Study of intracellular location of adenovirus protein IX with correlative light and electron microscopy based on miniSOG / 中华实验和临床病毒学杂志

Objective@#To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein IX.@*Methods@#MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM. MiniSOG-fused adenovirus IX gene (IXSOG) was cloned by PCR, and inserted into pcDNA3 plasmid to form pTPL-IXSOG, which was used to transfect 293 cells. IXSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG. The transfected cells were fixed in 2.5% glutaraldehyde in situ, stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location of IXSOG was studied by observing the sections under transmission electron microscope.@*Results@#Eukaryotic expression plasmid carrying IXSOG fusion gene was constructed. IXSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections. IXSOG fusion mainly formed punctate aggregations or inclusions in the nucleus.@*Conclusions@#The correlative light and electron microscopy method based on miniSOG was successfully established, and it could be used to study the intracellular localization of viral proteins.

Preparation of Recombinant Human Adenoviruses Labeled with miniSOG / 病毒学报

We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.

Characterizaiton of virus-like particles prepared by Bac-to-Bac system co-expressing HA and M1 ;protein of highly pathogenic avian influenza H5N1 / 中华微生物学和免疫学杂志

Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.

Inducing effect of clodronate-liposome on apoptosis of alveolar macrophages in rats with acute nectotizing pancreatitis / 中华肝胆外科杂志

Objective To study the effects of elodronate-liposome on inducing apoptosis of alve-olar macrophages from rats with acute neetotizing pancreatitis (ANP).Methods The AMs of eight rats with ANP were isolated, purified then incubated from broehoalveolar lavage by the differing rates of attachment of the various cell types in a forty-well cell culture plate.Then they were randomized in-to five groups including control group,blank liposome group( 50 μ1, 100 μ1),clodronate-liposome group (50μ1,100μ1).Values of OD were determined by MTT.AO fluorescence and haematoxylin dye were employed to determine the apoptosis of the AMs.Results There were no significant differences be-tween control group and blank liposome group(50 μ1, 100 μ1).Significant differences were found be-tween control group and clodronate-liposome group(50 μ1, 100 μ1).There were no marked differences between blank liposome group(50μ1, 100 μ1)and clodronate-liposome group(50 μ1,100μ1).AO fluo-rescence and haematoxylin dye were available to define the apoptosis of the AMs.Conclusion Clodr-onate-liposome can effectively induce the apoptosis of the AMs.

Preparation of human parvovirus B19 virus-like particles / 生物工程学报

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.

Application of Pseudoreplica Technique in Electron Microscope Observation of A? / 医学研究杂志

Objective To investigate the effects of application of pseudoreplica technique in electron microscope observation of A?.Methods With the modification of classical pseudoreplica technique,ADDLs were spotted and concentrated on the gel before negative staining.Results The structures of 5nm to10nm spherical granules and 20 to 30nm coils were visualized by transmission electron microscopy(TEM).Conclusion Pseudoreplica is rapid and effective in ADDLs condensation,negative staining especially in further ultra miero structure observation and research.

Effect of Urinastatin on Microcirculation of Extrapancreatic Organs in Rats with Acute Necrotizing Pancreatitis / 中国普外基础与临床杂志

Objective To explore the effects of urinastatin(UTI) on microcirculation of extra pancreatic organs in rats with acute necrotizing pancreatitis(ANP). Methods A total of 48 rats were randomized into control group, ANP group and UTI group. The model of ANP was established by uniform injection of 5% sodium taurocholate solution under pancreatic capsule, only injection of normal saline in control group. Then the rats of UTI group were injected with UTI through the femoral vein, the rats of ANP group and control group were injected with normal saline. The blood flow of lung, kidney and distal small intestine was measured by radioactive biomicrosphere technique at 2 h and 6 h after ANP.Results Compared with the control group, the blood flow of lung, kidney and intestine was decreased significantly in the ANP group at the 2 h and 6 h after ANP ( P

Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo.</p><p><b>METHODS</b>The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus.</p><p><b>RESULTS</b>Recombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100. Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation.</p><p><b>CONCLUSIONS</b>The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.</p>

Dynamic Changes of Serum TNF-?/IL-10 in Model Rats with Acute Necrotizing Pancreatitis and Study on the Intervention of Salvia Miltiorrhiza / 中国药房

OBJECTIVE:To study the dynamic changes of TNF-?/IL-10in model rats with acute necrotizing pancreatitis (ANP)and to study the intervention outcome of salvia miltiorrhiza.METHODS:A total of96rats were randomly divided into three groups(each with32rats):pancreatitis(P)group,salvia miltiorrhiza treatment(T)group and control group(C),the blood samples of rats in each group were taken to determine levels of TNF-?and IL-10and the ratio changes of TNF-?/IL-10.Meanwhile,pancreas tissue sample was collected for pathological scoring.RESULTS:Group P had significantly higher serum levels of TNF-?and IL-10than did group C(P