Objective To express and purify the E protein Domain Ⅲ(ED Ⅲ) of tick-borne encephalitis virus(TBEV) in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify ED Ⅲ gene fragment by PCR.Two ED Ⅲ gene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G_4S)_3 using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-2ED Ⅲ.After sequencing,pET-28a-2ED Ⅲ was transformed into E.coli BL21(DE3) competent cells,induced by IPTG and purified by Ni~(2+) affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of ED Ⅲ amino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2ED Ⅲ was identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV "Senzhang" strain(JQ650523.1) included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2ED Ⅲ protein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21 000 and a purity of 97.5%.The titer of rabbit anti-2ED Ⅲ serum polyclonal antibody was 1:10~7,which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of ED Ⅲ amino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV) and Dengue virus(DENY) was 36.56%,9.28% and 30.77%,respectively.Conclusion The TBEV envelope ED Ⅲ tandem recombinant expression plasmid pET-28a-2ED Ⅲ was successfully constructed.The expressed recombinant 2ED Ⅲ protein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily. It is highly related to the formation of metabolic syndrome and the glucose homeostasis, and therefore represents an important drug target against metabolic diseases and diabetes. In recent years, great progress has been made in the agonists, antagonists, and crystal structures of FXR. The diverse FXR ligands and their structure-activity relationship are reviewed in this article. The advances in the crystal structures of FXR in complex with different ligands are also introduced.

Objective:To express recombinant protein mIL-21-hIgGFc in 293E cells,and investigate its effect on CD8+T cell.Methods:Total RNA was extracted from the mouse spleen cells ,and then IL-21 gene was amplified by RT-PCR and inserted into expression vector PTT3-hIgGFc.PTT3-mIL-21-hIgGFc were transfected into 293E cells by calcium phosphate method.The supernatants were collected at 48 hours and 72 hours and concentrated by MOLLIPORE Labscale TM TFF system ( 5 kD membrane ).The mIL-21-hIgGFc fusion protein was purified with HiTrap TM Protein G column.The protein was quantified by SDS-PAGE and ELISA.The biological activity of the protein was determined by detecting the change of the phenotypes of CD 8+ T cells treated with the protein.Results: The constructed recombinant plasmid PTT 3-mIL-21-Fc was confirmed by sequencing.PTT3-mIL-21-Fc was transfected into 293E cells,mIL-21-Fc protein in culture supernatant was collected after 48 hours and 72 hours.The protein in cell su-pernatant reached a concentration of 787 ng/ml which was determined by ELISA.The protein was purified by Protein G chromatography column.P1A-specific T cells were treated with mIL-21-hIgGFc, and found that the CD44low CD62Lhi CD8+ population increased compared to the control.Conclusion:We built PTT3-mIL-21-hFc recombinant plasmid, expressed mIL21-hFc fusion protein in 293E cells,and purified by Protein G column.By treating mIL-21-hFc ,the antigen-primed CD8+T cells prefer to differentiate into CD44low CD62Lhi CD8+T cells which had been reported as a memory stem phenotype .This protein may be used to improve the effectness of adoptive T cell cancer therapy.

Objective To investigate the effect of gastrodin on rat vascular smooth muscle cell (VSMC) migration induced by platelet-derived growth factor-BB (PDGF-BB) and its possible mechanisms.Methods Enzyme digestion method wasused to obtain rataorticVSMCs and be purified bypassage.Immunofluorescence staining was used to identify VSMC marker proteins.A PDGF-BB induced cell migration model was established.Transwell chamber assay was used to evaluate the effect of gastrodin on PDGF-BB induced VSMC migration.Western blots were performed to detect the phosphohorylation levels of c-jun N-terminal kinase (JNK).Results The purity of primary cultured VSMC was more than 99％.The VSMC migrated number in the PDGF-BB group was 85.2 ± 3.486 per field.It was significantly more than 42.5 ± 1.927 per field in the control group (t =9.981,P＜0.001),and gastrodin was enable to make PDGF-BB induced the number of VSMC migration significantly reduce to 71.3 ± 1.783 per filed (t=3.550,P =0.002).Western blots analysis showed that gastrodin inhibited PDGF-BB induced JNK phosphorylation (0.190 ± 0.015 vs.0.190 ± 0.015; t =14.548,P =0.000).Conclusions Gastrodin inhibits PDGF-BB induced VSMC migration,its mechanisms may be associated with the inhibition of the JNK signaling pathway activation.

