目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳...目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳性的重组表达质粒转入感受态大肠杆菌中。分别在高温及低温条件下,少量诱导表达重组蛋白Tyr p 34以进行蛋白鉴定;大量诱导表达该重组蛋白并采用亲和层析法纯化,将纯化后的目的蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测并进行免疫印迹分析(Western blot)及生物信息学分析。结果 双酶切结果显示Tyr p 34基因与载体成功连接。少量表达后进行蛋白鉴定,显示目的基因在大肠杆菌中成功表达,Tyr p 34蛋白主要以可溶性蛋白的形式存在,在高温组中含量相对较多。大量表达后纯化的重组Tyr p 34蛋白分子量约为20 kDa。Western blot显示有明显条带,表明该重组基因所表达的蛋白可与腐食酪螨过敏患者的血清反应。生物信息学分析结果显示Tyr p 34基因大小为462 bp,编码153个氨基酸。氨基酸序列与NCBI公布的Tyr p 34氨基酸序列(登录号:ACL36923.1)同源性为99%。Tyr p 34蛋白有5个磷酸化位点,主要由无规则卷曲和α-螺旋构成。结论腐食酪螨Tyr p 34基因成功克隆并表达、纯化出重组蛋白,该蛋白具有免疫原性。通过Tyr p 34的生物信息学分析,有利于深入研究腐食酪螨过敏原的结构和理化性质。展开更多
The signaling events underlying oral mucosal inflammatory responses to P. gingivalis and its key endotoxin, lipopolysaccharide (LPS), relay primarily on the LPS engagement of Toll-like receptor-4 (TLR4), and the activ...The signaling events underlying oral mucosal inflammatory responses to P. gingivalis and its key endotoxin, lipopolysaccharide (LPS), relay primarily on the LPS engagement of Toll-like receptor-4 (TLR4), and the activation of IκB-kinase complex (IKK) and mitogen-activated protein kinases (MAPKs that exert their control over transcription factors implicated in the regulation of iNOS and COX-2 proinflammatory genes expression). Since spleen tyrosine kinase (Syk) has emerged recently as a major amplifier in the production of proinflammatory mediators, we investigated the process of recruitment and interaction of Syk with TLR4 in salivary gland acinar cells in response to P. gingivalis LPS. Our findings revealed that stimulation of the acinar cells with the LPS leads to protein kinase Cδ (PKCδ)-mediated phosphorylation of Syk on Ser which results in its localization with the membrane associated TLR4 complex and the activation through phosphorylation on Tyr. Further, our results support the involvement of Syk in the amplification of transcription factors involved in the assembly and expression of transcription complexes associated with the induction in COX-2 and iNOS genes. Therefore, our data suggest that PKCδ is a primary linchpin affecting the Syk recruitment to the membrane localized TLR4, and hence affects the efficiency of the kinase activation and the magnitude of oral mucosal inflammatory response to P. gingivalis.展开更多
Up-regulation in salivary gland acinar cell MMP-9 secretion in response to proinflammatory challenge by periodontopathic bacterium, P. gingivalis relays heavily on the factors that influence the protein processing at ...Up-regulation in salivary gland acinar cell MMP-9 secretion in response to proinflammatory challenge by periodontopathic bacterium, P. gingivalis relays heavily on the factors that influence the protein processing at the level of endoplasmic reticulum-to-Golgi trafficking, and occurs in concert with the changes in the stability dynamics of the major cytoskeleton polymeric structures, microtubules (MTs). In this study, we report that P. gingivalis LPS-elicited induction in the acinar cell MMP-9 secretion is accompanied by the enhancement in MT stabilization, while the modulatory influence of peptide hormone, ghrelin, is associated with MT destabilization. Further, we reveal that the changes in MT dynamics induced by the LPS and ghrelin occur through signal-regulated α-tubulin phosphorylation on Ser/Tyr. The LPS effect is reflected in a marked increase in PKCδ-mediated α-tubulin phosphorylation on Ser, whereas the modulatory influence of ghrelin on MT dynamics is manifested in by SFK-dependent phosphorylation of α-tubulin on Tyr. Moreover, we show that that the changes in MT dynamics, conferred by the LPS and ghrelin, affect the Golgi localization of GTP-Arf1 and the recruitment and activation of PKD2. The findings underscore the role of signal-regulated changes in MT stability dynamics through PKCδ/SFK-mediated α-tubulin phosphorylation on Ser/Tyr in controlling the salivary gland acinar cell MMP-9 secretion in response to P. gingivalis LPS as well as its modulation by ghrelin.展开更多
目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基...目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基因序列(GeneBank登录号AY710432.1)设计引物,PCR大量扩增目的基因。构建原核表达载体pET-32a(+)-Tyr p 13,转化感受态细胞E. coli Rosetta(DE3),用IPTG(异丙基-β-D-硫代半乳糖苷)诱导目的蛋白表达;层析纯化表达产物,免疫印迹(Western Blot)法检测纯化后的表达产物免疫学活性;通过生物信息学软件推测其抗原表位、构建进化树。结果克隆得到的序列经基因测序可知其长度约400 bp,其表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分子量约为14 kD,呈可溶性表达。免疫印迹结果显示,Tyr p 13能够与过敏患者血清IgE特异性结合,表明其具有过敏原性;表位分析进一步证实该过敏原具有免疫原性。结论经克隆表达、纯化后得到纯度较高的腐食酪螨Tyr p 13,为进一步开展临床特异性诊断和治疗提供参考。展开更多
