目的克隆TNF家族B细胞激活因子（B cell activating factor to the TNF family,BAFF）胞外区134～285（sBAFF134-285）氨基酸残基段缺失突变体（s△BAFF）的cDNA,并进行表达及纯化.方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向...目的克隆TNF家族B细胞激活因子（B cell activating factor to the TNF family,BAFF）胞外区134～285（sBAFF134-285）氨基酸残基段缺失突变体（s△BAFF）的cDNA,并进行表达及纯化.方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向PCR法,扩增缺失编码sBAFF的142～160位氨基酸的核苷酸序列.经测序证实后,克隆入原核表达载体pQE-80L.经IPTG诱导表达,SDS-PAGE和Western blot检测表达产物,Ni2＋-NTA柱层析纯化目的蛋白.结果经一步反向PCR扩增后得到401 bp的DNA片段,该片段序列与GenBank报道的编码人△BAFF的胞外区（s△BAFF）cDNA序列一致.含s△BAFF的表达载体在大肠杆菌中可表达出相对分子质量为18 000的蛋白质并以包涵体的形式存在,经Ni2＋-NTA柱层析纯化后得到高纯度的目的蛋白.结论已成功地制备出人s△BAFF蛋白,为其功能的研究创造了条件.展开更多
Background:B-cell activating factor(BAFF)is vital for B cell survival.Serum BAFF levels are elevated in thrombotic antiphospholipid syndrome,but little is known about levels in patients with positive antiphospholipid ...Background:B-cell activating factor(BAFF)is vital for B cell survival.Serum BAFF levels are elevated in thrombotic antiphospholipid syndrome,but little is known about levels in patients with positive antiphospholipid antibodies(aPLs)and previous adverse pregnancy outcomes(APOs).We aimed to analyze serum BAFF concentrations of these patients in early pregnancy along with different pregnancy outcomes.Methods:Thirty-six pregnant patients positive for aPLs and previous APOs(patient group),25 healthy pregnant females(HP group)and 35 healthy non-pregnant females(HNP group)from the Peking University Third Hospital,between October 2018 and March 2019,were enrolled in this study.Serum of HNP and serum of patients as well as HP in the first gestational trimester were collected.Enzyme-linked immunosorbent assay kits were used to measure serum BAFF and interferon-alpha(IFN-a)concentrations.Cytometric bead array analysis was used to measure serum concentrations of cytokines.The patient group was further divided into APOs and non-APOs(NAPOs)group,fetal loss and live birth group according to pregnancy outcomes.The Mann-Whitney U-test was used to assess significance between and within groups.Spearman rank-order was used to evaluate correlation coefficients between BAFF and related cytokines.Results:The serum BAFF level in HP group was significantly lower than HNP group(245.24[218.80,265.90]vs.326.94[267.31,414.80]pg/mL,Z=-3.966,P<0.001).The BAFF level was obviously elevated in patient group compared to that in HP group(307.77[219.86,415.65]vs.245.24[218.80,265.90]pg/mL,Z=-2.464,P=0.013).BAFF levels in APOs group tended to be higher than that in NAPOs group(416.52[307.07,511.12]vs.259.37[203.59,375.81]pg/mL,Z=-2.718,P=0.006).Compared to HP group,concentrations of IFN-a,interleukin(IL-6)and tumor necrosis factor were higher in patient group(33.37[18.85,48.12]vs.13.10[6.85,25.47]pg/mL,Z=-2.023,P=0.043;39.16[4.41,195.87]vs.3.37[2.92,3.90]pg/mL,Z=-3.650,P<0.001;8.23[2.27,64.46]vs.1.53[1.25,2.31]pg/mL,Z=-3.604,P<0.001,respectively).Serum BAFF levels had a positive correlation with the concentrations of both IL-6 and IL-10(IL-6:r=0.525,P=0.002;IL-10:r=0.438,P=0.012).Conclusions:Serum BAFF levels are increased in patients with positive aPLs and previous APOs as compared to healthy pregnant females and tend to be higher in individuals with current APOs.The BAFF levels have a positive correlation with serum IL-6 and IL-10.展开更多
Objective To investigate the expression of B cell activation factor of the TNF family （BAFF） gene between human normal and inflamed fallopian tubes. Methods Tissue samples of human normal fallopian tube （n=20） and...Objective To investigate the expression of B cell activation factor of the TNF family （BAFF） gene between human normal and inflamed fallopian tubes. Methods Tissue samples of human normal fallopian tube （n=20） and inflamed fallopian tube （n=20） were collected. The expression of BAFF gene was determined by the real- time reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry. Results BAFF immunostaining appeared on the cellular membrane and in the cytoplasm of tubal epithelial cells. Both BAFF protein and mRNA in normal fallopian tubes had lower levels than those in inflamed fallopian tubes （P〈0.01）. Conclusion BAFF protein and mRNA are present in human tubal tissues. BAFF gene in human inflamed fallopian tube would have a high expression.展开更多
目的:进化筛选与B淋巴细胞活化因子(B cell activating factor be longing to the TNF family,BAFF)特异结合的单链DNA适体,鉴定其结合力。方法:应用指数方式富集的配体系统进化(systemati cevolution of ligands by exponential enrich...目的:进化筛选与B淋巴细胞活化因子(B cell activating factor be longing to the TNF family,BAFF)特异结合的单链DNA适体,鉴定其结合力。方法:应用指数方式富集的配体系统进化(systemati cevolution of ligands by exponential enrich-ment,SELEX)技术,将随机序列寡核苷酸文库与BAFF分子相互作用,分离出结合的寡核苷酸配体,经反复扩增、筛选数个循环,获得重组人BAFF的单链DNA适体,应用Dot blotting鉴定其结合力,酶联法比较各适体序列对BAFF的相对结合力。结果:优化实验条件后成功进行了13轮次筛选,随机选取42个阳性克隆测序,各序列GC含量最高达75%,分为6类不同序列;Dotblotting证实这些适体均与BAFF相结合;比较各适体序列结合BAFF的光密度值,以序列44光密度值最高(0.752,P<0.05),具有对BAFF相对最高结合力。结论:首次成功获得特异结合BAFF的高亲和力的单链DNA适体,为研发防治肿瘤和自身免疫疾病新药奠定了基础。展开更多
基金the National Natural Science Foundation of China(No.81501390).
