Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

Objective To construct a qseC-deleted mutant strain of E.coli by Red recombination and to study the effect of qseC gene on biofilm formation in the mutants.Methods The chloramphenicolresistant gene flanked by homologues of target genes was amplified by PCR and electro-transformed into E.coli MC1000.When induced by L-arabinose,the plasmid pKD46 could express three recombinant proteins of λ-prophage,which led to the replacement of target gene(qseC) with chloramphenicol-resistant gene.Then the chloramphenicol-resistant gene was eliminated by FLP-promoted recombination events.The biofilm formation of wild-type and mutant strain was detected by crystal violet staining.Results The qseC-deleted mutant of E.coli was confirmed by various PCR and DNA sequencing.Gene qseC was completely deleted.There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000.The biofilm formation of wild-type and mutant strain was quantified by crystal violet staining.The absorbance determined with a plate reader at 570 nm was 1.00±0.15 and 0.47±0.10 respectively.Conclusion The qseC-deleted mutant of E.coli was constructed successfully.And the qseC gene plays an important role in regulation of biofilm formation in E.coli.

AIM: To establish a method for the quality standard of Chanfukang Granules (Radix Astragali, Herba Leonuri, Fructus Aurantii, Herba Agrimoniae, Radix Rehmanniae, etc.). METHODS: TLC was used for identification of Herba Leonuri, Fructus Aurantii, Herba Agrimoniae, Radix Rehmanniae. The content of astragaloside Ⅳ was determined by HPLC-ELSD. RESULTS: The TLC identification was highly specific and the spots clear and concentrated. The linear range of astragaloside Ⅳ was 0.336-2.016 ?g, r=0.999 4. The average recovery was 97.13% and RSD was 1.3%. CONCLUSION: The method is simple and accurate. It can be used for quality control of Chanfukang Granules.

OBJECTIVE:To optimize the ethanol mixed-extraction technology of Red ginseng and Salvia miltiorrhiza in Xin-likang granules. METHODS:L9(34)orthogonal test was adopted to optimize ethanol mixed-extraction technology with ethanol vol-ume fraction,amount of ethanol and extraction times as factors using weighting coefficient comprehensive score of the contents of ginsenoside Rg1,Re and Rb1 in R. ginseng and the contents of tanshinone ⅡA and salvianolic acid B in S. miltiorrhiza as index;and the verification test was detected. RESULTS:Optimal mixed-ethanol extraction technology was as follows as 6-fold 70% etha-nol,reflux extracting for 3 times,2 h each time. In verification test,average contents of ginsenosides Rg1,Re,Rb1 in R. ginseng and those of tanshinone ⅡA and salvianolic acid B in S. miltiorrhiza were 3.963 8,0.757 3,4.986 2,0.964 7,27.662 5 mg/g,re-spectively,and comprehensive score was 0.96 (RSD=1.26%,n=3). CONCLUSIONS:Ethanol extraction technology of R. gin-seng and S. miltiorrhiza in Xinlikang granules optimized by multiple indexes comprehensive score combined with orthogonal test is stable,reasonable and feasible.

ObjectiveTo evaluate the clinical efficacy of acupuncture at Waiguan (TE5) and Zhigou (TE6) in treating post-stroke hand spasm.MethodSixty patients with post-stroke hand spasm graded≥Ⅰand≤Ⅲby the Modified Ashworth Scale (MAS) were randomized into a treatment group (30 cases) and a control group (30 cases). The treatment group was intervened by acupuncture at Waiguan and Zhigou plus rehabilitation training, while the control group was by dry rehabilitation training alone. The acupuncture and rehabilitation were performed once a day, 5 sessions a week, totally for 3 months. The change of hand spasm degree was observed by using MAS; the Fugl-Meyer Assessment (FMA) was adopted to observe the change of hand function; the Modified Barthel Index (MBI) was used to observe the change of the activities of daily living (ADL).ResultAfter treatment, there was a significant difference in comparing the MAS score between the two groups (P<0.05); there was a significant difference in comparing the FMA score between the two groups after treatment (P<0.05); after intervention, there was a significant difference in comparing the MBI score between the two groups (P<0.05).ConclusionAcupuncture at Waiguan and Zhigou can significantly improve the hand spasm sate after stroke; acupuncture plus rehabilitation can substantially improve the hand function and ADL of the patients, and can produce a more significant efficacy compared to dry rehabilitation training.

