Objective To analyze the complete genome sequence of a coxsackievirus B 3 (CVB3) strain KM06/2009 and its genetic variation , evolution and cardiovirulence .Methods Eight clones with overlapped gene fragments covering the complete viral genome ( excluding the poly-A tail) were obtained by RT-PCR and sequenced .The nucleotide ( nt ) and amino acid ( aa ) sequences of the eight clones were aligned with sequences of other known CVB 3 clinical strains .Phylogenetic and pairwise alignment analyses based on the genome and complete VP 1 regions were conducted by using Mega 4.1,RDP3 and SimPlot3.5.1 softwares.The RNA secondary structure of CVB3 stem loopⅡ (SLⅡ) was determined by using Mfold web server.Results The complete genome of CVB3 strain KM06/2009 was 7401 nt in length, consisting of 743 nt and 100 nt on 5′untranslated region (UTR) and 3′UTR,respectively.The entire open reading frame contained 6558 nt, encoding a 2185 aa polyprotein.There was no nucleotide deletion or insertion in the coding region,but some changes of amino acid were unique .KM06/2009 strain showed 81.4%and 95.7%identities in nucleotide and amino acid sequences respectively as compared with CVB 3 prototype Nancy strain.It shared 88.4%-98.1%nucleotide and 98.1%-99.4%amino acid homology with the other Chinese clinical strains isolated at the same period .KM06/2009 strain and CVB3 GA strain without cardiovirulence shared 80.7%homology in nucleotide and 96.4% in amino acid.The phylogenetic analysis indicated that the significant spatial and temporal clustering was detected in CVB 3 isolate.CVB3 KM06/2009 strain showed a strong cardiovirulence tendency as indicated by the RNA secondary structure of CVB 3 SL Ⅱ. Conclusion CVB3 KM06/2009 isolate showed a strong cardiovirulence tendency in comparison with other CVB3 clinical isolates based on the bioinformatics analysis .

Objective To analyze the genetic characteristics of the complete genome of a human echovirus 30 (Echo30) KM/A363/09 strain isolated in Yunnan, China in 2009.Methods Primers specif-ic for Echo30 were designed .The extracted RNA was amplified by using RT-PCR.Seven fragments covering the complete viral genome were sequenced and the complete sequences were aligned with other sequences of enterovirus reference strains downloaded from Genbank . By using Mega5.1, Geneious, RDP3 and SimPlot3.5.1 softwares, the phylogenetic and recombination analysis were carried out .Results The com-plete nucleotide sequence of KM/A363/09 isolate was 7425 bp in length, encoding 2194 amino acids.KM/A363/09 isolate was highly similar with Bastianni prototype strain showing the homology of 81.2%in nucle-otide and 95.8%in amino acid.The eight Echo30 isolates shared 81.2%-88.6%homologies in nucleotide sequences and 95.8%-97.8%in amino acid sequences .Phylogenetic analysis showed that the KM/A363/09 strain belonged to one clade of Echo 30 in China.The genetic recombination of KM/A363/09 isolate was detected in the non-structural region .Conclusion KM/A363/09 isolate belongs to one clade of Echo 30 in China indicating that the evolution of Echo 30 has occurred in China .

Objective To analysis of the detection result of amniotic fluid chromosome which in NIPT high-risk pregnant women.Methods Amniotic fluid cells via amniotic cavity puncture were cultured and analyzed,the chromosome karyotypes were observed.Results The highest positive predictive value of NIPT was for trisomy 21(85.00%),then trisomy 18(75.00%),sex chromosome abnormalities(68.00%),other chromosome abnormalities(41.67%),trisomy 13 (25.00%).Conclusion The highest accuracy of NIPT was shown in detection of Down''s syndrome by NIPT.NIPT was screening test which is effective and noninvasive in prenatal diagnosis.Amniotic fluid Chromosomal karyotype analysis was the gold standard in the diagnosis of fetal chromosomal disease.

OBJECTIVETo investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice.

METHODSThe E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit.

RESULTSThe percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups.

CONCLUSIONIntragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

Animals ; Bifidobacterium ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Spleen ; cytology ; Th1 Cells ; cytology ; Thymus Gland ; cytology ; Tumor Necrosis Factor-alpha ; blood

Objective:To get norovirus (NoV) GII.6 P protein through prokaryotic expression and prepare the polyclonal antibody.Methods:NoV GII.6 P region gene was amplified and cloned into prokaryotic expression vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein GII.6 P was expressed by induced Isopropyl β-D-Thiogalactoside (IPTG) and then purified with Ni-NTA Affinity Column. The binding ability of recombinant GII.6 P was determined by oligosaccharide binding assay and the polyclonal antibody serum was prepared by immunizing BALB/c mice. The titer of GII.6 P polyclonal antibody was determined by ELISA, and the specificity of the antibody was detected by western blot (WB). The effectiveness of GII.6 P polyclonal antibody was assessed. Results:The recombinant GII.6P-pET28a plasmid was constructed successfully and the recombinant GII.6 P protein was expressed with relative molecular mass of 40 ×10 3. The purity of GII.6P protein was more than 90% after purification. The oligosaccharide binding showed that the GII.6 P protein binds to B, le b and H2, but does not bind to A, H1, and le a type; the titer of GII.6 P polyclonal antibody was 1∶160 000. WB indicated that the antibody had high specificity and the cross experiments did not show affinity to GII.4. Conclusions:The GII.6P protein has been expressed successfully and the GII.6 P polyclonal antibody with high titer was prepared, which provides an effective tool for detection and vaccine development for NoV GII.6.

OBJECTIVETo analyze the biochemical and pathological changes in mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet combined with low-dose streptozotocin (STZ) injections.

METHODSC57BL/6J mice were divided randomly into normal control group (NC group), high-fat diet group (HC group) and high-fat diet plus STZ group (HC+STZ group). The mice were fed on normal chow or a high-fat diet for 1 month before two introperitoneal injections of STZ (40 mg/kg) or citrate buffer with an interval of 24 h as appropriate. Fasting blood glucose (FBG) was detected every week for 4 weeks, and oral glucose tolerance test (OGTT) was performed one month after the injections, after which the biochemical profiles, islet and liver were evaluated by immunohistochemical and pathological analysis.

RESULTSIn HC+STZ group, FBG was above the cutoff value (13.89 mmol/L) in 75% of the mice at 1 week after STZ injections and in all the mice at two weeks except for the death of 1 mouse, with a success rate of modeling of 91.3%. FBG in HC group, though slightly higher than that in NC group, remained normal (6.8 mmol/L). The body weight in HC+STZ and HC groups was significantly higher than that in NC group after feeding but without obvious increases after the injections (P<0.01). Blood glucose in HC+STZ group at 0.5 to 2 h after OGTT and the area under curve (AUC) were higher than those in NC and HC groups (P<0.01); the AUC in HC group was a also higher than that in NC group (P<0.05). Plasma creatinine was significantly higher in HC+STZ group than in NC (P<0.01) and HC (P<0.05) groups. Insulin secretion by the islets decreased obviously in HC+STZ and HC group. The mice in HC+STZ group showed atrophy, fibrosis, and vacuolization in the islets with mild fatty liver but no visible renal pathologies.

CONCLUSIONHigh-fat diet and low-dose STZ injections can induce T2DM in mice with very similar biochemical and pathological changes to human T2DM and with such complications as fatty liver.

Animals ; Blood Glucose ; Body Weight ; Diabetes Mellitus, Type 2 ; physiopathology ; Diet, High-Fat ; Fatty Liver ; physiopathology ; Glucose Tolerance Test ; Insulin ; Insulin Resistance ; Islets of Langerhans ; pathology ; Kidney ; pathology ; Mice ; Mice, Inbred C57BL ; Streptozocin

Objective To investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice. Methods The E.coli-Bifidobacterium shuttle expression vector containing hIFN-αgene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010/ml) was prepared after induction with 0.2%L-arabinose for hIFN-αexpression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γand TNF-αwere assayed using mouse cytokine FlowCytomix Kit. Results The percentages of CD3+CD8+and CD4+CD8+cells in the thymus, CD3+CD4+, CD3+CD8+and CD4+CD8+cells in the spleen, and CD3+CD8+cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γalso increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups. Conclusion Intragastric administration of hIFN- α- transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

Objective To investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice. Methods The E.coli-Bifidobacterium shuttle expression vector containing hIFN-αgene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010/ml) was prepared after induction with 0.2%L-arabinose for hIFN-αexpression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γand TNF-αwere assayed using mouse cytokine FlowCytomix Kit. Results The percentages of CD3+CD8+and CD4+CD8+cells in the thymus, CD3+CD4+, CD3+CD8+and CD4+CD8+cells in the spleen, and CD3+CD8+cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γalso increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups. Conclusion Intragastric administration of hIFN- α- transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.