Objective To explore the expression of novel protein kinase C ε ( PKCε) in normal prostate ( NP) tissue, benign prostate hyperplasia ( BPH) ,pericancerous ( PC) tissue and prostate cancer ( Pca) ,and study its corre...Objective To explore the expression of novel protein kinase C ε ( PKCε) in normal prostate ( NP) tissue, benign prostate hyperplasia ( BPH) ,pericancerous ( PC) tissue and prostate cancer ( Pca) ,and study its correlation with the grade and stage of Pca. Methods Ten NP slides,ten BPH slides,ten PC slides and 43 Pca展开更多
Objective:A novel protein was first purified from Panax quinquefolius L.(AGNP),and in vitro antioxidant activities of AGNP were first studied in this work.Methods:AGNP was purified by Ion-exchange chromatography and G...Objective:A novel protein was first purified from Panax quinquefolius L.(AGNP),and in vitro antioxidant activities of AGNP were first studied in this work.Methods:AGNP was purified by Ion-exchange chromatography and Gel-filtration chromatography.The chemical characterizations of AGNP were tested by sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE),high-pressure gel-filtration chromatography,MALDI-TOF-MS and HPLC.In vitro antioxidant effects were tested in simple antioxidant assay including 2,2-diphenylpicrylhydrazyl radical scavenging,superoxide radical(O2^-)scavenging,hydroxyl radical(OH)scavenging,and ferric-reducing ability.Results:The results showed which the content of AGNP measured by Bradford method was 2.42 mg/m L and the subunit molecular weight of AGNP measured by sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE)was 15 kD.The AGNP molecular weight was 15,114 Da both of SDS-PAGE and mass spectrum purity.The result of high-pressure gel-filtration chromatography demonstrated that the molecular weight of AGNP was 31,086 Da,which implied that AGNP was a homodimer.The in vitro Antioxidant results indicated that AGNP had obvious effects to remove the free radicals in vitro.Conclusion:In conclusion,AGNP had more powerful antioxidant capacity and it can be used as an effective natural antioxidant to alleviate oxidative stress.展开更多
Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cel...Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.展开更多
Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion tha...Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion that accumulation of Aβis a primary cause of AD pathogenesis（Huang and Mucke,2012）.Amyloid precusor protein（APP）processing is dependent on its subcelluar trafficking pathway：Aβis derived from APP by proteolyric processing.展开更多
To clone,express and purify C12orf49 recombinant protein.To prepare rabbit anti-C12orf49 protein polyclonal antibody and further elucidate its biological function.Methods PCR was applied to amplify the gene C12orf49 i...To clone,express and purify C12orf49 recombinant protein.To prepare rabbit anti-C12orf49 protein polyclonal antibody and further elucidate its biological function.Methods PCR was applied to amplify the gene C12orf49 in vitro.pET-32a(+)-C12orf49,the recombinant protein prokaryotic expression vector,was transformed into E.coli.IPTG was used as an inductive agent to obtain C12orf49 recombinant protein,then the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)and Western blot.Specific polyclonal antibody was derived from rabbits that was immunized by recombinant protein.ELISA and Western blot were used to detect its titer and specificity,respectively.MTT cell proliferation experiment was carried out to observe the effect of protein on proliferation of HepG2 cells.Results The C12orf49 recombinant protein was expressed in a large quantity.Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000.And a good specificity of the antibody was confirmed by Western blot.C12orf49 recombinant protein might have an advanced effect on the proliferation of HepG2 cells.Conclusions Using C12orf49 recombinant protein,we can obtain the polyclonal antibody with great titer and good specificity.Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.展开更多
文摘Objective To explore the expression of novel protein kinase C ε ( PKCε) in normal prostate ( NP) tissue, benign prostate hyperplasia ( BPH) ,pericancerous ( PC) tissue and prostate cancer ( Pca) ,and study its correlation with the grade and stage of Pca. Methods Ten NP slides,ten BPH slides,ten PC slides and 43 Pca
基金financial support from the Jilin Provincial Key Laboratory of Biomacromolecules of Chinese Medicine, Local Standard Project of Chinese Medicinal Materials in Jilin Province (No. PZ-2016-03 and No. JLPZGF-2018-004)The Science and Technology Development Plan Project of Jilin Province (No. 20190101010JH, No. 20190304095YY)+1 种基金The National Key Research and Development Program of China (2017YFC1702106)the National Traditional Chinese Medicine Standardization Project of China (No. ZYBZH-C-JL-22 and No. ZYBZH-C-HEB-11)
文摘Objective:A novel protein was first purified from Panax quinquefolius L.(AGNP),and in vitro antioxidant activities of AGNP were first studied in this work.Methods:AGNP was purified by Ion-exchange chromatography and Gel-filtration chromatography.The chemical characterizations of AGNP were tested by sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE),high-pressure gel-filtration chromatography,MALDI-TOF-MS and HPLC.In vitro antioxidant effects were tested in simple antioxidant assay including 2,2-diphenylpicrylhydrazyl radical scavenging,superoxide radical(O2^-)scavenging,hydroxyl radical(OH)scavenging,and ferric-reducing ability.Results:The results showed which the content of AGNP measured by Bradford method was 2.42 mg/m L and the subunit molecular weight of AGNP measured by sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE)was 15 kD.The AGNP molecular weight was 15,114 Da both of SDS-PAGE and mass spectrum purity.The result of high-pressure gel-filtration chromatography demonstrated that the molecular weight of AGNP was 31,086 Da,which implied that AGNP was a homodimer.The in vitro Antioxidant results indicated that AGNP had obvious effects to remove the free radicals in vitro.Conclusion:In conclusion,AGNP had more powerful antioxidant capacity and it can be used as an effective natural antioxidant to alleviate oxidative stress.
