is expressed successfully in insect cells, the study lay the foundation for further developing HCV vaccine.
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Objective: To study the relationship between human herpes vi rus 6 (HHV-6) infection and oral squamors cell carcinoma. Methods: The serum anti-HHV-6 antibody titers from 16 cases of oral squamors cell carcinoma (OSCC) and 16 control subjects were detected by indirect immunofluores cence assay. HHV-6 DNA in peripheral blood mononuclear cells was amplified by P CR and the specificity was confirmed by Southern-blot hybridization with an int ernal probe oligonuclotide. An immunohistochemical staining was used to detect H HV-6 antigen in OSCC tissues. Results: Positive expression of s erum HHV-6 IgG antibody was found in all the cases of OSCC and in 12 of the 16 controls. Geometric mean titer of OSCC group and control group was 1∶118 and 1 ∶64 respectively (P
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The cultured HUVE cells were infected with HFRS veruses. At different intervals after in-fection cytotoxicity of NK and LAK by 4 hours Cr release method. It was found that the infec-tion of endothelial cells inhibit the lytic activity of NK and LAK cells. Theinhibition. It is sug-gested that the deceased susceptibility of HFRS virus infected endothelial cells to NK and LAKcells may protect the viruses from the cellular immunity during the course of HFRS.
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A ELISA indirect sandiwich method using McAb against for HFRS virus and the roughly purified antigen has been developed for detections of IgM and IgG antibodies in sera from HFRS patients.The specificity of this technique was proved by the sera cross test,antibody-blocking test and 2-Mercaptoethnol method.The IgM antibodies of 121 sera from HFRS patients were examined by the ELISA and IFAT.The positive rates were 90.1% and 83.5%,respectively and the difference was significant(p