Contribution of 3CD Region to the Virulence of Enterovirus 71 / 生物医学与环境科学（英文）

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.

Therapeutic effect of lacrimal balloon surgery on lacrimal duct obstruction in infants / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To evaluate the clinical efficacy and safety of balloon dilatation in infants with lacrimal passage obstruction.METHODS:Totally 86 patients (116 eyes) with lacrimal duct obstruction from July 1, 2015 to June 30, 2016 were randomly divided into two groups according to the digital table.The observation group (43 cases, 60 eyes) were operated with balloon dilatation and the control group (43 cases, 56 eyes) were treated with duct exploratory operation.The patients were followed up for 6mo to compare the efficacy.RESULTS:At the 6mo postoperatively, the primary cure rate and total cure rate in the observation group were significantly higher than those in the control group.There was significant difference between the two groups (P<0.05).CONCLUSION:Balloon dilatation operation is safe, and its clinical efficacy is better than lacrimal duct exploratory operation, is an effective way to treat lacrimal duct obstruction in infants.

Expression of miRNA-630 in bladder urothelial carcinoma and its clinical significance / 华中科技大学学报（医学）（英德文版）

Many studies informed that microRNAs (miRNAs) could function as diagnostic and prognostic indicators in several cancers. The aims of this study were to explore the expression of miR-630 in bladder urothelial carcinoma and its clinical significance for the evaluation of cancer prognosis. A total of 116 patients with bladder urothelial carcinoma were obtained in this retrospective study between May, 2012 and Sep. 2015. Quantitative real-time PCR (qRT-PCR) was conducted to evaluate the expression level of miR-630. The chi-square test was used to examine the associations between miR-630 expression and the clinicopathological features. The Kaplan-Meier method was conducted to explore the survival status of urothelial carcinoma patients. The log-rank test was used to analyze differences in survival rate. The results showed an obvious increase in miR-630 expression from normal bladder to bladder urothelial carcinoma (P=0.027). Additionally, patients with higher miR-630 expression had significantly shorter disease-free survival (DFS) (P=0.043) and overall survival (OS) (P=0.038) than those with lower miR-630 expression. Furthermore, multivariate analysis revealed that up-regulation of miR-630 was an independent prognostic factor for both DFS (P=0.042) and OS (P=0.046). It was demonstrated that miR-630 may be a novel and valuable prognostic factor for bladder urothelial carcinoma.

Effects of HIV-1 tat on secretion of TNF-α and IL-1β by U87 cells in AIDS patients with or without AIDS dementia complex / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.</p><p><b>METHODS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.</p><p><b>RESULTS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.</p><p><b>CONCLUSION</b>Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.</p>

Expression and bioassay of rubella virus E1-374 glycoprotein in yeast cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein.</p><p><b>METHODS</b>The cDNA of protein E1-374 was cloned into the expression vector pGAPZalphaA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein.</p><p><b>RESULTS</b>SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 10(3). Antiserum (1:100) and E1-374 (5.5 microg/ml) was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0.36%-12.45%.</p><p><b>CONCLUSION</b>Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.</p>

Analysis of genetic diversity of HIV-1 nef genes from a patient with AIDS dementia complex / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the genetic diversity of HIV-1 nef genes from a patient with AIDS dementia complex(ADC) , so as to research the amino acid variability and the pathogenesis of ADC.</p><p><b>METHODS</b>The nef gene was amplified with PCR from genomic DNA which was extracted from spleen and different brain tissues(basal ganglia, frontal gray matter, meninges, temporal lobe)of a patient who died of ADC. PCR products were cloned into the pMD19-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced and confirmed with BLAST. HIV-1 nef sequences were processed with BioEdit and MEGA4 to do Neighbor-Joining tree, p-Distances, and values of ds/dn.</p><p><b>RESULTS</b>The samples were all identified as HIV-1 B and genetic variation exists in HIV-1 nef gene isolated from different tissues compared with HXB2. In addition,part of the changes were different between periphery and brain.</p><p><b>CONCLUSION</b>Variations exist in the HIV-1 nef gene extracted from the ADC patient and the variations from peripheral and central nerve tissues were different,these variations may change the function of Nef,and it needs more research.</p>