文摘Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.
文摘Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion that accumulation of Aβis a primary cause of AD pathogenesis（Huang and Mucke,2012）.Amyloid precusor protein（APP）processing is dependent on its subcelluar trafficking pathway：Aβis derived from APP by proteolyric processing.
文摘To clone,express and purify C12orf49 recombinant protein.To prepare rabbit anti-C12orf49 protein polyclonal antibody and further elucidate its biological function.Methods PCR was applied to amplify the gene C12orf49 in vitro.pET-32a(+)-C12orf49,the recombinant protein prokaryotic expression vector,was transformed into E.coli.IPTG was used as an inductive agent to obtain C12orf49 recombinant protein,then the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)and Western blot.Specific polyclonal antibody was derived from rabbits that was immunized by recombinant protein.ELISA and Western blot were used to detect its titer and specificity,respectively.MTT cell proliferation experiment was carried out to observe the effect of protein on proliferation of HepG2 cells.Results The C12orf49 recombinant protein was expressed in a large quantity.Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000.And a good specificity of the antibody was confirmed by Western blot.C12orf49 recombinant protein might have an advanced effect on the proliferation of HepG2 cells.Conclusions Using C12orf49 recombinant protein,we can obtain the polyclonal antibody with great titer and good specificity.Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.
文摘【目的】由新型鹅星状病毒(novel goose astrovirus,nGAstV)引起的雏鹅痛风病对我国养鹅业造成了重大经济损失。通过构建nGAstV纳米抗体(nanobody,Nb)噬菌体展示文库,获得识别nGAstV ORF2蛋白的特异性Nb,可为建立nGAstV抗体检测方法及研究nGAstV ORF2蛋白结构和功能奠定基础。【方法】使用蔗糖密度梯度离心方法纯化在LMH细胞中增殖的nGAstV,RT-PCR鉴定nGAstV并通过细胞病变测定nGAstV滴度。用0.06%甲醛溶液灭活纯化的nGAstV作为免疫原,免疫两周岁羊驼。首次免疫时用灭活纯化的nGAstV与等量弗氏完全佐剂乳化后免疫,第2—5次免疫用灭活纯化的nGAstV与等量弗氏不完全佐剂乳化。每间隔两周免疫一次,每次免疫剂量均为50μg。第5次免疫14 d后,通过间接ELISA方法测定羊驼血清中针对nGAstV的IgG抗体效价。待IgG抗体效价达到构建噬菌体抗体展示文库标准时,分离羊驼外周血淋巴细胞(peripheral blood lymphocyte,PBL),然后提取PBL总RNA将其反转录为cDNA,利用巢氏PCR方法扩增重链抗体重链可变区(variable domain of the heavy chain of heavy chain antibodies,VHH)基因,将其构建至pComb噬菌体载体并结合噬菌体展示技术构建nGAstV Nb噬菌体展示文库,计算该文库库容量并分析其多样性。nGAstV作为靶抗原,通过三轮富集淘选初步获得与nGAstV反应的重组噬菌体Nb阳性克隆,将其克隆至pcDNA3.1-Fc真核表达载体并进行测序分析,将测序成功且序列不同的质粒转染至HEK-293F细胞中表达,并通过聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达情况。以nGAstV为靶标抗原,利用间接ELISA方法和Western blot试验对表达成功的Nb进行特异性、反应活性验证,并以nGAstV ORF2蛋白为靶标抗原,利用间接ELISA方法对Nb进行亲和力验证,获得生物学活性较好的Nb。【结果】RT-PCR结果显示,nGAstV增殖成功;Reed-Muench法分析nGAstV滴度达4.38 Log10TCID50/mL。羊驼经5次免疫nGAstV后,通过间接ELISA方法测得其血清中针对nGAstV的抗体效价达到1:64000;利用巢氏PCR方法扩增出VHH并成功构建库容量为3.8×108CFU/mL的nGAstV Nb噬菌体展示文库;遗传进化树分析显示,该噬菌体Nb库多样性良好。三轮富集淘选后,初步获得39株与nGAstV反应的重组噬菌体纳米抗体阳性克隆,其中有25株序列不同;SDS-PAGE鉴定结果显示,共有10株Nb在HEK-293F细胞中表达成功。通过ELISA、Western blot验证进一步获得8株与nGAstV ORF2蛋白特异性反应的Nb,且1株Nb生物学活性最好。【结论】首次筛选到与nGAstV ORF2蛋白特异性反应的Nb,为nGAstV的基础研究和检测方法提供材料。
