Objective To construct,express and identify the anti-idiotypic antibody single chain variable fragment(scFv) against Edwardsiella tarda.Methods By using RT-PCR method,the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody(mAb) 1E11 against Edwardsiella tarda were cloned and joined with a(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH was constructed.It was then cloned into vector plasmid pET-28a,expressed in Escherichia coli BL21(DE3),and confirmed by SDS-PAGE,Western blot and ELISA.Results The recombinant scFv could be expressed in E.coli BL21(DE3) in a fusion protein pattern.The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded.The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv had the binding activity to the antigen.Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21(DE3),providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

Educational reform of pathophysiology oriented to clinical application is to pass the physician qualifica -tion examination .One of essential approach is to implement pathophysiology teaching with the translational medical philosophy and promote the harmonious development of physician -patient relationship with the utilization of the de-velopment and changes of disease in the teaching process .In that way, the pathophysiology in basic and clinical medicine is worthy of the name of “bridge”, and ultimately achieves the goal of “the transformation and develop-ment of the cultivation of clinical application talents”.

With comparison of three different methods for the marking of amines compound, an optimal deri vatization method was selected.5-(2-Hydroxyethyl) benzoacridine (HBA) reacts with coupling agent N,N'-carbonyldiimidazole(CDI) to form an activated amide intermediate 2-(benzoacridin) ethyl-imidazole-1-carbox-ylate(BAEIC).BAEIC, which is dual-sensitive probe, reacts preferably with amino compounds at 80 ℃ in the presence of 4-dimethylaminopyridine(DMAP) catalyst in N,N-dimethylformamide(DMF) solvent to give the corresponding sensitively fluorescent derivatives with an excitation maximum at λ_(ex) of 280 nm and an emis sion maximum at λ_(em) of 510 nm.BAEIC-amine derivatives simultaneously exhibited high ionization potential with percent ionization (changing from 5.62% to 58.08% in aqueous acetonitrile and from 2.14% to 56.58% in aqueous methanol.Derivatives were not only sensitive to fluorescence but also to MS ionizable potential.The fluorescence detection limits(5/iV = 3) were 0.12-0.59 μg/L.The online APCI-MS detection limits were 1.9-14 μg/L(S/N=5).

AIM: To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS: The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS: Compared with control group, incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein, and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels, increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation, and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells, and reduced Akt protein phosphorylation and NO production.CONCLUSION: Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.

Objective To investigate the effects of puerarin on the expression of human umbilical vein endothelial cells (HUVECs) tissue factor (TF) and tissue factor pathway inhibitor (TFPI) induced by oxidized low-density lipoprotein (ox-LDL).Methods After HUVECs were incubated with different concentrations of puerarin and 50 mg/L ox-LDL,the expression of TF and TFPI mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blot respectively.Results Compared with control,treatment with ox-LDL caused the augment of TF mRNA and protein expression (P<0.01),and the decrease of TFPI mRNA and protein expression.However,50,100,and 200 μmol/L puerarin blunted the augment of TF mRNA and protein expression and weakened the inhibition of TFPI mRNA and protein expression induced by ox-LDL(P<0.01).Conclusions Puerarin reduces HUVECs TF and TFPI mRNA and protein induced by ox-LDL.

Objective To study the application value of MSCT in preoperative evaluation of rectal cancer before surgery.Methods Clinical materials of 146 patients with colonoscopy and clinical proved rectal cancer were recruited.MSCT were performed with plain and triphasic dynamic contrast enhancement before surgery.The value of MSCT was evaluated by comparing the results of pre-operative evaluation with the surgical results.Results 130 cases accepted surgical treatment:3 cases of transanal local excision,28 cases of Miles operation,3 cases of Hartmann operation,96 cases of Dixon.Preoperative evaluation of MSCT:33 cases of Miles,4 cases of Hartmann,88 cases of Dixon,5 cases of local excision of the anus.The accuracy on MSCT preoperative evaluation was 84.62%. MSCT preoperative evaluation consistency was consistent with postoperative outcome(k=0.653,χ2=225.352,P=0.000).Conclu-sion It is high accuracy for MSCT in preoperative evaluation of rectal cancer,and it can provide important significance for preopera-tive selection of surgical procedures.

Objective To explore the weight for the training target of Applied nursing undergraduate. And find the degree of importance for the indexes of the target , thus highlighting theApplied training objective characteristics. Methods Developed by Bloom's teaching objectives classification theory and educational objectives theory to refined indexes, using Delphi method to establish the Applied nursing undergraduate training objectives. By the importance assignment from experts on each index, using analytic hierarchy process (AHP) method to construct the judgment matrix. Then calculate the weight of each index weight, and test its logical consistency. Results The weight of theattitudes and valueswas 0.56, andknowledgeandabilitywere 0.22. For the secondary indicators, in the values dimension,professional attitudeandideological qualityhad relatively high weight.Under the knowledge dimension, the professional knowledge had the highest weight; and for the ability dimension,communication skillsandapplicationswere heavily maximum. All the random consistency rates (CR) for the indexes were less than 0.10, so there was no logical confusion. ConclusionsAppliednursing undergraduate talents need to combine theory and practice quickly into clinical work. By weight setting, this study provides basis to further quantify and highlight the Applied nursing undergraduate training features, and provides the basis for the application-oriented undergraduate nursing personnel reforms.

Objective To evaluate the value of ultrasound in the diagnosis of congenital intestinal malrotation and midgut vovulus.Methods The findings of ultrasound of 161 children with clinically suspected intestinal malrotation were analysed retrospectively,and were compared with the results of the operation and/or the upper gastrointestinal contrast study.Results The positive sign of ultrasound for congenital intestinal malrotation was that the superior mesenteric vein(SMV) being located at the left of the superior mesentery artery(SMA),or being directly anterior to the SMA,and the positive sign for midgut vovulus was clockwise whirlpool sign (clockwise rotation of the small bowel,its mesentery and the SMV around the SMA).The sensitivity of ultrasound for congenital intestinal malrotation was 82％,and the specificity was 94％.Those for midgut vovulus were 94％ and 94％ respectively.Conclusions Special sign as the superior mesenteric vein(SMV) being located at the left of the superior mesentery artery(SMA) strongly indicate congenital intestinal malrotatione,and the clockwise whirlpool sign means great possibility of midgut vovulus.

Objective To develop the Applied nursing undergraduate students training framework objective, clear the entry of applied training target for undergraduate nursing students. Methods Through literature review and panel discussions, Delphi expert consultation questionnaires were preliminary designed. 23 experts were issued via email and paper questionnaires, conducted two expert advice and analysis. Results A total of 20 valid questionnaires were recovered after two consultation. The expert advice of good scientific reliability:experts had positive reply, and the authority level was 0.857, Kendall were 0.130-0.293, it showed a good level of coordination (P < 0.05). By two rounds of expert advice, three capacity index target level for Applied nursing undergraduate students training were gotten: attitudes, values and basic literacy; knowledge of the target; the ability. The secondary capacity index for target had 15 items, and the third capacity index had 63 items. Conclusions TheAppliednursing undergraduate students training objectives and targets were refined through Delphi which provide the basis for the Applied nursing undergraduate training model and curriculum reform.