The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.

K88ac genes, heat-stable enterotoxin I (ST1) mutant genes and heat-labile enterotoxin B subunit (LTB) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac-ST1-LTB fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac-ST1-LTB fusion protein was highly expressed in recombinant strain BL21 (DE3)(pXKST3LT5) and the expression level of the K88ac-ST1-LTB fusion protein was about 75.53% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro. These sera antibodies were able to neutralize the biological activity of native ST1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain.
Animals ; Bacterial Toxins ; genetics ; immunology ; isolation & purification ; Electrophoresis, Polyacrylamide Gel ; methods ; Enterotoxins ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Gene Expression ; Genetic Engineering ; Mice ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Recombination, Genetic ; Sodium Dodecyl Sulfate

Objective To investigate the sedative and hypnotic interaction between remifentanil and propofol by target-controlled infusion (TCI) during induction of anesthesia.Methods Third-two ASA Ⅰ or Ⅱpatients,aged 22-63 yr,body mass index 18-25 kg/m2,scheduled for elective surgery under general anesthesia,were randomly divided into 4 groups(n=8 each).Group Ⅰ only received TCI pmpofol.GroupⅡ,Ⅲ,and Ⅳreceived a target concentration of 2,4 or 6 ng/ml remifentanil respectively.While the blood-effect site concentrations of remifentanil were equilibrated,patients received TCI of propefol,with an initial target concentration of 0.5μg/ml.After the blood-effect site concentrations of propofol were equilibrated then with 0.5μg/ml increments until the loss consciousness was achieved.The eyelash reflex and state of consciousness were assessed and radial arterial blood sample 6 ml was taken every 3 min to determine the remifentanil and propofol concentrations in blood.Propofol and remifentanil concentrations in blood were measured by reversed-phase high-performance liquid chromatography and high-performance liquid chromatography with ultraviolet detection respectively.The sedative and hypnotic interaction between propofol and remifentanil was determined with a pharmacodynamie interaction model by regression analysis and determined using the isobolographic method.Results Propofol concentrations in blood were lower in group Ⅱ,Ⅲ and Ⅳ than group Ⅰ(P<0.05).The propofol concentratopms in blood were significantly decreased in trun with the increase in the remifentanil concentrations in blood in group Ⅱ-Ⅳ(P<0.05).At loss of eyelash reflex and loss of consciousness of patients,the pharmacodynamic interaction model by curve fitting was superior to linear regression (P<0.05).At loss of eyelash reflex of patients,the curve fitting result showed EC50,prop=2.77μg/ml and EC50,rem=26.67 ng/ml,and the isobolographic method equation is ECprop/2.77+ECrem/26.67=0.69.At loss of consciousness of patients,the curve fitting result showed EC50,prop==3.76μg/ml and EC50,rem=31.56ng/ml,and the isobolographic method equation is Ecprop/3.76+Ecrem/31.56=0.65.Conclusion Remifentanil (Cp 2-6 ng/ml) and propofol by TCI shows a synergistic type of pharmacodynamic interaction on the sedative and hypnotic during induction of anesthesia.

Gene engineering is the main course of biological engineering. It should be adapted to the demand of innovation spirit, practice ability and comprehensive quality of students. Educational reform of gene engineering conducted by constructing system of theory and practice, optimizing course teaching content, strengthening practice teaching content, using modern teaching technology, strengthening web course construction and improving teaching methods. We pay attention to impart specialty knowledge and learning methods to students. Its aim was to increase teaching effects and meet the demands of bioengineering specialty and qualified personal training in 21 century.

