The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) an...The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.展开更多
AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transfor...AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.展开更多
Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to ex...Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.展开更多
文摘The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.
文摘AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.
基金Fund supported by the Healthcare Technology Plan of Zhejiang Provincial Health Bureau(No.2016KYB292)the Technology Plan of Science and Technology Bureau of Jiaxing,Zhejiang province(No.2016AY23054)~~
文摘Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.
