Saponin from Tupistra chinensis Bak Inhibits NF-κB Signaling in Sarcoma S-180 Cell Mouse Xenografts / 华中科技大学学报（医学）（英德文版）

This study examined the effect of saponins from Tupistra chinensis Bak (STCB) on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms.Cell proliferation was assessed by MTT assay.Cell cycle distribution was determined by flow cytometry.Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 μg/mL 5-fluorouracil (5-Fu) as a positive control.The activity of nuclear factor (NF)-κB was detected by gel mobility shift assay.The mRNA level of NF-κB was determined by real-time quantitative RT-PCR.The results showed that in vitro STCB inhibited the growth of S-18 0 cells in a concentration-dependent manner,which was accompanied by cell cycle arrest at S-phase.In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis.Moreover,STCB inhibited the activity of NF-κB p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts.It was concluded that STCB inhibits the proliferation and cell cycle progression of S-180 cells by suppressing NF-κB signaling in mouse xenografts.Our findings suggest STCB is a promising agent for the treatment of sarcoma.

Saponin from Tupistra chinensis Bak Inhibits NF-κB Signaling in Sarcoma S-180 Cell Mouse Xenografts / 华中科技大学学报（医学）（英德文版）

This study examined the effect of saponins from Tupistra chinensis Bak (STCB) on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms.Cell proliferation was assessed by MTT assay.Cell cycle distribution was determined by flow cytometry.Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 μg/mL 5-fluorouracil (5-Fu) as a positive control.The activity of nuclear factor (NF)-κB was detected by gel mobility shift assay.The mRNA level of NF-κB was determined by real-time quantitative RT-PCR.The results showed that in vitro STCB inhibited the growth of S-18 0 cells in a concentration-dependent manner,which was accompanied by cell cycle arrest at S-phase.In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis.Moreover,STCB inhibited the activity of NF-κB p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts.It was concluded that STCB inhibits the proliferation and cell cycle progression of S-180 cells by suppressing NF-κB signaling in mouse xenografts.Our findings suggest STCB is a promising agent for the treatment of sarcoma.

Efficient soluble expression and purification of influenza A nucleoprotein in Escherichia coli / 病毒学报

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.

Immune protective effectiveness of seasonal influenza spilt vaccine against homologous and heterogonous subtypes of influenza virus in mice / 病毒学报

To investigate the seasonal influenza split vaccine's immune protective effectiveness against the homologous and heterogonous subtypes of influenza A virus challenge and the relationship between the protective effectiveness and hemagglutination inhibition (HI) antibody titer in mice. Two components of H1N1 and H3N2 in Chinese 2008-2009 seasonal influenza spilt vaccine, were derived from vaccine strain A/Brisbane/59/2007 (H1N1)-like virus and A/Brisbane/10/2007 (H3N2)-like virus respectively, and were used to immune BALB/c mice. Firstly, different doses of the vaccines were used to immunize mice and the HA immunization dosage that can induce the HI antibody titer of 40 in mice was identified; Secondly, H1N1 vaccine immunized mice were challenged with different doses of influenza virus mouse adaptation strains of A/Brisbane/59/2007 (H1N1)-like virus (MA) (referred to as A1 virus, well matched-strain in the homologous subtype) and A/Purto Rico/8/34 (H1N1) (referred to as PR8 virus, poor matched-strain in the homologous subtype) respectively, and H3N2 vaccine immunized mice were challenged with H1N1 influenza virus of A1 strain (Heterogonous subtype), body weight changes and survival rates were observed to explore the immune protective effectiveness of influenza split vaccine against the homologous and heterogonous subtypes of influenza A virus in mice. Results indicated that HI antibody titers were elevated as the HA protein immunization dosages increased from 0.15 microg, 0.5 microg, 1.5 microg, 5 microg to 15 microg in mice, and 1.5 microg HA of the seasonal influenza split vaccine could induced HI antibody titer of 40 in mice; 3LD50, 10LD50, 30LD50, 100LD50, 300LD50,1000LD50 and 3000LD50 of influenza virus strain A1 were used to challenge the H1N1 immunization mice, 1.5 microg HA of H1N1 vaccine could 100% protect mice against challenge with 1000LD50 of matched and homologous subtype of influenza virus strains A1, mice immunized with 15 microg HA of H1N1 vaccine even could 100% protect mice against challenge with 3000LD50 of influenza virus strains A1; but mice immunized with both the 1.5 microg and 15 microg HA of H1N1 vaccine were all sacrificed when challenged with 3LD50 of the mismatched and homologous subtype of influenza virus strain PR8, and mice immunized with the high dosage of 15 microg HA of H3N2 vaccine also were all sacrificed when challenged with 3LD50 of the heterogonous subtype of influenza virus strain A1. These results suggest that 1.5 microg HA of seasonal influenza split vaccine could induced HI antibody titer of 40 after one dose in mice, this dosage of HA can effectively protect mice against matched homologous subtype of influenza virus strain, but hardly to protect mice against mismatched homologous or heterogonous subtype of influenza virus strain. These results provide materials for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.