Research progress on ebola virus glycoprotein / 病毒学报

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

Analysis of evolution features of whole genome of influenza virus H3N2 in Qingdao between year 2007 and 2011 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the evolution features of whole-genome of influenza virus H3N2 prevalent in Qingdao from year 2007 to 2011.</p><p><b>METHODS</b>The RNA of 58 strains of influenza virus H3N2 prevalent in Qingdao between 2007 and 2011 was extracted and all segments amplified by RT-PCR. The sequence was then detected and assembled by software Sequencer. A total of 589 strains of influenza virus H3N2 with more than 300 amino acid recorded by GenBank were selected. The phylogeny and molecular features of all gene segments were analyzed by software Mega 5.0, referred by the heavy chain of hemagglutinin (HA1).</p><p><b>RESULTS</b>Hemagglutinin (HA) genes of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 formed a single trunk of phylogenetic tree. Every prevalent strain originated in last season. The analysis of the evolution of whole genome found that reassortment virus strains were prevalent between year 2009 and 2010, but between 2010 and 2011 there were two series of prevalent strains, which showed complicated reassortment. Compared with the vaccine strains, the variant amino acids of protein of virus HA1 between year 2007 and 2011 were 8, 6, 6, 8 and 11, involving 13 antigenic sites. The sequence analysis of M2 protein showed that the isolated influenza virus H3N2 mutated in amino acid site 31, from serine to asparagine (S31N). HA1 gene of influenza virus H3N2 isolated in Qingdao between 2007 and 2011 shared the similar phylogenetic tree with the globally prevalent strain. The comparison of the sequence and the analysis of the antigenicity found co-infection between H3N2 and A/H1N1 in the strain A/Qingdao/F521/2011.</p><p><b>CONCLUSION</b>The evolution features of all segments of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 were complicated.</p>

Progress on mechanism of cell apoptosis induced by rubella virus / 病毒学报

Rubella virus (RV), a member of the family Togaviridae, can induce apoptosis of host cells in vitro. Protein kinases of the Ras-Raf-MEK-ERK pathway and PI3K-Akt pathway play essential roles in virus multiplication, cell survival and apoptosis. Proteins p53 and TAp63 that bind to specific DNA sequences stimulate Bax in a manner to produce functional pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation. In this review, the molecular mechanisms of RV-induced cell apoptosis, including RV-infected cell lines, pathological changes in cell components and apoptosis signaling pathways are summarized.

Study on functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase protein / 病毒学报

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.

Cloning and eukaryotic expression of HIV-1B gp120 genes from a patient with AIDS dementia complex / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.</p><p><b>METHODS</b>Using the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.</p><p><b>RESULTS</b>HIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.</p><p><b>CONCLUSION</b>All the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.</p>

Analysis of genetic diversity and amino acid sequence of HIV-1tat in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the diversity of HIV-1 tat gene in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC, so as to research HIV evolution, the mechanism of CNS invasion and the pathogenesis of ADC.</p><p><b>METHODS</b>The tat gene was amplified with nested PCR from genomic DNA which was extracted from spleen and basal ganglia of one non-ADC patient with a wide range of cerebral artery atherosclerosis and one ADC patient. PCR products were cloned into the PGEM-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid motifs were all done, so as to research the diversity of HIV-1 tat gene in CNS and peripheral tissue.</p><p><b>RESULTS</b>Gene mutation of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between CNS and peripheral tissue in ADC patient were greater than the non-ADC patient. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.</p><p><b>CONCLUSIONS</b>The compartmentation of tat gene in CNS and peripheral tissue of the two patients was different, the reason may be related to the pathway of HIV into the CNS, the relationship between HIV gene mutation in CNS and ADC still need more investigation.</p>

HIV-1B gp120 genes from one patient with AIDS dementia complex can affect the secretion of tumor necrosis factor and interleukin 1β in glial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>HIV-1 infected and immune-activated macrophages and microglia secrete neurotoxins, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which play major role in the neuronal death. It has been shown that different HIV-1 variants have varying abilities to elicit secretion of TNF-α by peripheral blood mononuclear cell (PBMC); however, whether the difference of gp120 gene could affect the secretion of TNF-α and IL-1β by glial cells is unknown. The aim of this study was to explore the association between gene diversity and induction of neurotoxic cytokines.</p><p><b>METHODS</b>In this study, we constructed retroviral vectors MSCV-IRES-GFP/gp120 using HIV-1 gp120 genes isolated from four different tissues of one patient who died of AIDS dementia complex (ADC). Recombinant retroviruses produced by cotransfection of MSCV-IRES-GFP/gp120, pCMV-VSV-G and pUMVC into 293T cells were collected and added into U87 glial cells. Concentrations of TNF-α and IL-1β secreted by transduced U87 cells were assayed with ELISA separately.</p><p><b>RESULTS</b>The four HIV-1 gp120 were in the different branch of the neighbor-joining tree. Compared to the pMIG retrovirus (gp120-negative) or U87 cells, all the gp120-positive recombinant retroviruses induced more TNF-α (P < 0.01) and IL-1β (P < 0.01). In addition, compared with the L/MIG retrovirus, all the three brain gp120-positive recombinant retroviruses induced less TNF-α (P < 0.01) and IL-1β (P < 0.01).</p><p><b>CONCLUSIONS</b>HIV-1 gp120 could induce U87 cells secret more TNF-α and IL-1β again. The more important is that difference of HIV-1 gp120, especially cell-tropism may account for the different ability in eliciting secretion of TNF-α and IL-1β, which might supply a novel idea helping understand the pathogenesis of ADC.</p>