Objective To investigate the infection status and drug suscepetibility of mycoplasma from 6 573 patients with non-gonococcal urethritis ,and to provide the scientific bases for the clinical application of antibiotics .Methods Mycoplasma detection kit was used to detect ureaplasma urealyticum (Uu) and mycoplasma hominis(Mh) and the drug susceptibility .All the patients were divided into two groups :Chinese group and foreigner group .Results Among 5 675 Chinese patients ,2 985 patients were infected by mycoplasma(52 .6% ) .The infection rate of Uu was 2 312(40 .7% ) .35 .2% patients were male ,and 61 .4% patients were female .In 898 foreign patients ,440 patients were infected by mycoplasma(49 .0% ) .The infection rate of Uu was 327(36 .4% ) .32 .2% pa-tients were male ,and 59 .5% patients were female .In Chinese patients infected by Uu ,the susceptibility rates to MIN ,DOX ,JOS and CLA were 96 .7% ,96 .2% ,93 .7% ,89 .7% ,respectively .In foreign patients ,the susceptibility rates to MIN ,DOX ,JOS ,and CLA were 98 .9% ,98 .4% ,95 .8% ,92 .1% .Conclusion The mycoplasma infection rate of Chinese patients is higher than foreign patients .In both groups ,Uu infection is the main type .Female patients are more than male patients .The drug sensitivity rate in for-eign group is higher than that in Chinese group .mycoplasma are sensitivity to MIN ,DOX ,JOS .

Objective To study the effect of epinephrine on biofilm formation of the qseC-deleted mutant of Escherichia coli on biomaterial.Methods The strains used in this study are Escherichia coli MC1000 and MC1000AqseC.LB was used for all the experiments.To determine the effect of epinephrine on motility,halos were measured in LB medium at 37℃ in the presence of epinephrine(50 μmol/L).LB with epinephrine and without epinephrine were used,and then the experiment of bacterial biofilm formation on PVC material was taken.The relative amount of biofilm was estimated.The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope( CLSM),and the surface structure of biofilm formation was observed by scanning electron microscope(SEM).Results The mutant strain formed less biofilm than the wild-type strain in LB.The increment in motility of wild-type strain due to epinephrine addition was shown,but mutant strain is unaffected.Similarly,biofilm formation of the wild-type strain was increased by epinephrine,but epinephrine did not affect the biofilm formation of the qseC mutant.The CLSM and SEM showed that epinephrine stimulated biofilm formation of wild-type strain on PVC materials,but had no effect on qseC-deleted mutant strain.Conclusion Epinephrine increases Escherichia coli biofilms on biomaterials through qseC.

Objective To investigate the role of quorum sensing Escherichia coli regulator C (qseC) in intestinal bacterial translocation in rats subjected to hemorrhagic shock.Methods Thirty Sprague-Dawley rats,weighing 250-300 g,were randomly divided into 5 groups (n =6 each):control group (group C),MC1000-sham shock group (group M-SS),MC1000qseC-sham shock group (group △-SS),MC1000-hemorrhagic shock group (group M-HS),and MC1000△ qseC-hemorrhagic shock group (group △-HS).The rats drank 150 μg/ml of disinfect water containing streptomycin in 3 consecutive days to inhibit the autochthonous flora in the intestinal tract.From 4th day,the rats were fed with Escherichia Coli MC1000 or MC1000△ qseC 1 ml/100 g by gastric perfusion once a day for another 3 consecutive days in the other 4 groups,while the rats were fed with normal saline instead in group C.Hemorrhagic shock was induced by blood-letting.The mesenteric lymph node (MLN),spleen and liver specimens were obtained at 24 h after operation for bacterial culture and the bacteria were identified.Bacterial translocation from gut to MLN,spleen and liver was observed and the number of bacteria in MLN,spleen and liver tissues were counted.Results The rate of bacterial translocation was significantly higher,and the number of bacterial colonies in MLN,spleen and liver tissues and the total number of bacterial colonies were significantly larger in groups M-HS and △-HS than in group C,and in group M-HS than in groups M-SS and △-SS (P ＜ 0.05).The rate of bacterial translocation was significantly lower,and the number of bacterial colonies in MLN,spleen and liver tissues and the total number of bacterial colonies were significantly smaller in group △-HS than in group MHS.Conclusion QseC is involved in the intestinal bacterial translocation following hemorrhagic shock in rats.

AIM: To explore the role of Akt/NF-?B pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-?B. RESULTS: Mesangial cells cultured in vitro had a low level NF-?B activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-?B was markedly increased(0.35?0.06 vs 0.75?0.16, P

AIM: To investigate expression and function of CD40 ligand by peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). METHODS: Expression of CD40 ligand by PBMCs in patients with SLE and control were examined by flow cytometric analysis before and after stimulated by phytohemagglutinin(PHA)and depressed by Dexamethasone(Dex). The correlation between expression of CD40 ligand and SLE activity index(SLEDAI) was analysed in patients with SLE. RESULTS: The expression of CD40 ligand by PBMCs in patients with active SLE was higher than that in patients with inactive SLE and control. Though the expression of CD40 ligang by PBMCs could be stimulated by PHA in three groups, it was the highest in patients with active SLE. Dex depressed the expression of CD40 ligand by PBMCs significantly in patients with SLE , but not in control. There was high positive correlation between expression of CD40 ligand and SLEDAI in patients with active and inactive SLE. CONCLUSION: Increased expression of CD40L by PBMCs in patients with SLE may play an important role in pathogenesis of SLE.