文摘Background:B-cell activating factor(BAFF)is vital for B cell survival.Serum BAFF levels are elevated in thrombotic antiphospholipid syndrome,but little is known about levels in patients with positive antiphospholipid antibodies(aPLs)and previous adverse pregnancy outcomes(APOs).We aimed to analyze serum BAFF concentrations of these patients in early pregnancy along with different pregnancy outcomes.Methods:Thirty-six pregnant patients positive for aPLs and previous APOs(patient group),25 healthy pregnant females(HP group)and 35 healthy non-pregnant females(HNP group)from the Peking University Third Hospital,between October 2018 and March 2019,were enrolled in this study.Serum of HNP and serum of patients as well as HP in the first gestational trimester were collected.Enzyme-linked immunosorbent assay kits were used to measure serum BAFF and interferon-alpha(IFN-a)concentrations.Cytometric bead array analysis was used to measure serum concentrations of cytokines.The patient group was further divided into APOs and non-APOs(NAPOs)group,fetal loss and live birth group according to pregnancy outcomes.The Mann-Whitney U-test was used to assess significance between and within groups.Spearman rank-order was used to evaluate correlation coefficients between BAFF and related cytokines.Results:The serum BAFF level in HP group was significantly lower than HNP group(245.24[218.80,265.90]vs.326.94[267.31,414.80]pg/mL,Z=-3.966,P<0.001).The BAFF level was obviously elevated in patient group compared to that in HP group(307.77[219.86,415.65]vs.245.24[218.80,265.90]pg/mL,Z=-2.464,P=0.013).BAFF levels in APOs group tended to be higher than that in NAPOs group(416.52[307.07,511.12]vs.259.37[203.59,375.81]pg/mL,Z=-2.718,P=0.006).Compared to HP group,concentrations of IFN-a,interleukin(IL-6)and tumor necrosis factor were higher in patient group(33.37[18.85,48.12]vs.13.10[6.85,25.47]pg/mL,Z=-2.023,P=0.043;39.16[4.41,195.87]vs.3.37[2.92,3.90]pg/mL,Z=-3.650,P<0.001;8.23[2.27,64.46]vs.1.53[1.25,2.31]pg/mL,Z=-3.604,P<0.001,respectively).Serum BAFF levels had a positive correlation with the concentrations of both IL-6 and IL-10(IL-6:r=0.525,P=0.002;IL-10:r=0.438,P=0.012).Conclusions:Serum BAFF levels are increased in patients with positive aPLs and previous APOs as compared to healthy pregnant females and tend to be higher in individuals with current APOs.The BAFF levels have a positive correlation with serum IL-6 and IL-10.
文摘Objective To investigate the expression of B cell activation factor of the TNF family （BAFF） gene between human normal and inflamed fallopian tubes. Methods Tissue samples of human normal fallopian tube （n=20） and inflamed fallopian tube （n=20） were collected. The expression of BAFF gene was determined by the real- time reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry. Results BAFF immunostaining appeared on the cellular membrane and in the cytoplasm of tubal epithelial cells. Both BAFF protein and mRNA in normal fallopian tubes had lower levels than those in inflamed fallopian tubes （P〈0.01）. Conclusion BAFF protein and mRNA are present in human tubal tissues. BAFF gene in human inflamed fallopian tube would have a high expression.
文摘目的:进化筛选与B淋巴细胞活化因子(B cell activating factor be longing to the TNF family,BAFF)特异结合的单链DNA适体,鉴定其结合力。方法:应用指数方式富集的配体系统进化(systemati cevolution of ligands by exponential enrich-ment,SELEX)技术,将随机序列寡核苷酸文库与BAFF分子相互作用,分离出结合的寡核苷酸配体,经反复扩增、筛选数个循环,获得重组人BAFF的单链DNA适体,应用Dot blotting鉴定其结合力,酶联法比较各适体序列对BAFF的相对结合力。结果:优化实验条件后成功进行了13轮次筛选,随机选取42个阳性克隆测序,各序列GC含量最高达75%,分为6类不同序列;Dotblotting证实这些适体均与BAFF相结合;比较各适体序列结合BAFF的光密度值,以序列44光密度值最高(0.752,P<0.05),具有对BAFF相对最高结合力。结论:首次成功获得特异结合BAFF的高亲和力的单链DNA适体,为研发防治肿瘤和自身免疫疾病新药奠定了基础。