Molecular characteristics of the full-length genome of two new Japanese encephalitis virus isolated from mosquitoes in Hubei province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To sequence and analyze the complete genome of two new Japanese encephalitis virus (JEV) strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area.</p><p><b>METHODS</b>RT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of Clustal X (1.83), MegAlign, Mega (4.0) and Genedoc (3.2).</p><p><b>RESULTS</b>The genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein, the homology of nucleotide and amino acid sequence between two isolates were 98.2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype I. Two new strains were most closely related to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08-55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV.</p><p><b>CONCLUSION</b>Two new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.</p>

Effects of the site-directed mutagenesis at nsP2-726Pro on replicon vector derived from XJ-160 virus / 病毒学报

To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --> Leu, nsP2-726Pro --> Ser, nsP2-726Pro --> Val or nsP2-726Pro --> Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --> Val or nsP2-726Pro --> Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.

Effects of disulfide bridges in glycoprotein E1 on the membrane fusion activity of rubella virus / 病毒学报

To reveal the effects of disulfide bridges in rubella virus glycoprotein E1 on the membrane fusion activity, the recombinant plasmid pBSK-SPE2E1 and site-directed mutagenesis to mutate 11 cysteines individually in the ectodomain of E1 to remove a disulfide bridge from the wild-type E1 were constructed. All mutants and the wild-type plasmid were expressed on BHK-21 cell. Giemsa Staining was used to show the polykaryon formed in the transfected BHK-21 cells. The cell surface expression efficiency of the plasmids was assayed with fluorescence-activated cell sorter (FACS). Hemadsorption was performed to detect the receptor recognition activity of the recombinant plasmids. The results showed that all the 10 disulfide bridges in the ectodomain of E1 played an important role in the process of the membrane fusion. The removal of any disulfide bridge resulted in the loss of the fusion activity. The disulfide formed by the 5th and the 8th cysteine might be critical for the interaction of E1 and E2. While the disulfide bridges formed by the 3rd, the 4th, and the 13th might influence the membrane fusion activity of E1 directly.

Sequencing and analysis of the full coding sequence of Batai virus isolated in China / 病毒学报

By RT-PCR and TAIL-PCR, the full coding region of Batai virus isolated in China (YN92-4 strain)was sequenced for the first time. According to the results, the genome of the virus contained three segments S, M and L of 947, 4,371 and 6,860 nucleotides, respectively. The S segment coded a nucleoprotein of 234 amino acids and a nonstructural protein of 102 amino acids, the M and L segments coded a precursor protein of 1 ,435 amino acids and RNA polymerase of 2,239 amino acids, respectively. Compared with the full coding sequence of Batai viruses isolated out of China, the S and M segments of YN92-4 and ON-7/B/01 showed the highest homology in nucleotide and amino acid sequenes with similarity of 97.7% (100%) and 95.7% (98%), respectively. Since there was no full coding sequence information on the L segment in GenBank for the reference, the L segment of YN92-4 was compared with that of Bunyamwera virus and the homology of nucleotide and amino acid was 73.5%and 81.6%, respectively. Phylogenetic analysis showed YN92-4 strain was clustered into one group with the prototype of Batai virus (MM2222). The results suggested that the YN92-4 strain had no occurrance of genetic reassortment (like Ngari virus) and was close to the Batai virus (ON-7/B/01 strain) isolated from cattle serum in Japan.

Analysis of genetic diversity and biological functional site of HIV-1 gp120 from a patient with AIDS dementia complex / 病毒学报

To explore the relationship between the genetic diversity and biological functional site of human immunodeficiency virus HIV-1 gp120 and the pathogenesis of AIDS dementia complex (ADC), the full length sequences of gp120 gene was amplified with PCR from genomic DNA which was extracted from lymphoid and different brain department (periaortic lymphoid, temporal gray/white matter junction, periventricular tissue, choroids plexus, occipital white matter and occipital gray/white matter junction.) of a patient who died of ADC. PCR products were cloned into the pGEM-T vector and positive clones were sequenced. The analysis of neighbor-joining tree, N-glycosylation sites, values of ds/dn, and loop were then all performed. The samples were all identified as HIV-1 B and genetic variation existed in HIV-1 gp120 isolated from different tissues. Compared with standard HIV-1B gp120, biological functional sites of HIV-1 gp120 isolated from the patient changed to some extent. In addition, there were differences in some biological functional sites of HIV-1 gp120 between lymphoid and brain. Therefore, genetic diversity and alterations of some biological functional sites of HIV-1 gp120 might be associated with the pathogenesis of ADC.

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells.</p><p><b>METHODS</b>We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells.</p><p><b>RESULTS</b>The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly.</p><p><b>CONCLUSION</b>With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.</p>

Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.</p><p><b>METHODS</b>Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.</p><p><b>RESULTS</b>The purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.</p><p><b>CONCLUSIONS</b>Ag85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.</p>