文摘目的 克隆腐食酪螨Tyr p 34基因,表达、纯化出重组蛋白,并鉴定其免疫原性。方法 提取腐食酪螨的总RNA,通过反转录聚合酶链式反应获得其cDNA片段。根据引物和cDNA进行PCR扩增,通过酶切连接构建表达载体pET-28a(+)-Tyr p 34,并将鉴定为阳性的重组表达质粒转入感受态大肠杆菌中。分别在高温及低温条件下,少量诱导表达重组蛋白Tyr p 34以进行蛋白鉴定;大量诱导表达该重组蛋白并采用亲和层析法纯化,将纯化后的目的蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测并进行免疫印迹分析(Western blot)及生物信息学分析。结果 双酶切结果显示Tyr p 34基因与载体成功连接。少量表达后进行蛋白鉴定,显示目的基因在大肠杆菌中成功表达,Tyr p 34蛋白主要以可溶性蛋白的形式存在,在高温组中含量相对较多。大量表达后纯化的重组Tyr p 34蛋白分子量约为20 kDa。Western blot显示有明显条带,表明该重组基因所表达的蛋白可与腐食酪螨过敏患者的血清反应。生物信息学分析结果显示Tyr p 34基因大小为462 bp,编码153个氨基酸。氨基酸序列与NCBI公布的Tyr p 34氨基酸序列(登录号:ACL36923.1)同源性为99%。Tyr p 34蛋白有5个磷酸化位点,主要由无规则卷曲和α-螺旋构成。结论腐食酪螨Tyr p 34基因成功克隆并表达、纯化出重组蛋白,该蛋白具有免疫原性。通过Tyr p 34的生物信息学分析,有利于深入研究腐食酪螨过敏原的结构和理化性质。
文摘The signaling events underlying oral mucosal inflammatory responses to P. gingivalis and its key endotoxin, lipopolysaccharide (LPS), relay primarily on the LPS engagement of Toll-like receptor-4 (TLR4), and the activation of IκB-kinase complex (IKK) and mitogen-activated protein kinases (MAPKs that exert their control over transcription factors implicated in the regulation of iNOS and COX-2 proinflammatory genes expression). Since spleen tyrosine kinase (Syk) has emerged recently as a major amplifier in the production of proinflammatory mediators, we investigated the process of recruitment and interaction of Syk with TLR4 in salivary gland acinar cells in response to P. gingivalis LPS. Our findings revealed that stimulation of the acinar cells with the LPS leads to protein kinase Cδ (PKCδ)-mediated phosphorylation of Syk on Ser which results in its localization with the membrane associated TLR4 complex and the activation through phosphorylation on Tyr. Further, our results support the involvement of Syk in the amplification of transcription factors involved in the assembly and expression of transcription complexes associated with the induction in COX-2 and iNOS genes. Therefore, our data suggest that PKCδ is a primary linchpin affecting the Syk recruitment to the membrane localized TLR4, and hence affects the efficiency of the kinase activation and the magnitude of oral mucosal inflammatory response to P. gingivalis.
文摘Up-regulation in salivary gland acinar cell MMP-9 secretion in response to proinflammatory challenge by periodontopathic bacterium, P. gingivalis relays heavily on the factors that influence the protein processing at the level of endoplasmic reticulum-to-Golgi trafficking, and occurs in concert with the changes in the stability dynamics of the major cytoskeleton polymeric structures, microtubules (MTs). In this study, we report that P. gingivalis LPS-elicited induction in the acinar cell MMP-9 secretion is accompanied by the enhancement in MT stabilization, while the modulatory influence of peptide hormone, ghrelin, is associated with MT destabilization. Further, we reveal that the changes in MT dynamics induced by the LPS and ghrelin occur through signal-regulated α-tubulin phosphorylation on Ser/Tyr. The LPS effect is reflected in a marked increase in PKCδ-mediated α-tubulin phosphorylation on Ser, whereas the modulatory influence of ghrelin on MT dynamics is manifested in by SFK-dependent phosphorylation of α-tubulin on Tyr. Moreover, we show that that the changes in MT dynamics, conferred by the LPS and ghrelin, affect the Golgi localization of GTP-Arf1 and the recruitment and activation of PKD2. The findings underscore the role of signal-regulated changes in MT stability dynamics through PKCδ/SFK-mediated α-tubulin phosphorylation on Ser/Tyr in controlling the salivary gland acinar cell MMP-9 secretion in response to P. gingivalis LPS as well as its modulation by ghrelin.
文摘目的克隆腐食酪螨第13组变应原基因(Tyr p 13),表达纯化其重组蛋白,并进行免疫学特性鉴定,利用生物信息学软件分析该过敏原抗原表位等信息。方法挑取腐食酪螨,提取螨总RNA。逆转录-聚合酶链式反应(RTPCR)扩增cDNA,根据已知的Tyr p 13基因序列(GeneBank登录号AY710432.1)设计引物,PCR大量扩增目的基因。构建原核表达载体pET-32a(+)-Tyr p 13,转化感受态细胞E. coli Rosetta(DE3),用IPTG(异丙基-β-D-硫代半乳糖苷)诱导目的蛋白表达;层析纯化表达产物,免疫印迹(Western Blot)法检测纯化后的表达产物免疫学活性;通过生物信息学软件推测其抗原表位、构建进化树。结果克隆得到的序列经基因测序可知其长度约400 bp,其表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分子量约为14 kD,呈可溶性表达。免疫印迹结果显示,Tyr p 13能够与过敏患者血清IgE特异性结合,表明其具有过敏原性;表位分析进一步证实该过敏原具有免疫原性。结论经克隆表达、纯化后得到纯度较高的腐食酪螨Tyr p 13,为进一步开展临床特异性诊断和治疗提供参考。
