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 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.

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.

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 evaluate the relationship between quartz's deposit and expression of NF-κB in non-small cell lung cancer (NSCLC) lung tissues in Xuanwei, Yunnan Province, and to clarify the role of quartz in Xuanwei NSCLC's carcinogenic mechanism. Methods As research objects, the lung tissues of NSCLC and lung benign lesions after surgical resection were collected from July 2009 to September 2015 at the Third Affiliated Hospital of Kunming Medical University. Firstly, the transmission electron microscopic (TEM) with energy dispersive X-ray analyzer (EDS) was used for observation of crystalline deposit and local pathological changes. Secondly, expression level of NF-κB had been analysed and a correlation analysis with particle size of SiO2 crystal in the same lung sample was made. Results The occurrence rates of quartz in Xuanwei NSCLC lung tissues were above non-Xuanwei NSCLC and benign lung tissues (P<0.01);the average particle size of SiO2 crystal was (226 ± 120) nm × (237 ± 163) nm in Xuanwei NSCLC group and it was smaller than the other two groups; In Xuanwei NSCLC group, the expression level of NF-κB was significantly higher than non-Xuanwei and benign lung tissues (P< 0.01), but there was no significant difference between cancer tissues and normal lung tissues in the group (P>0.05). The expression level of NF-κB was generally increasing when quartz 's size became smaller. Conclusion Quartz 's deposit may play a certain role in carcinogenic mechanism of lung cancer in Xuanwei, the smaller the particle size, the greater the cytotoxicity.

OBJECTIVE:To establish a method for the content determination of berberine hydrochloride in the Jiangtang xiao-zhi tablets. METHODS:HPLC was conducted with the Symmetry C18 column. The mobile phase was acetonitrile-0.05 mol/L sodium dihydrogen phosphate(pH adjusted to 3.0 using phosphoric acid)(24∶76,V/V)at the flow rate of 1.0 ml/min. The detection wave-length was 345 nm,the column temperature was room temperature and injection volume was 10 μl. RESULTS:The linear range of berberine hydrochloride was 0.522-4.698 μg(r=0.999 9);RSDs of precision,stability and reproducibility tests were no more than 0.72%;the average recovery was 97.79%(RSD=2.09%,n=6). CONCLUSIONS:The method is simple,accurate,sensitive and reproducible,and can be used for the content determination of berberine hydrochloride.

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.

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.

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