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@#This study aimed to investigate the effects of fusion proteins GnRH-GRP(G3G6)and HSP65-STEAP1(HST1)on dendritic cells(DC)and the sensitization of DCs to B16F10 melanoma. The fusion proteins G3G6 and HST1 were obtained using the previous engineering strains in our laboratory. Group by unsensitized DC(US-DC), the G3G6 fusion protein sensitized DC, the HST1 fusion protein sensitized DC(HST1-DC)and the combined sensitized DC(GH-DC), the mouse bone marrow-derived DCs were sensitized with fusion protein to obtain the fusion protein sensitized DC vaccines. B16F10 melanoma cells were transplanted into C57BL/6J male mice to construct a melanoma model(1×106 cells per mouse), and DC vaccine was injected for treatment. The antitumor efficacy of DC vaccine was explored by in vitro and in vivo experiments. Flow cytometry analysis showed that the fusion protein can effectively stimulate DC into differentiation and maturation; in the animal experiment, the inhibition rate of melanoma treated with G3G6-DC was 35. 75%, that of HST1-DC group and combination group were 34. 03% and 55. 74%. It was initially proved that both G3G6-DC and HST1-DC can effectively inhibit the growth of transplanted tumors of melanoma B16F10 cells in mice, and the combination therapy is superior to the single therapy.
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Objective To investigate the significance of heat shock protein 60 (HSP60) and heat shock protein 65 (HSP65) on prognosis acute coronary syndrome (ACS) within one year.Methods Eightynine hospitalized patients were collected from department of Cardiovascular disease,the people's hospital of Wuxi city affiliated of Nanjing Medical University and the Second People's Hospital Wuxi City from November 2009 to February 2011,and divided into ACS group (n =50),stable angina pectoris (SAP) group (n =19) and nonCHD group(n =20).HSP60,HSP65 levels in human serum were measured at the time of admission.The followup records of all patients were established to observe the occurrence of coronary events during one year,and analyzed its relationship between with HSP60,HSP65.Results Eighty-four cases were successful followed-up,and lost cases were 5.Eighteen patients occurred cardiovascular events within one year,and their content of serum HSP60 and HSP65 were significantly higher than that of without cardiovascular events (HSP60:(1026.19 ± 253.47) ng/L vs.(845.75 ± 138.52) ng/L,t =2.49,P ＜ 0.05 ; HSP65:(2573.95 ± 768.75) ng/L vs.(2076.38 ± 385.46) ng/L,t =2.58,P ＜ 0.05).In ACS group,the level of serum HSP60 and HSP65 of the patients occurred cardiovascular events was significant higher than that of without cardiovascular events,and there was significant difference(HSP60:(1162.73 ±249.14) ng/L vs.(892.55 ±204.62) ng/L,t =2.19,P ＜ 0.05 ; HSP65:(2714.39 ± 738.44) ng/L vs.(2136.85 ± 472.62) ng/L,t =2.65,P ＜ 0.05).COX regression analysis showed that HSP65 was an independent risk factor for recent cardiovascular events in patients with ACS (RR =1.002,95％CI 1.000-1.004,P =0.035).Conclusion The detection of HSP60,HSP65 in prognostic coronary artery disease prognosis has important value,and HSP65 was an independent risk predictor of ACS in recent cardiovascular events within one year.
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A fused functional gene of human OPG and Mhsp65 was amplified by PCR,and cloned into the prokaryotic expression vector pET-28a.The BL21(DE3) strain of E.coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG.Result of SDS-PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body.The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis.The purified and refolded fusion protein could inhibit osteoclast proliferation and bone absorption in vitro.The results of mouse ear swelling assay and expressions of TNF-?,IFN-? and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity.The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.
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AIM: To improve the prevent efficacy of peptide p277 in autoimmune diabetes. METHODS: The recombinant expression plasmid pET28-Hsp65-6×p277 was constructed by inserting 6×p277 which were amplified by PCR into the vector pET28-Hsp65. The plasmid pET28-Hsp65-6×p277 was transformed into E.coli BL21 (DE3) and the fusion protein (Hsp65-6×p277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified and then used to immunize 4-week old female NOD mice with three times of i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at monthly interval. The concentrations of blood glucose and antibodies were measured by automatic analyzer. RESULTS: Administration with the Hsp65-6×p277 to NOD mice could prevent the development of diabetes. CONCLUSION: The fusion protein Hsp65-6×p277 might be further developed to a vaccine against insulin-dependent diabetes mellitus.