The 50% effective dose (ED50a) of seasonal spilt influenza vaccine in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the seasonal influenza spilt vaccine's immunogenicity and the 50% effective dose (ED50a) of hemagglutin (HA) that can make 50% of the mice hemagglutination inhibition antibody (HI) titers to 40.</p><p><b>METHODS</b>The 2008-2009 seasonal influenza spilt vaccine's two components, with HA from H1N1 and H3N2 influenza virus respectively, were used as a model. Mice were immunized once or twice with different doses, and the HI antibody titers were tested to determine the immunization procedure and to evaluate the immugenicity of seasonal influenza spilt vaccine in mice; Consequently, HI antibody response kinetics of the two components were observed to determine the time point when the HI antibody titer reached the peak point; Finally, mice were immunized with different doses of HA to evaluate the ED50a that can make 50% of mice HI titers reach 40.</p><p><b>RESULTS</b>Immunization procedures study showed that one-dose of seasonal influenza vaccine induced the HI antibody titers ranged from 10 to 120, while two-dose of influenza vaccine improved the HI antibody titer 10-100 times as compared with one dose; antibody kinetics study suggested that the time point of HI antibody produced to peak is 28-35 days post one dose immunization; and the ED50a detection results indicated that one dose of 1.5 microg HA could make 50% of the mice HI antibody titer reach 40.</p><p><b>CONCLUSION</b>Seasonal influenza spilt vaccine is very immunogenic in mouse; the time point of HI antibody produced to peak is 28-35 days post one dose immunization; and the ED50a of HA is 1.5 microg, which can make 50% of the mice HI titer reach 40. The experimental results provided foundation for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.</p>

The virus isolation analysis of the H5N1 subtype human avain influenza cases in mainland China from 2005 to 2009 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyse the correlation between the virus isolation and the specimen collection of the H5N1 human high pathogenic avain influenza cases in Mainland China.</p><p><b>METHODS</b>The specimens were collected in Mainland China from 2005.10 to 2009.3 and the H5N1 viruses were isolated by passage in embryonated chicken eggs.</p><p><b>RESULTS</b>Most specimens were obtained within 14 days after disease onset. For the specimens collected within 7 days, the isolation rate was relatively high and the difference of the positive rate between different years was lower than those specimens collected after 7 days. Most of the samples in our study were collected from the upper or lower respiratory tract with few from blood, feces, et al. The isolation rate of lower respiratory specimens was higher and the difference of the positive rate between different years was relatively lower than those from upper respiratory specimens.</p><p><b>CONCLUSION</b>We suggest that the samples should be collected from lower respiratory tract during the acute phase to get the higher isolation rate.</p>

Expression of influenza A3 virus (H3N2) M2 gene in vaccinia virus Tiantan strain / 病毒学报

To construct a recombinant vaccinia virus RVJ1175M2 expressing influenza A3 virus (H3N2) M2 gene, full length gene encoding influenza virus (H3N2) M2 protein was amplified with PCR and cloned into plasmid pJSC1175 which was used for homologous recombination with vaccinia virus Tiantan strain. Along with this, a recombinant vaccinia virus RVJ1175M2 containing the M2 gene was subsequently constructed. It was identified by PCR that the gene of M2 protein was inserted into the TK locus of vaccinia virus Tiantan strain correctly and M2 protein was expressed by recombinant vaccinia virus RVJ1175M2 effectively. Two electrophoretic bands of M2 protein expressed by the infected HeLa cells, one of 15kD and the other of 13kD in accordance with related documents, was deteced by Western-blot. M2 protein distributing on the surface of the infected cells was demonstrated by immunofluorescence and flow cytometry. The results suggested that recombinant vaccinia virus RVJ1175M2 could express M2 protein effectively, this laid a foundation for comparative research on the immune effect of universal vaccine of influenza virus with different kinds of vaccine expressing M2 protein.

Development of an enzyme-linked immunosorbent assay for the diagnosis of lethal avian influenza virus infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a convenient method for diagnosis of lethal avian influenza virus infection.</p><p><b>METHODS</b>Ninety-six-well microtiter plates were coated with 100 microl of H5N1 protein (0.1 microg/ml) diluted in coating buffer. These plates were used for enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Four of the 23 suspected borderline cases of lethal avian influenza virus infection were positive by the ELISA, and all the healthy controls (n=234) were negative. These results were confirmed by hemagglutination-inhibition (HI) and neutralization tests, and results were totally consistent (100 percent) with each other among the three methods.</p><p><b>CONCLUSION</b>The ELISA may be used in the screening for lethal avian influenza virus infection.</p>

Establishment of a mouse model of primary biliary cirrhosis by AMA M2 autoantigen injection / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To establish a primary biliary cirrhosis (PBC) model by AMAM2 autoantigen injection into C57BL/6 mice.</p><p><b>METHODS</b>Mice of the model group were immunized intraperitonealy with 200 microl of purified recombinant AMAM2 autoantigen in complete Freund's adjuvant (CFA). Mice immunized with bovine serum albumin and CFA in the same way were used as negative controls. Sixty-six weeks later, mice were sacrificed and their sera were collected. Sera samples were assayed for AMAM2 autoantibody, alkaline phosphatase (ALP), ALT and total bilirubin (TBil). Their liver, stomach, muscle and kidney tissues were sectioned and stained using HE to observe the pathological changes.</p><p><b>RESULTS</b>Antibodies to AMAM2 autoantigen were readily induced in the model group. The mice in the model group had no significant changes in the level of serum ALT and TBil but had an obvious increase of ALP (P<0.05). The stomach, muscle and kidney tissues showed no evident damage while the livers had obvious pathological changes, including bile duct degeneration or proliferation, and mononuclear cell infiltration.</p><p><b>CONCLUSION</b>The AMAM2 autoantigen-induced PBC animal model was successfully established in C57BL/6 mice in our experiment and its characteristic biochemical and pathology are quite similar to that in the early stage of human PBC. This model may provide a useful experimental approach for further study of the pathogenesis and clinical treatment of human PBC.</p>