OBJECTIVE: To investigate the expression of matrix metalloproteinase-3 after brain contusion and its applicability for estimating the age of brain contusion. METHODS: Rats had been divided into three groups: control group, sham operation group and brain contusion group. The expression of matrix metalloproteinase-3 at different time was detected by immunohistochemistry and Western blot. RESULTS: By the immunohistochemistry, no staining was observed in control and sham operation groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually in 24 hours and peaked 5 days after contusion, then started to decrease, 14 days after contusion still could be observed. By the Western blot analysis, no expression of MMP-3 was detected in control and sham groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually and maximized 5 days after contusion, then started to decrease, 14 days after contusion still could be found. CONCLUSION Time-order expression of MMP-3 could be used for estimating the age of brain contusion in forensic pathology.
Animals ; Blotting, Western ; Brain Injuries/enzymology* ; Forensic Pathology ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 3/genetics* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors

Aim To investigate the potential mechanism of Zushima in the treatment of rheumatoid arthritis (RA). Methods Through PubMed literature mining, combined with tcmsp, TCMIP, Batman TCM, Pub- Chem, Swiss target prediction, GeneCards and other databases, the active ingredients, action targets and RA targets of Zushima were obtained. The active ingredient RA target pathway network was constructed by u- sing Cytoscape 3. 7. 2 software. The protein-protein interaction network was constructed by string database. David online tool was used to analyze the enrichment of GO and KEGG pathway. The molecular docking was verified in autodocking Vina and other software. Results Through screening, 22 chemical constituents were obtained, and 273 potential targets were identi fied. GO (P <0.05) enriched 1 376 GO items, mainly involving cell surface receptor connection signal transduction, plasma membrane part, nucleotide binding and so on. KEGG ( P < 0. 05) pathway analysis showed that 58 signaling pathways were involved in the focal adhesion pathway, chemokine signaling pathway, MAPK signaling pathway and so on. Conclusions The multi-component and multi-target mechanism of Zushima in treating rheumatoid arthritis can be studied by network pharmacology method, which provides scientific basis for experimental research and clinical application of Zushima.

Silibinin, a natural flavonoid antioxidant isolated from extracts of the milk thistle herb, has recently been identified as having anti-hepatotoxic and anticancer properties. In this paper, we investigated the effects of silibinin on behavior and neuroplasticity in mice subjected to chronic unpredictable mild stress (CUMS). After 5 consecutive weeks of CUMS, the mice were treated with silibinin (100 mg/kg, 200 mg/kg and 400 mg/kg by oral gavage) for 3 consecutive weeks. The results showed that silibinin administration significantly alleviated the CUMS-induced depressive-like behavior, including the total number of squares crossed and the frequency of rearing in the open field test, the immobility time in the tail suspension test and the forced swimming test. Furthermore, silibinin treatment increased the levels of brain-derived neurotrophic factor (BDNF), serotonin (5-HT) and norepinephrine (NE) in the prefrontal cortex and hippocampus. Our study provides new insight into the protective effects of silibinin on the depressive status of CUMS mice, specifically by improving neuroplasticity and neurotransmission.
Animals ; Brain-Derived Neurotrophic Factor ; Depression ; Hindlimb Suspension ; Hippocampus ; Mice* ; Milk Thistle ; Neuronal Plasticity ; Norepinephrine ; Physical Exertion ; Prefrontal Cortex ; Serotonin ; Synaptic Transmission

We isolated and identified the genotypes and molecular characteristics of the imported B3 measles virus (MeV) in the Chinese mainland. The Vero/SLAM cell line was used to isolate the viruses. Reverse transcription-polymerase chain reaction was undertaken to amplify the 450 nucleotide acids of the 3-terminal of the nucleoprotein gene. A phylogenetic tree was constructed and similarities in homology assessed. Results suggested that the Shanghai isolates MVi/Shanghai. CHN/38. 13/02 [B3] and MVi/Shanghai. CHN/40. 13/02[B3] were clustered within the same genotype group as the World Health Organization (WHO) B3 genotype reference strain. The number of differences in nucleotide acids between the two Shanghai isolates was one. The homology of nucleotide acids between the Shanghai isolates and the WHO B3 genotype reference strain (MVi/Ibadan. NGA/0.97/1/B3) was 98%. Comparative results from the Measles Nucleotide Surveillance system suggested that the sequences of Shanghai isolates and the 2013 vi- ruses from Australia, Japan, Korea, Hong Kong China, Philippines and Iran were identical. This is the first time that the B3 genotype of MeV in the Chinese mainland has been isolated since 1993. These data can be used to create a "baseline" of genetic information for measles viruses in China, and help to trace the transmission of measles viruses in China and the rest of the world.
China ; Genotype ; Humans ; Measles ; virology ; Measles virus ; classification ; genetics ; isolation & purification