The aim of this study was to observe whether necroptosis is involved in the process of cardiac hypertrophy induced by pressure overload. SD rats underwent transverse abdominal aortic constriction (TAC) operation for establishing cardiac hypertrophy model. The structure and function of the left ventricle of rats were evaluated via echocardiography, left ventricular mass index, the expression of markers of cardiac hypertrophy and histological detection. Real-time PCR and Western blot were used to measure the gene and protein expression of receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3, the necroptosis markers) respectively. Four weeks after TAC operation, rat model for cardiac hypertrophy was established. The experimental data showed that the gene and protein expressions of RIPK1 and RIPK3 in the rat heart hypertrophic tissues after TAC for 4 weeks were increased significantly compared with those in the sham group. HE staining showed cardiomyocytes injury and hypertrophy in the hearts of TAC rat models. By transmission electron microscope, we observed that mitochondria of cardiomyocytes were damaged seriously in the TAC models. Treatment with losartan used, the selective antagonist of angiotensin II type I receptor could improve the cardiac function of TAC rats. Moreover, losartan treatment decreased the expression of RIPK1 and RIPK3 in heart tissues of TAC rats. The results suggest that necroptosis occurrs in the process of cardiac hypertrophy with pressure overload, and losartan could alleviate the cardiac hypertrophy and inhibit necroptosis.
Animals ; Apoptosis ; Cardiomegaly ; pathology ; Disease Models, Animal ; Echocardiography ; Heart ; physiopathology ; Losartan ; pharmacology ; Myocytes, Cardiac ; Pressure ; Protein-Serine-Threonine Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptor-Interacting Protein Serine-Threonine Kinases ; metabolism

The aim of this study is to construct specific shRNA expressing plasmids, and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation (H/R). RIPK1 and RIPK3 are the key kinases mediating the process of necroptosis. Using recombinant DNA technology, we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively. We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively, before we treated them with H/R stimulation. Then, we measured the relevant genes and proteins by real-time PCR and Western blot. Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury. The results showed that inhibition of ripk1 or ripk3 gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.
Animals ; Apoptosis ; Cell Hypoxia ; Cell Line ; Gene Expression ; Myocytes, Cardiac ; pathology ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; Rats ; Real-Time Polymerase Chain Reaction ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; Transfection

Objective To investigate the correlation between serum homocysteine (Hcy) and macrophage migration inhibitory factor(MIF) with carotid atherosclerosis and between serum Hcy and MIF ,and to study whether serum Hcy influence the carotid atherosclerosis formation by MIF .Methods 258 inpatients and outpatients were performed the color ultrasound examination to ob-serve the carotid arterial vascular anatomic form ,endomembrane circumstance ,plaque and the plaque echo nature ,and the carotid in-tima-media thickness(IMT) was measured .According to the results of color ultrasound ,the cases were divided into three groups :IMT normal group(control group) ,thickening group and plaque group .According to the plaque echo characteristics ,the plaque group was redivided into two subgroups :stable plaque group and unstable plaque group .Serum Hcy and MIF levels and biochemical parameters were measured simultaneously in all cases .The differences of serum Hcy and MIF levels were compared between groups .The correlation coefficients among serum Hcy levels ,MIF levels and IMT were calculated .Results The serum Hcy and MIF levels in the control group ,thickening group and plaque group were increased in turn ,the difference among groups was statisti-cally significant(P<0 .01);which in the unstable plaque group was significantly higher than that in the stable plaque group ,the difference between them was statistically significant (P<0 .01) .The positive correlation was found among serum Hcy levels ,serum MIF levels and IMT (r=0 .584 ,0 .562 ,0 .607 ,P<0 .01) .Conclusion The serum Hcy and MIF levels are closely related with the carotid atherosclerosis degree and the plaque stability ;the serum Hcy and MIF levels are positively correlated with the carotid arte-rial IMT ;serum Hcy is positively correlated with the MIF level ,Hcy may cause the carotid atherosclerosis formation via MIF .

It's important that the university teachers apply proper linguistic strategies in the Traditional Chinese medicine courses for foreign students,which can reduce the non-native language ability limitation to teaching effect to a certain extent,which can let students better understand and grasp the contents.This paper summarize the influence of linguistic strategies on teaching effect,such as signposting,signalling importance,back channeling,repetition,questioning,commenting,which is the important foundation and powerful guarantee of ideal teaching effects of EMI.

It has been well established that immune surveillance plays critical roles in preventing the occurrence and progression of tumor. More and more evidence in recent years showed the host anti-tumor immune responses also play important roles in the chemotherapy and radiotherapy of cancers. Our previous study found that tumor- targeting therapy of anti-HER2/neu mAb is mediated by CD8(+) T cell responses. However, we found here that enhancement of CD8(+) T cell responses by combination therapy with IL-15R/IL-15 fusion protein or anti-CD40, which are strong stimultors for T cell responses, failed to promote the tumor therapeutic effects of anti-HER2/neu mAb. Analysis of tumor microenviornment showed that tumor tissues were heavily infiltrated with the immunosuppressive macrophages and most tumor infiltrating T cells, especially CD8(+) T cells, expressed high level of inhibitory co-signaling receptor PD-1. These data suggest that tumor microenvironment is dominated by the immunosuppressive strategies, which thwart anti-tumor immune responses. Therefore, the successful tumor therapy should be the removal of inhibitory signals in the tumor microenvironment in combination with other therapeutic strategies.
Animals ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Breast Neoplasms ; drug therapy ; immunology ; pathology ; Cell Line, Tumor ; Female ; Humans ; Immune Tolerance ; immunology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Receptor, ErbB-2 ; immunology ; Tumor Microenvironment ; immunology

With the development of the internationalization of Chinese medicine education,higher requirements are demanded for the faculty of foreign students.How to explore a suitable way of teaching foreign students to study TCM is the problem for all the foreign education teachers.Based on the concept of Gamification,we apply the game elements or mechanism effectively in teaching which can active the classroom atmosphere,improve students' interest and enhance the effect of learning.