BACKGROUNDIn cardiology, it is controversial whether different therapy strategies influence prognosis after acute coronary syndrome. We examined and compared the long-term outcomes of invasive and conservative strategies in patients with non-ST-segment elevation myocardial infarction (NSTEMI) and characterized the patients selected for an invasive approach.

METHODSA total of 976 patients with acute NSTEMI were collected from December 2006 to October 2012 in the First Affiliated Hospital of Dalian Medical University Hospital. They are divided into conservative strategy (586 patients) and invasive strategy (390 patients) group. Unified follow-up questionnaire was performed by telephone contact (cut-off date was November, 2013). The long-term clinical events were analyzed and related to the different treatment strategies.

RESULTSThe median follow-up time was 29 months. Mortality was 28.7% (n = 168) in the conservative group and 2.1% (n = 8) in the invasive management at long-term clinical follow-up. The secondary endpoint (the composite endpoint) was 59.0% (n = 346) in the conservative group and 30.3% (n = 118) in the invasive management. Multivariate analysis showed that patients in the conservative group had higher all-cause mortality rates than those who had the invasive management (adjusted risk ratio [RR] = 7.795; 95% confidence interval [CI]: 3.796-16.006, P < 0.001), and the similar result was also seen in the secondary endpoint (adjusted RR = 2.102; 95% CI: 1.694-2.610, P < 0.001). In the subgroup analysis according to each Thrombolysis in Myocardial Infarction risk score (TRS), log-rank analysis showed lower mortality and secondary endpoint rates in the invasive group with the intermediate and high-risk patients (TRS 3-7).

CONCLUSIONSAn invasive strategy could improve long-term outcomes for NSTEMI patients, especially for intermediate and high-risk ones (TRS 3-7).

Acute Coronary Syndrome ; mortality ; pathology ; therapy ; Aged ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; mortality ; pathology ; therapy ; Prognosis ; Retrospective Studies

Throat swabs collected from patients whose serum was measles IgM negative and rubella IgM positive during 2001-2011 were used to conduct cell culture for rubella virus. After identification of cell culture with RT-PCR, nucleotide of gene E1 of rubella virus was amplified and sequenced, followed by molecular epidemiological analysis. A total of 31 rubella viruses were isolated from 60 throat swabs. Compared 27 isolates with the WHO reference strains of all genotypes, phylogenetic tree was constructed based on the amplified 739 nucleotide fragment. These isolates belonged to two different genotypes respectively. Isolates 11009, 11052 and 11106 in 2011 belonged to genotype 2B, and others belonged to genotype 1E. Most of mutations were nonsense mutation, and sequence of amino acid was highly conserved. Amino acid sequence of most isolates of genotype 1E was identical, which suggested rubella viruses from same transmission chain might be transmitted continually since 2001. Rubella virus genotype 2B was found to be popular for the first time in Shanghai in 2011. The nucleotide sequences of these genotype 2B isolates showed 99% identity compared with that of isolates recently from Vietnam, Japan and Argentina. The resources of these strains were not confirmed due to the absence of rubella virus surveillance before.
Adolescent ; Adult ; Amino Acid Sequence ; Child ; Child, Preschool ; China ; epidemiology ; Humans ; Infant ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Rubella ; epidemiology ; virology ; Rubella virus ; classification ; genetics ; isolation & purification ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; Young Adult